The study of HIV-infected “controllers” who are able to maintain low levels of plasma HIV RNA in the absence of antiretroviral therapy (ART) may provide insights for HIV cure and vaccine strategies. Despite maintaining very low levels of plasma viremia, controllers have elevated immune activation and accelerated atherosclerosis. However, the degree to which low-level replication contributes to these phenomena is not known. Sixteen asymptomatic controllers were prospectively treated with ART for 24 weeks. Controllers had a statistically significant decrease in ultrasensitive plasma and rectal HIV RNA levels with ART. Markers of T cell activation/dysfunction in blood and gut mucosa also decreased substantially with ART. Similar reductions were observed in the subset of “elite” controllers with pre-ART plasma HIV RNA levels below conventional assays (<40 copies/mL). These data confirm that HIV replication persists in controllers and contributes to a chronic inflammatory state. ART should be considered for these individuals (ClinicalTrials.gov NCT01025427).
HIV-infected “controllers” are rare individuals who are HIV-seropositive but are able to maintain low levels of plasma HIV RNA in the absence of antiretroviral therapy (ART). There has been intense interest in characterizing these unique individuals because they have been considered as a potential model for a “functional cure” of HIV. Previously, our group has shown that controllers have elevated levels of T cell activation and accelerated atherosclerosis, suggesting that very low levels of viral replication may lead to disproportionately high levels of immune activation. However, the degree to which viral replication contributes to these outcomes is not known. We therefore conducted the first, prospective study of ART initiation in a cohort of asymptomatic HIV-infected controllers, in order to determine the virologic and immunologic effects of treating controllers with ART. Controllers had a significant decreases in ultrasensitive plasma HIV RNA, rectal HIV RNA, and markers of T cell activation/dysfunction in blood and gut mucosa with ART. Similar reductions were observed in the subset of “elite” controllers with extremely low pre-ART plasma HIV RNA levels (<40 copies/mL). These data suggest that HIV replication persists in controllers and contributes to a chronic inflammatory state.
Citation: Hatano H, Yukl SA, Ferre AL, Graf EH, Somsouk M, Sinclair E, et al. (2013) Prospective Antiretroviral Treatment of Asymptomatic, HIV-1 Infected Controllers. PLoS Pathog 9(10): e1003691. doi:10.1371/journal.ppat.1003691
Editor: Guido Silvestri, Emory University, United States of America
Received: May 24, 2013; Accepted: August 23, 2013; Published: October 10, 2013
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: 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, R01 AI057020), the National Cancer Institute (K23 CA157929), the Delaney AIDS Research Enterprise (DARE; U19 AI0961090), California HIV/AIDS Research Program (ID08-SF-004), the American Foundation for AIDS Research (106710-40-RGRL, 107170-44-RGRL, and 108073-50-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 manuscript.
Competing interests: HH and SGD have received research support from Merck, Inc., and Gilead, Inc. UO has received research support from Merck, Inc. SAY, ALF, EHG, MS, ES, MAM, TL, KH, RH, SP, PB, PWH, JNM, JMM, RPT, MPB, BLS, and JKW have no conflicts to declare. The study drug was provided by Merck, Inc., and Gilead, Inc., at no cost to this study. Reagents were provided by Gen-Probe/Hologic and Ortho Clinical Diagnostics at no cost to this study. This does not alter our adherence to all PLoS Pathogens policies on sharing data and materials.
HIV-infected “controllers” are individuals who are HIV-seropositive but are able to maintain low levels of plasma HIV RNA in the absence of antiretroviral therapy (ART) . These individuals are rare, comprising less than 1–7% of the HIV-infected population, depending upon the plasma HIV RNA criteria that are used to define the group , , . Most controllers have evidence of strong host immune responses, which have been widely assumed to be responsible for durable viral control. Because knowledge regarding these protective immune responses might lead to novel interventions aimed at preventing or curing HIV infection, there has been intense interest in further characterizing these unique individuals.
