Antiretroviral therapy (ART) decreases HIV-associated morbidity and mortality but does not completely restore health [1,2]. A small proportion of HIV-infected individuals (‘controllers’) are able to maintain low plasma viremia in the absence of ART [3–5]. They present a unique opportunity to better understand HIV persistence and viral control. Multiple studies have examined the potential virologic and host factors associated with viral control [6–9]. We and others have previously shown that most controllers have detectable plasma viremia and cell-associated RNA and DNA in peripheral blood mononuclear cells (PBMCs) if ultrasensitive assays are used [10–12]; and a significant proportion of controllers are infected with replication-competent virus [13,14] and not with virus that contains significant genetic defects . However, these studies have thus far been limited to measurements in blood. Given the increasing recognition that the interactions between the host and virus during low-level viremic states are more apparent in tissues than in blood [16–19], we measured HIV DNA and RNA in gut-associated lymphoid tissue (GALT) of controllers, and compared these measurements with those in untreated and ART-suppressed noncontrollers.
Patients were identified from the University of California San Francisco (UCSF) SCOPE cohort. Colorectal biopsies were obtained from five untreated noncontrollers (plasma RNA >10 000 copies/ml), five ART-suppressed noncontrollers (plasma RNA <40 copies/ml for ≥12 months), and nine untreated controllers (plasma RNA ≤1000 copies/ml for ≥12 months). All patients provided written informed consent. This study was approved by the UCSF Committee on Human Research.
For each patient, 30 colorectal biopsy specimens were obtained 10–20 cm from the anal verge using 3 mm jumbo forceps. Eighteen to 24 biopsy pieces were placed directly into 10 ml RPMI-1640 media containing fetal calf serum (15%), penicillin (100 U/ml), streptomycin (100 μg/ml), and L-glutamine (2 mmol/l). Biopsy pieces were dissociated to a cell suspension by collagenase digestion and mechanical disruption . One aliquot of cells was set aside for flow cytometry and stained with CD45-FITC, CD3-APC and CD4-PE (BD biosciences) for 15 min at 25°C. Propidium iodide was added to stain nonviable cells and samples were run on an Accuri C6; the total number of viable mononuclear cells, and the proportion and absolute number of viable CD45+ leukocytes and CD4+ T cells was determined. Another aliquot of cells was frozen at −80°C for subsequent nucleic acid extraction.
Total DNA was extracted from rectal cells using Trireagent (BD Bioscience) and further purified using the QIAgen Pure Gene kit. DNA concentrations and purity were assessed using a ND-1000 Spectrophotometer (NanoDrop). Three replicates of up to 500 ng of DNA were assayed for HIV DNA using a modification of a published TaqMan PCR assay that uses primers (HXB2 positions 522–543, 626–643) and probe (559–584) from the long terminal repeat (LTR)region . Reaction volume was 50 μl with 10 pmol of each primer, 10 pmol of probe, and 25 μl of 2× TaqMan Gene Expression Master Mix (Applied Biosystems). Cycling conditions were: 50°C for 2 min, 95°C for 10 min, then 60 cycles of 95°C for 15 s and 59°C for 1 min. External standards were prepared from DNA extracted from known numbers of 8E5 cells (NIH AIDS Reagent Program). HIV DNA copy numbers were normalized to cellular input into the PCR, as determined by DNA mass (assuming 1 μg total DNA corresponds to 160 000 cells). Results were further normalized by the percentage of all cells that were CD3+CD4+ by flow cytometry and expressed as copies/106 CD4+ T cells.
