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Comparison of Gut-Associated Lymphoid Tissue HIV RNA and DNA Levels in HIV-infected Controllers, Non-controllers, and HAART-suppressed Individuals: 'Untreated non-controllers have reservoir....lymphoid tissues are an active reservoir of HIV in controllers.....treatment should be considered for these individuals'
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Reported by Jules Levin
CROI 2012 March 5-8 Seattle WA
Steven Yukl1,2, Elizabeth Sinclair1, Kara Harvill1, Lee Gilman1,
Rebecca Hoh1, Peter W. Hunt1, Jeffrey N. Martin1, Diane Havlir1, Joseph K. Wong1,2, Steven G. Deeks1, and Hiroyu Hatano1 1University of California, San Francisco; 2San Francisco Veterans Affairs Medical Center; San Francisco, CA, USA
ABSTRACT
Background: HIV+ individuals who are able to maintain very low levels of plasma HIV RNA in the absence of ART ("controllers") have provided insights into novel mechanisms of viral control, and are a model for a functional cure. We studied the degree to which HIV RNA and DNA is present in gut-associated lymphoid tissue (GALT) of controllers, non-controllers, and HAART-suppressed subjects.
Methods: Colorectal biopsies were obtained from 9 treatment-na•ve controllers (median plasma viral load 58 copies/mL), 5 treatment-na•ve non-controllers (median viral load = 33,000 copies/mL), and 8 HAART-suppressed non-controllers (median viral load <40 copies/mL). Rectal mononuclear cells were isolated using collagenase, assayed for HIV RNA and DNA by real-time PCR, and normalized to CD4+ T content as determined by flow cytometry.
Results: The median peripheral CD4+ T cell counts were 708, 424, and 478 cells/mm3 for the controllers, non-controllers, and HAART-suppressed subjects, respectively. Controllers had lower levels of HIV RNA in GALT compared to non-controllers (33 vs 43,121 copies per mil CD4+ T cells, p = 0.003), but similar levels compared to HAART-suppressed subjects (33 vs 454 copies per mil CD4+ T cells). In addition, controllers had lower levels of HIV DNA in GALT compared to non-controllers (4733 vs 1.2x106 copies per mil CD4+ T cells, p = 0.003), and a trend toward lower HIV DNA in GALT compared to HAART-suppressed subjects (4733 vs 21,824 copies per mil CD4+ T cells, p = 0.10). Interestingly, the ratio of HIV RNA:DNA in GALT was similar in controllers (0.04) and non-controllers (0.04), but slightly higher than that in HAART-suppressed subjects (0.02).
Conclusions: Untreated non-controllers have an extremely large reservoir of HIV DNA in the rectum (average of 1 copy for every CD4+ T cell). In addition, despite being able to maintain very low levels of plasma HIV RNA in the absence of ART, HIV-infected controllers have readily measurable levels of HIV RNA and DNA in GALT, and a ratio of HIV RNA:DNA comparable to that in non-controllers. These data confirm that the lymphoid tissues are an active reservoir of HIV in controllers, and that treatment should be considered for these individuals.
Background
· HIV-infected "controllers" (seropositive individuals who are able to maintain very low levels of plasma HIV RNA in the absence of antiretroviral therapy) have provided insights into novel mechanisms of viral control, and are a model for a functional cure.
· To date, quantification of the viral reservoir in controllers has been limited to studies in blood (plasma and PBMCs). Increasing evidence in non-controllers suggests that the analysis of tissue reservoirs is more critical for understanding viral persistence.
· We therefore studied the degree to which HIV RNA and DNA is present in gut-associated lymphoid tissue (GALT) of untreated controllers, and compared them to untreated non-controllers and HAART-suppressed non-controllers.
Results
·Rectal CD4+ T-cell content (% of all cells) was higher in controllers (median 7.1%) than in untreated non-controllers (4.1%, p=0.019), or a historical cohort of long-term HAART-suppressed subjects (2.7%, p=0.001) (Figure 1).
·Rectal HIV DNA (per 106 CD4+ T cells) was lower in controllers (median 4.7x103 copies) than in untreated non-controllers (1.2x106 copies, p<0.001), short-term HAART-suppressed subjects (1.2x105 copies, p=0.018), or a historical cohort of long-term HAART-suppressed subjects (2.2x104 copies, p=0.059) (Figure 2).