Multiple groups have examined how HIV is controlled by these individuals , , 7,8,9. More recently, our group has focused on defining the potential clinical consequences of long-term, host-mediated, virologic control. We and others have shown that: (1) the vast majority of controllers have stable low-level viremia , ; (2) controllers have elevated levels of microbial translocation and T cell activation compared to HIV-negative and ART-suppressed individuals , ; (3) a minority (7–10%) of controllers with high levels of T cell activation progress immunologically to AIDS despite preservation of virologic control ; and (4) controllers have accelerated measures of atherosclerosis compared to HIV-negative individuals, even after adjustment for traditional cardiovascular risk factors , . Collectively, these data suggest that very low levels of viral replication may lead to disproportionately high levels of immune activation in HIV-infected controllers, which may lead to an increased risk of AIDS- and non-AIDS defining events. However, the degree to which viral replication contributes to these outcomes is not known. No prospective ART studies have been performed in controllers, because it has generally been assumed that most controllers do not need ART due to their ability to control plasma viremia to very low levels.
We therefore conducted the first, prospective study of antiretroviral therapy in a cohort of asymptomatic HIV-infected controllers, in order to determine the virologic and immunologic effects of treating controllers with ART. We also measured changes in biomarkers of inflammation and coagulation. Multiple biomarkers (e.g., high sensitivity C-reactive protein and D-dimer) remain elevated in both untreated and treated non-controllers , and have been shown to be strongly predictive of morbidity and all-cause mortality in ART-treated non-controllers , , . We therefore examined whether ART initiation led to a reduction in biomarkers of inflammation and coagulation in controllers, in order to assess whether low-level viral replication has any potential immunologic and clinical consequences in these individuals.
Sixteen asymptomatic controllers were prospectively treated with open-label raltegravir+tenofovir/emtricitabine for 24 weeks. Controllers were defined by the following inclusion criteria: (1) HIV-seropositive; (2) ART untreated; and (3) plasma HIV RNA <1,000 copies/mL for ≥12 months. Exclusion criteria included: (1) known rheumatologic conditions (e.g., systemic lupus erythematosus), because of the potential for biologic false-positive testing on HIV antibody tests; (2) known kidney disease; (3) known bone disease, including pathologic fractures; (4) chronic hepatitis B infection, because of the potential risk of liver abnormalities after starting and stopping tenofovir/emtricitabine in patients with chronic hepatitis B infection; (5) serious illness requiring hospitalization or parental antibiotics within the preceding 3 months; and (6) pregnant or breastfeeding women.
Subjects were seen every four weeks. Plasma and peripheral blood mononuclear cells (PBMCs) were collected and detailed interviews were conducted at the majority of visits. Thirteen out of 16 subjects consented to undergo 3 serial colorectal biopsies at weeks −2, 6, and 22. Five out of 16 subjects also underwent leukapheresis at weeks −4 and week 21 in order to obtain large PBMC samples for measurement of integrated HIV DNA. Adherence to study drug was measured at every study visit by self-report and pill-count. An independent Data Monitoring Committee comprised of three individuals from the scientific community met at 12, 24, 48, and 60 weeks after the enrollment of the first subject, and at 60 weeks after the enrollment of the last subject.
Baseline Characteristics (Table 1)
All subjects had a baseline plasma HIV RNA level <1,000 copies/mL in the absence of ART. The median baseline plasma HIV RNA level using a standard assay (Abbott Real Time assay, lower limit of detection <40 copy/mL) was 77 copies/mL; 4/16 subjects had an “undetectable” (<40 copies/mL) baseline plasma HIV RNA level with this assay. The median baseline plasma HIV RNA level using an ultrasensitive “single copy assay” (lower limit of detection <0.3 copy/mL) was 23 copies/mL. The median baseline age was 49 years; most subjects (88%) were men. The median baseline CD4+ and CD8+ T cell counts were 616 and 897 cells/mm3, respectively; the median baseline CD4+ to CD8+ T cell count ratio was 0.71. The median nadir CD4+ T cell count was 590 cells/mm3. The median self-reported duration of known HIV diagnosis was 10 years.