Total RNA was extracted from rectal cells using Trireagent (BD Bioscience), treated with DNase [2.5 μl RNase-free DNase (QIAgen) and 10 μl buffer RDD in a total of 100 μl for 15 min at 25°C], and purified with the QIAgen RNeasy protocol with minor modifications (precipitation with 700 μl of 100% EtOH and washing with RPE). RNA concentrations and purity were assessed using a ND-1000 Spectrophotometer. Three replicates of up to 500 ng of RNA were assayed for total processive HIV RNA transcripts using primers and probe from the LTR region (as above). Reaction volume was 50 μl with 10 pmol of each primer, 10 pmol of probe, 25 μl of TaqMan RNA-to-Ct 1-Step mix (Applied Biosystems), and 1.25 μl of 40× RT. Cycling conditions were: 48°C for 20 min, 95°C for 5 min, then 60 cycles of 95°C for 15 s and 59°C for 1 min. Genomic HIV RNA standards were prepared from lab stocks of NL4–3 virions by extracting RNA and quantifying HIV RNA via replicate measurements using the Abbot Real Time assay. HIV RNA copy numbers were normalized to cellular input into the PCR, as determined by RNA mass (assuming that 1 ng RNA correspond to 1000 cells ), which has been shown to correlate with levels of GAPDH RNA . Results were further normalized by the percentage of all cells that were CD3+CD4+ by flow cytometry and expressed as copies/106 CD4+ T cells.
All statistical analyses were conducted with GraphPad Prism version 5.04. Virologic parameters were compared between unmatched groups using the Wilcoxon rank sum test.
The median plasma RNA was 3.3 × 104 copies/ml for untreated noncontrollers, less than 40 copies/ml for ART-suppressed noncontrollers (median duration of viral suppression 8.6 years), and 58 copies/ml for controllers (Table 1).
Rectal CD4+ T-cell content (percentage of total rectal cells) was higher in controllers (median 7.1%) than in untreated noncontrollers (4.1%, P = 0.019) or ART-suppressed patients (4.3%, P = 0.007) (Fig. 1a).
Rectal HIV DNA/106 rectal cells (‘tissue burden’) was lower in controllers (median 44 copies/106 rectal cells) than in untreated noncontrollers (4558 copies/106 rectal cells; P = 0.0033) or ART-suppressed patients (254 copies/106 rectal cells, P = 0.0112). Similarly, when normalized to 106 CD4+ T cells (‘HIV per CD4+ T cell’), rectal HIV DNA was lower in controllers (median 496 copies/106 CD4+ T cells) than in untreated noncontrollers (117 483 copies/106 CD4+ T cells, P = 0.001) or ART-suppressed patients (6116 copies/106 CD4+ T cells, P = 0.004) (Fig. 1b).
Rectal HIV RNA/106 rectal cells was lower in controllers (median 2 copies/106 rectal cells) than in untreated noncontrollers (694 copies/106 rectal cells, P = 0.001), but the difference between controllers and ART-suppressed patients (70 copies/106 rectal cells) did not reach statistical significance (P = 0.1119). However, when normalized to 106 CD4+ T cells, rectal HIV RNA was lower in controllers (median 19 copies/106 CD4+ T cells) than in noncontrollers (15 210 copies/106 CD4+ T cells, P = 0.001) or ART-suppressed patients (1625 copies/106 CD4+ T cells, P = 0.0599) (Fig. 1c).
Rectal HIV RNA/DNA ratios (a measure of average transcription per infected cell) were not statistically different between the three groups (median 0.19 controllers, 0.25 untreated noncontrollers, and 0.29 ART-suppressed patients).
Given the potential limitations of ART [1,2,23], there is a growing interest in developing curative approaches in which viral control is maintained in the absence of any therapy. HIV-infected controllers may prove to be an informative model for developing such strategies. We performed extensive virologic measurements in a cohort of controllers, focusing for the first time on GALT, in which much of the viral reservoir is presumed to reside.
First, we observed that untreated noncontrollers have an extremely high burden of HIV DNA in the rectum, corresponding to an average of one copy for every 10 CD4+ T cells. This calculation, which assumes that most or all of the HIV DNA is in CD4+ T cells and is evenly distributed, should be verified in sorted and terminally diluted CD4+ T cells. However, if this approximation is true, it suggests that in most untreated noncontrollers, a large proportion of CD4+ T cells in the rectum may be infected with HIV.