· Rectal HIV RNA (per 106 CD4+ T cells) was lower in controllers (median 35 copies) than in untreated non-controllers (2.8x104 copies, p=0.001). However, there was no statistical difference between controllers and HAART-suppressed subjects (438 copies, p=0.86) (Figure 3).
· Rectal HIV RNA/DNA ratios were similar in controllers (median 0.07) and untreated non-controllers (0.04, p=1.0). However, HAART-suppressed subjects tended to have lower RNA/DNA ratios (0.003) than controllers (p=0.064) (Figure 4).
· All of the above findings were also observed when HIV RNA and DNA levels were expressed as copies/106 rectal cells (instead of copies/106 CD4+ T cells).
Conclusions
· Untreated non-controllers have an extremely large reservoir of HIV DNA in the rectum (average of one copy for every CD4+ T-cell).
· Despite being able to maintain very low levels of plasma HIV RNA in the absence of antiretroviral therapy, HIV-infected controllers have readily measurable levels of HIV RNA and DNA in GALT.
· As expected, controllers had higher rectal CD4+ T-cell numbers, lower HIV DNA, and lower HIV RNA when compared to untreated non-controllers. However, HIV RNA/DNA ratios did not differ between controllers and untreated non-controllers. These data suggest that the mechanism(s) of elite control result in a lower total frequency of HIV-infected cells, but does not affect the average HIV transcription rate per infected cell.
· Controllers had lower rectal HIV DNA even when compared to HAART-suppressed non-controllers, but rectal HIV RNA did not differ between the two groups, and the average transcription per infected cell (HIV RNA/DNA ratio) tended to be lower in the HAART-suppressed non-controllers. These data suggest that compared to the mechanism(s) of elite control, antiretroviral therapy has a proportionally greater impact on HIV transcription but does not reduce the total HIV reservoir to the level of controllers.
· The observed HIV RNA/DNA ratios in controllers is consistent with (but not proof of) ongoing cycles of viral replication. Treatment studies are currently underway to define the role of viral replication and the effects of HAART in these individuals.
Methods
· Patient Characteristics: Colorectal biopsies were obtained from (Table 1):
· Controllers: 9 untreated controllers (plasma HIV RNA <75 copies/mL)
· Non-controllers (NC): 5 untreated non-controllers (plasma HIV RNA >10,000 copies/mL)
· HAART-suppressed:11 (3 short-term and 8 long-term) HAART-suppressed non-controllers (plasma HIV RNA <75 copies/mL)
· Isolation of rectal cells: 18-24 colorectal biopsy pieces were obtained from each subject and separated into single cells through 3 rounds of collagenase digestion, mechanical disruption (blunt 16g needle), and cell straining (70μm). One aliquot of cells was set aside for flow cytometry, while another was frozen for HIV RNA and DNA extraction.
· Rectal Cell-associated HIV DNA by Real Time PCR: Total DNA was extracted from rectal cells using Trireagent (using the back-extraction buffer) and purified using the QIAgen Pure Gene kit. DNA concentrations were assessed using the ND-1000 Spectrophotometer. Three replicates of up to 500ng of DNA from each sample were assayed for HIV DNA using a modification of a published real time TaqMan PCR assay that uses primers and probe from the LTR region (HxB2 positions 522-43 and 626-43). HIV DNA copy numbers were measured in triplicate and normalized to cellular input into the PCR, as determined by DNA concentration (assuming 1 μg total DNA=160,000 cells). To account for variation in the number of CD4+ T-cells in different samples, results were further normalized by the percent of all cells that were CD3+CD4+ by flow cytometry and expressed as copies/106 CD4+ T-cells.
· Rectal Cell-associated HIV RNA by qRT-PCR: Total RNA was extracted from rectal cells using Trireagent, treated with DNase, and purified with QIAgen RNeasy columns. RNA concentrations were assessed using the ND-1000 Spectrophotometer. Three replicates of up to 500ng of RNA from each sample were assayed for total processive HIV RNA transcripts using primers and probe from the LTR region (as above). HIV RNA copy numbers were measured in triplicate and normalized to cellular input into the PCR, as determined by RNA concentration (assuming that 1ng RNA=1000 cells), and to the percent of all cells that were CD3+CD4+ by flow cytometry and expressed as copies/106 CD4+T-cells.
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