Antiretroviral therapy was well tolerated and all subjects completed 24 weeks of ART. No significant adverse events occurred during the study. The majority of controllers (11/16) elected to continue ART after the 24-week study period. Five out of 16 subjects elected to discontinue ART after the 24-week study period at various times (median 10.0 weeks, interquartile range [IQR] 1.0 to 19.0 weeks) after the end of the treatment study. They have subsequently been followed for a median 63.0 (IQR 47.4 to 66.3) weeks after discontinuing ART, and at the time of last follow-up the plasma HIV RNA level using a standard assay (Abbott Real Time assay, lower limit of detection <40 copy/mL) was a median <40 (IQR<40 to 73) copies/mL. Of the 5 subjects who elected to discontinue ART after the end of the treatment study, 4/5 of the subjects had an “undetectable” pre-ART plasma HIV RNA level at baseline using a standard assay (Abbott Real Time assay, lower limit of detection <40 copy/mL).
CD4+ T Cell Counts
Controllers did not have a statistically significant increase in peripheral CD4+ T cell counts (mean 1.00-fold increase in CD4+ T cells at week 24, 95% confidence interval [CI] 1.05-fold decrease to 1.06-fold increase, p = 0.93) (Fig. 1A). Similarly, controllers did not have a statistically significant increase in %CD3+CD4+ T cells in the rectum (mean +0.4%, 95% CI −0.8% to 1.6%, p = 0.50) (Fig. 1B).
Thin lines indicate data for each individual subject. The thick line indicates the estimated mean value over time from mixed effects linear regression. P-values refer to change from baseline at each referenced time-point.
Ultrasensitive Plasma HIV RNA and HIV Antibody Levels
Despite having low pre-ART plasma HIV RNA levels by conventional assays, controllers had an early and persistent decrease in ultrasensitive plasma HIV RNA levels after initiation of ART (mean 66-fold decrease in S/Co at week 24, 95% CI 155 to 28-fold decrease, p<0.001) (Fig. 2A). In addition, we examined change in HIV antibody levels as a surrogate measure of antigenic stimulation and viral persistence , , , , . Controllers also had an early and persistent decrease in HIV antibody levels (mean −7.2 S/Co at week 24, 95% CI −9.6 to −4.8, p<0.001) (Fig. 2B).
Thin lines indicate data for each individual subject. The thick line indicates the estimated mean value over time from mixed effects linear regression. P-values refer to change from baseline at each referenced time-point. S/Co = signal/cutoff ratio.
Cell-Associated HIV RNA and Total and Integrated HIV DNA
At baseline, the median (IQR) levels of cell-associated HIV RNA and total HIV DNA in PBMCs were 6.9 (3.5, 45.7) S/Co per million CD4+ T cells and 57 (34, 138) copies/million CD4+ T cells, respectively. In PBMCs, controllers did not have a substantial decrease in cell-associated HIV RNA (mean 1.20-fold decrease in S/Co per million CD4+ T cells, 95% CI 2.4-fold decrease to 1.62-fold increase, p = 0.58) or total HIV DNA (mean 1.22-fold decrease in copies/million CD4+ T cells, 95% CI 1.95-fold decrease to 1.32-fold increase, p = 0.41) at week 24. However, controllers did have an early and persistent decrease in rectal cell-associated HIV RNA after initiation of ART, with a mean decrease of 0.61 log10 copies/million CD4+ cells, which corresponded to a 4.1-fold decrease (95% CI 12.0 to 1.40-fold decrease, p = 0.010) at week 22 (Fig. 3A). There was a similar trend towards a decrease in rectal total HIV DNA, with a mean decrease of 0.28 log10 copies/million CD4+ cells, which corresponded to a 1.91-fold decrease (95% CI 5.1-fold decrease to 1.38-fold increase, p = 0.19) at week 22 (Fig. 3B). We also measured integrated HIV DNA levels in PBMCs obtained through leukapheresis in 5 controllers. In these subjects, there was a statistically significant decrease in integrated HIV DNA after initiation of ART, with a mean decrease of 0.32 log10 copies/million PBMCs, which corresponded to a 2.1-fold decrease (95% CI 2.7 to 1.13-fold decrease, p = 0.027) at week 21 (Fig. 4).