Second, we observed that HIV-infected controllers have readily measurable levels of HIV DNA and RNA in the rectum, despite being able to maintain very low levels of plasma RNA in the absence of ART. As expected, controllers had higher rectal CD4+ T-cell numbers and lower rectal HIV DNA and RNA levels compared with untreated noncontrollers. However, we did not detect a difference in HIV RNA/DNA ratios. These data suggest that in controllers, the mechanisms of viral control result in a lower total frequency of HIV-infected cells but may not reduce the average HIV transcription rate per infected cell.
Controllers also had lower rectal HIV DNA and RNA levels compared with ART-suppressed noncontrollers, but HIV RNA/DNA ratios were similar between the two groups. These data suggest that compared with the mechanisms responsible for ‘natural’ host-mediated control of viral replication, long-term ART does not reduce the total HIV reservoir to the level of controllers. Strong, polyfunctional mucosal responses are at least partially responsible for the ability of controllers to limit the total HIV reservoir size [24,25].
Finally, it was notable that the degree of heterogeneity in rectal HIV DNA and RNA measurements appeared to be greater in controllers, compared with untreated noncontrollers or ART-suppressed patients. This is consistent with previous immunologic/virologic studies of controllers. Although grouped into the same phenotypic group based upon suppression of plasma viremia to low levels, multiple studies have shown that controllers are in fact a rather heterogeneous group. For example, although controllers are enriched for several HLA class I alleles, not all controllers have protective HLA alleles [7,8]. Similarly, replication-competent virus has been recovered from a significant proportion [13,14], but not all, HIV-infected controllers . The observed heterogeneity in rectal HIV DNA and RNA measurements undoubtedly reflects that multiple factors are contributing to ‘natural’ viral control.
Our study has several limitations, including a relatively small sample size and variability in duration of HIV infection, immunodeficiency, and ART regimen for ART-suppressed patients. Mucosal sampling was limited to the colorectum, and HIV measurements were limited to unsorted cells. Future studies should characterize the viral reservoir in controllers in other important locations within the gut (including ileum)  and other lymphoid tissues (lymph nodes), and should describe the relationship between reservoir size and immunologic correlates. Finally, our HIV assays measured total HIV DNA and RNA and did not allow us to distinguish between unintegrated and integrated forms of DNA, or between genomic and messenger RNA. Recent reports have shown that controllers have large excesses of unintegrated HIV DNA and 2-LTR circles in PBMCs, suggesting that they may have an intrinsic ability to block HIV integration [27,28].
Despite the apparent ability of controllers to limit the spread of infection (as measured by rectal HIV DNA), the paradoxically high relative level of transcription (as demonstrated by HIV RNA/DNA ratios that were similar to untreated noncontrollers) suggests that controllers may have productive infection and/or ongoing viral replication that could theoretically be reduced by ART. We have previously shown that controllers have higher levels of immune activation compared with ART-suppressed patients , some controllers with high levels of immune activation progress immunologically to AIDS despite maintenance of virologic control , and controllers have higher levels of atherosclerosis compared with HIV-negative patients . Thus, the ability to control HIV to low levels may be incomplete and/or dissociated from control of immune activation or end-organ damage. Prospective treatment studies are currently underway by our group to define the role of viral replication and the virologic and immunologic effects of ART in these individuals.
H.H. conceived and designed the study, recruited and enrolled study participants, conducted statistical analyses, and wrote the article. M.S. and P.W.H. obtained gut biopsy samples and edited the article. E.S. processed gut biopsy samples and edited the article. K.H., L.G., M.C., and R.H. recruited study participants. J.N.M. and S.G.D. provided conceptual advice and edited the article. J.K.W. and S.A.Y. performed HIV RNA and DNA analyses on gut biopsy samples and edited the article.