Thin lines indicate data for each individual subject. The thick line indicates the estimated mean value over time from mixed effects linear regression. P-values refer to change from baseline at each referenced time-point.
Change in integrated DNA levels before and after 21 weeks of antiretroviral therapy was measured using the paired t-test with bias-corrected and accelerated non-parametric confidence intervals. PBMCs = peripheral blood mononuclear cells.
T Cell Activation/Dysfunction
Markers of T cell activation/dysfunction in blood and gut also decreased substantially with ART. In PBMCs, controllers had a mean decrease of 1.9% in %CD38+HLA-DR+ CD4+ T cells (95% CI −2.8% to −0.9%, p<0.001) and a mean decrease of 9.0% in %CD38+HLA-DR+ CD8+ T cells (95% CI −12.4% to −5.6%, p<0.001) at week 24 (Fig. 5). Controllers also had a mean decrease of 1.6% in %PD-1+ CD4+ T cells (95% CI −3.1% to −0.1%, p = 0.04) and a mean decrease of 4.5% in %PD-1+ CD8+ T cells (95% CI −6.4% to −2.6%, p<0.001) in PBMCs at week 24. In the rectum, controllers had a trend towards a decrease in %CD38+HLA-DR+ CD4+ T cells (mean −0.9%, 95% CI −2.3% to +0.5%, p = 0.20) and a statistically significant mean decrease of 12.2% in %CD38+HLA-DR+ CD8+ T cells (95% CI −21.9% to −2.5%, p = 0.014) at week 22 (Fig. 6).
Thin lines indicate data for each individual subject. The thick line indicates the estimated mean value over time from mixed effects linear regression. P-values refer to change from baseline at each referenced time-point.
Thin lines indicate data for each individual subject. The thick line indicates the estimated mean value over time from mixed effects linear regression. P-values refer to change from baseline at each referenced time-point. GALT = gut-associated lymphoid tissue.
At baseline, the median (IQR) levels of high sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), soluble CD14 (sCD14), and D-dimer were 1.20 (0.55, 3.03) ug/mL, 1.71 (1.28, 4.42) pg/mL, 1696 (1446, 1971) ng/mL, and 0.37 (0.28, 0.56) ug/mL, respectively. After ART initiation, there was a trend towards a decrease in hsCRP, with a mean 1.74-fold decrease (95% CI 3.2-fold decrease to 1.04-fold increase, p = 0.069) at week 4, and a mean 1.67-fold decrease (95% CI 3.0-fold decrease to 1.09-fold increase, p = 0.093) at week 24 (Fig. 7). We also observed similar trends in IL-6 (mean 1.34-fold decrease, 95% CI 2.1-fold decrease to 1.19-fold increase, p = 0.22), sCD14 (mean −44.2, 95% CI −138.6 to +50.3, p = 0.36), and D-dimer (mean 1.30-fold decrease, 95% CI 2.1-fold decrease to 1.25-fold increase, p = 0.29) levels after 24 weeks of ART, although these trends did not reach statistical significance.
Thin lines indicate data for each individual subject. The thick line indicates the estimated mean value over time from mixed effects linear regression. P-values refer to change from baseline at each referenced time-point. hsCRP = high sensitivity C-reactive protein.
HIV-Specific T Cell Responses
At baseline, the median (IQR) levels of percentage of Gag-specific IFNγ+IL2+ CD4+ and CD8+ T cell responses in PBMCs were 0.10% (0.03%, 0.17%) and 1.53% (0.37%, 2.55%), respectively. Controllers did not have a substantial change in the percentage of Gag-specific IFNγ+IL2+ CD4+ T cell responses in PBMCs (mean 1.07-fold decrease, 95% CI 1.40-fold decrease to 1.22-fold increase, p = 0.62), although there was a trend towards a decrease in the percentage of Gag-specific IFNγ+IL2+ CD8+ T cell responses in PBMCs (mean 1.28-fold decrease, 95% CI 1.68-fold decrease to 1.03-fold increase, p = 0.075) at week 24. At baseline, the median (IQR) levels of percentage of total (IFNγ, IL-2, TNFα, and/or CD107a) Gag-specific CD4+ or CD8+ in the rectum were 0.56% (0%, 1.3%) and 0.22% (0.08%, 0.32%), respectively. Controllers did not have a substantial change in the percentage of total Gag-specific CD4+ (mean 1.26-fold increase, 95% CI 1.82-fold decrease to 2.9-fold increase, p = 0.58) or CD8+ (mean 1.61-fold decrease, 95% CI 3.6-fold decrease to 1.37-fold increase, p = 0.24) T cell responses in the rectum at week 22.
At baseline, 4/16 controllers had an “undetectable” pre-ART plasma HIV RNA level using a standard assay (Abbott Real Time assay, lower limit of detection <40 copy/mL). Despite having this extremely low pre-ART plasma HIV RNA level, this subset of so-called “elite” controllers had a statistically significant decrease in ultrasensitive plasma HIV RNA levels after initiation of ART (mean 14-fold decrease in S/Co at week 24, 95% CI 115 to 1.74-fold decrease, p = 0.013) (Fig. 8A). Similarly, this subset of controllers had a statistically significant decrease in HIV antibody levels (mean −4.2 S/Co at week 24, 95% CI −7.9 to −0.5, p = 0.027) (Fig. 8B). Finally, we observed similar trends in immune activation in these 4 controllers after initiation of ART. There was a mean decrease of 6.0% in %CD38+HLA-DR+ CD8+ T cells in PBMCs at week 24 (95% CI −13.0% to +0.10%, p = 0.091, Fig. 8C) and a mean decrease of 24.3% in %CD38+HLA-DR+ CD8+ T cells in the rectum at week 22 (95% CI −54.1% to +5.6%, p = 0.11, Fig. 8D).
Change in ultrasensitive plasma HIV RNA, HIV antibody levels, and T cell activation in peripheral blood and rectum in 4/16 controllers with baseline plasma HIV RNA <40 copies/mL. Thin lines indicate data for each individual subject (n = 4). The thick line indicates the estimated mean value over time from mixed effects linear regression for the entire cohort (n = 16). The y-axes are on the same scale as Figures 2, 5, and 6. P-values refer to change from baseline at each referenced time-point. S/Co = signal/cutoff ratio. GALT = gut-associated lymphoid tissue.
In this first prospective study of antiretroviral therapy initiation in asymptomatic HIV-infected controllers, 24 weeks of ART was safe and well-tolerated. Despite being able to maintain very low plasma HIV RNA levels in the absence of ART, controllers had readily measurable levels of HIV RNA and DNA in the gut. Antiretroviral therapy led to statistically significant decreases in ultrasensitive plasma HIV RNA levels, HIV antibody levels, rectal cell-associated HIV RNA, and immune activation in the blood and gut. Collectively, these data suggest that HIV in most controllers is replication-competent , , , and that host rather than virologic factors account for the remarkable degree of viral control in these unique individuals. We also observed a statistically significant decrease in levels of integrated HIV DNA with ART, while total HIV DNA levels remained stable. These findings may be due to an excess of unintegrated HIV DNA in controllers, as previously reported by our group .
We observed that measures of immune activation/dysfunction decreased as measures of virologic burden and HIV antigenic stimulation decreased with ART. We also observed a trend towards a decrease in hsCRP (a measure of systemic inflammation) with ART; similar trends were observed with IL-6, sCD14, and D-dimer. These biomarkers have been shown to be strong, consistent, and independent predictors of increased morbidity and mortality in HIV infection , , . Because the confidence intervals were wide, however, we cannot assess with certainty whether the observed decrease in hsCRP levels has any clinical relevance; it would be important to pursue these findings in future, larger studies. Taken together, however, these data suggest that there may be immunologic consequences to even very low levels of viral replication. This latter finding may have important implications for HIV-infected non-controllers as well , , , , .