This work was supported by grants from the National Institute of Allergy and Infectious Diseases (R01 AI087145, K23 AI075985, K24 AI069994, R56 AI091573, R01 NS051132), the National Cancer Institute (K23 CA157929), the Delaney AIDS Research Enterprise (DARE; U19 AI0961090), California HIV/AIDS Research Program (grant number ID08-SF-004), American Foundation for AIDS Research (106710–40-RGRL), the UCSF/Gladstone Institute of Virology & Immunology CFAR (P30 AI027763), the UCSF Clinical and Translational Research Institute Clinical Research Center (UL1 RR024131), the Center for AIDS Prevention Studies (P30 MH62246), the CFAR Network of Integrated Systems (R24 AI067039), and the U.S. Department of Veterans Affairs (1 IK2 CX000520–01, 5101BX001048). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article.
Conflicts of interest
H.H. and S.G.D. have received research support from Merck, Inc., and Gilead, Inc. M.S., E.S., K.H., L.G., M.C., R.H., P.W.H., J.N.M., J.K.W., S.A.Y.: No conflicts of interest.
1. Phillips AN, Neaton J, Lundgren JD. The role of HIV in serious diseases other than AIDS
2. Kuller LH, Tracy R, Belloso W, De Wit S, Drummond F, Lane HC, et al. Inflammatory and coagulation biomarkers and mortality in patients with HIV infection
. PLoS Med
3. Hubert JB, Burgard M, Dussaix E, Tamalet C, Deveau C, Le Chenadec J, et al. Natural history of serum HIV-1 RNA levels in 330 patients with a known date of infection. The SEROCO Study Group
4. Lambotte O, Boufassa F, Madec Y, Nguyen A, Goujard C, Meyer L, et al. HIV controllers: a homogeneous group of HIV-1-infected patients with spontaneous control of viral replication
. Clin Infect Dis
5. Lefrere JJ, Mariotti M, Morand-Joubert L, Thauvin M, Roudot-Thoraval F. Plasma human immunodeficiency virus RNA below 40 Copies/mL is rare in untreated persons even in the first years of infection
. J Infect Dis
6. Emu B, Sinclair E, Favre D, Moretto WJ, Hsue P, Hoh R, et al. Phenotypic, functional, and kinetic parameters associated with apparent T-cell control of human immunodeficiency virus replication in individuals with and without antiretroviral treatment
. J Virol
7. Emu B, Sinclair E, Hatano H, Ferre A, Shacklett B, Martin JN, et al. HLA class I-restricted T-cell responses may contribute to the control of human immunodeficiency virus infection, but such responses are not always necessary for long-term virus control
. J Virol
8. Pereyra F, Addo MM, Kaufmann DE, Liu Y, Miura T, Rathod A, et al. Genetic and immunologic heterogeneity among persons who control HIV infection in the absence of therapy
. J Infect Dis
9. Pereyra F, Jia X, McLaren PJ, Telenti A, de Bakker PI, Walker BD, et al. The major genetic determinants of HIV-1 control affect HLA class I peptide presentation
10. Hatano H, Delwart EL, Norris PJ, Lee TH, Dunn-Williams J, Hunt PW, et al. Evidence for persistent low-level viremia in individuals who control human immunodeficiency virus in the absence of antiretroviral therapy
. J Virol
11. Pereyra F, Palmer S, Miura T, Block BL, Wiegand A, Rothchild AC, et al. Persistent low-level viremia in HIV-1 elite controllers and relationship to immunologic parameters
. J Infect Dis
12. O’Connell KA, Brennan TP, Bailey JR, Ray SC, Siliciano RF, Blankson JN. Control of HIV-1 in elite suppressors despite ongoing replication and evolution in plasma virus
. J Virol
13. Blankson JN, Bailey JR, Thayil S, Yang HC, Lassen K, Lai J, et al. Isolation and characterization of replication-competent HIV-1 from a subset of elite suppressors
. J Virol