Importantly, our study also shifts the field's working definition of a “functional cure.” On one hand, our data suggest that a complete block of viral replication is not necessary to achieve long-term virologic control. However, natural long-term virologic control appears to be coming at an immunologic and/or clinical “cost,” at least as defined by increased levels and manifestations of immune activation. Thus, although further study of controllers is warranted, untreated HIV-infected controllers may not represent the best model of a functional cure, if we believe that a cure should require a disease-free (and not just treatment-free) state.
Several limitations of our study deserve comment. First, this was a small pilot study and our findings should be replicated in a larger study of HIV-infected controllers with a longer duration of follow-up. In our study of controllers who had relatively high baseline CD4+ T cell counts, 24 weeks of ART did not appear to confer a CD4+ T cell count benefit. In studies of HIV-infected non-controllers, a greater absolute decrease in plasma HIV RNA levels during the early period after ART initiation has been shown to be a consistent predictor of an early increase in CD4+ T cell counts (with much of the increase assumed to be due to redistribution) . In our study, although we did observe a significant decrease in ultrasensitive plasma HIV RNA levels with initiation of ART, the absolute change was small compared to that observed in HIV-infected non-controllers; this may have partially accounted for the limited changes in peripheral CD4+ T cell counts. It is possible that with a much longer duration of follow-up, an increase in CD4+ T cell count may have been observed. Second, there was a trend towards a decrease in the percentage of Gag-specific IFNγ+IL2+ CD8+ T cell responses in PBMCs, although a similar trend was not observed in the rectum. This observation raises the possibility that initiation of ART in controllers may reduce host mechanisms of virologic control, leading to rebound in viremia if ART is discontinued. However, in the 5 subjects who elected to discontinue ART after the 24-week study period, there was no evidence of rebound in plasma viremia after discontinuation of ART. Nevertheless, the long-term safety of ART in controllers should be confirmed. Third, we enrolled a relatively heterogeneous group of controllers. As we and others have shown, however, controllers are a heterogeneous group with varying levels of steady-state viremia; there appears to be a continuum of viremia across controllers , , , . In order to determine whether there is a differential effect of ART on a spectrum of controllers, we enrolled individuals whose baseline plasma HIV RNA levels spanned from nearly 0 to 1000 copies RNA/mL for at least 12 months (median duration of HIV diagnosis 10 years, IQR 4.5 to 24 years). Thus, our study included controllers who had both low-level but detectable and undetectable pre-ART plasma HIV RNA levels using conventional assays. Remarkably, however, even amongst the latter group of “elite” controllers who had undetectable pre-ART plasma HIV RNA levels at baseline, we observed a statistically significant decrease in ultrasensitive plasma HIV RNA levels and HIV antibody levels, and a trend towards a decrease in immune activation with ART. Fourth, although 24 weeks of ART significantly decreased levels of CD4+ and CD8+ T cell activation, it did not normalize them to levels observed in HIV-uninfected individuals . Thus, at least in HIV-infected controllers, low-level viral replication is unlikely to be the only factor contributing to immunologic disease. The potential role of other factors that might contribute to immune activation—including co-infections and substance abuse—could not be addressed in this pilot study, but might be addressed in future studies with larger cohorts. It would also be important to systematically assess the individual and potentially synergistic contributions of ART and lifestyle modifications towards decreasing inflammation, immune activation, and clinical disease in HIV-infected controllers . Finally, it is worth noting that there may be multiple pathways to virologic control, some of which may represent an appropriate model of a “functional cure” and may not receive an additional benefit from ART.