14. Lamine A, Caumont-Sarcos A, Chaix ML, Saez-Cirion A, Rouzioux C, Delfraissy JF, et al. Replication-competent HIV strains infect HIV controllers despite undetectable viremia (ANRS EP36 study)
15. Miura T, Brockman MA, Brumme CJ, Brumme ZL, Carlson JM, Pereyra F, et al. Genetic characterization of human immunodeficiency virus type 1 in elite controllers: lack of gross genetic defects or common amino acid changes
. J Virol
16. Chun TW, Nickle DC, Justement JS, Meyers JH, Roby G, Hallahan CW, et al. Persistence of HIV in gut-associated lymphoid tissue despite long-term antiretroviral therapy
. J Infect Dis
17. Yukl SA, Gianella S, Sinclair E, Epling L, Li Q, Duan L, et al. Differences in HIV burden and immune activation within the gut of HIV-positive patients receiving suppressive antiretroviral therapy
. J Infect Dis
18. Yukl SA, Shergill AK, McQuaid K, Gianella S, Lampiris H, Hare CB, et al. Effect of raltegravir-containing intensification on HIV burden and T-cell activation in multiple gut sites of HIV-positive adults on suppressive antiretroviral therapy
19. Hatano H, Jain V, Hunt PW, Lee TH, Sinclair E, Do TD, et al. Cell-based measures of viral persistence are associated with immune activation and programmed cell death protein 1 (PD-1)-expressing CD4+ T cells
. J Infect Dis (in press)
20. Shacklett BL, Yang O, Hausner MA, Elliott J, Hultin L, Price C, et al. Optimization of methods to assess human mucosal T-cell responses to HIV infection
. J Immunol Methods
21. Kumar AM, Borodowsky I, Fernandez B, Gonzalez L, Kumar M. Human immunodeficiency virus type 1 RNA Levels in different regions of human brain: quantification using real-time reverse transcriptase-polymerase chain reaction
. J Neurovirol
22. Fischer M, Huber W, Kallivroussis A, Ott P, Opravil M, Luthy R, et al. Highly sensitive methods for quantitation of human immunodeficiency virus type 1 RNA from plasma, cells, and tissues
. J Clin Microbiol
23. Lodwick RK, Sabin CA, Porter K, Ledergerber B, van Sighem A, Cozzi-Lepri A, et al. Death rates in HIV-positive antiretroviral-naive patients with CD4 count greater than 350 cells per microL in Europe and North America: a pooled cohort observational study
24. Ferre AL, Hunt PW, Critchfield JW, Young DH, Morris MM, Garcia JC, et al. Mucosal immune responses to HIV-1 in elite controllers: a potential correlate of immune control
25. Ferre AL, Lemongello D, Hunt PW, Morris MM, Garcia JC, Pollard RB, et al. Immunodominant HIV-specific CD8+ T-cell responses are common to blood and gastrointestinal mucosa, and Gag-specific responses dominate in rectal mucosa of HIV controllers
. J Virol
26. Kloosterboer N, Groeneveld PH, Jansen CA, van der Vorst TJ, Koning F, Winkel CN, et al. Natural controlled HIV infection: preserved HIV-specific immunity despite undetectable replication competent virus
27. Graf EH, Mexas AM, Yu JJ, Shaheen F, Liszewski MK, Di Mascio M, et al. 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
. PLoS Pathog
28. Buzon MJ, Seiss K, Weiss R, Brass AL, Rosenberg ES, Pereyra F, et al. Inhibition of HIV-1 integration in ex vivo-infected CD4 T cells from elite controllers
. J Virol
29. Hunt PW, Brenchley J, Sinclair E, McCune JM, Roland M, Page-Shafer K, et al. Relationship between T cell activation and CD4(+) T cell count in HIV-seropositive individuals with undetectable plasma HIV RNA levels in the absence of therapy
. J Infect Dis
30. Hsue P, Hunt PW, Martin JN, Schnell A, Kalapus C, Deeks SG. Role of ART, viral replication, and HIV infection in atherosclerosis.
15th Conference on Retroviruses and Opportunistic Infections. Boston, MA; 2008.