In summary, 24 weeks of ART was safe and well-tolerated in chronically HIV-infected controllers. Antiretroviral therapy in controllers led to significant decreases in ultrasensitive plasma and rectal HIV RNA, HIV antibody levels, and markers of immune activation/dysfunction in blood and gut, confirming that HIV replication persists in controllers and contributes to a chronic inflammatory state. We acknowledge that this was a small pilot study and that our findings would be ideally replicated in a larger, randomized, clinical-endpoint study. However, the relative rarity of HIV-infected controllers may make such a study impractical, if not impossible. In the absence of such a study, clinicians will need to weigh the potential benefits of ART (suggested by the changes in immune activation and biomarkers observed in our study) with the potential risks and costs associated with long-term antiretroviral therapy.
Materials and Methods
All subjects provided written informed consent. This study was approved by the University of California San Francisco (UCSF) Committee on Human Research.
Ultrasensitive Plasma HIV RNA
The isothermal Transcription Mediated Amplification (TMA) assay (Aptima, Gen-Probe/Hologic) was used to measure ultrasensitive plasma HIV RNA levels at weeks 0, 4, 12, and 24. This is a nucleic acid-amplification test that has been FDA-approved for the early detection of HIV infection in blood donors , , . It is a highly specific and sensitive assay, with a singlicate 50% detection limit of 3.6–14 copies/mL , . The assay was performed in triplicate on 0.5 mL plasma (1.5 mL total plasma), improving the overall 50% detection limit to <5 copies/mL. The output is a signal/cutoff (S/Co) ratio (range 0–30), with S/Co<1.0 = “negative” and S/Co≥1.0 = “positive.” Ultrasensitive plasma HIV RNA levels were also measured at weeks 0 and 12 with a “single copy assay” (lower limit of detection <0.3 copy/mL), using a median 7.3 mL of plasma .
Plasma HIV Antibody Levels
A “de-tuned” or less-sensitive enzyme immunoassay (LS-VITROS) was used to measure HIV antibody levels at weeks 0, 4, 12, and 24. The VITROS (Ortho-Clinical Diagnostics) is an FDA-approved diagnostic assay for the detection of IgM/IgG antibodies to HIV-1/-2. The less-sensitive modification tests 1∶400 dilutions of plasma and calculates a S/Co ratio (range 0–80), and has been validated as a method to identify early HIV infection .
Cell-Associated HIV RNA and Total HIV DNA (PBMCs)
Cell-associated HIV RNA and total HIV DNA were measured from PBMCs at weeks 0, 4, and 24. Cell-associated HIV RNA was measured using modifications of published methods (Aptima, Gen-Probe/Hologic) , . The output is a S/Co ratio (range 0–30), with S/Co<1.0 = “negative” and S/Co≥1.0 = “positive.” All S/Co ratios were normalized to per million CD4+ T cells. Total HIV DNA was measured using modifications of published methods with an overall sensitivity of 1 copy/3 µg of DNA (450,000 PBMCs) , , , . All total HIV DNA levels were normalized to per million CD4+ T cells.
Integrated HIV DNA (PBMCs)
Integrated HIV DNA was measured from PBMCs at weeks −4 and 21. DNA was prepared (Qiagen Mid) and integrated HIV DNA was measured using a published repetitive sampling method because integration levels are known to be low in controllers , . At least 42 Alu-gag PCR reactions were performed with 150,000 diploid genomes per PCR, for a total of 6.3 million diploid genomes assayed per subject.
T Cell Immunophenotyping and Cytokine Flow Cytometry (PBMCs)
PBMCs were isolated from whole blood, cryopreserved, and stored at the UCSF AIDS Specimen Bank. Markers of T cell activation/dysfunction and antigen-specific T cell responses were measured at weeks 0, 4, and 24 at the UCSF Core Immunology Laboratory, using published methods that have been optimized and validated for cryopreserved PBMCs . Briefly, cryopreserved PBMCs were rapidly thawed in warm media, counted on an Accuri C6 (BD Biosciences) with the Viacount assay (Millipore), and washed and stained the same day (T cell immunophenotyping) or rested overnight (cytokine flow cytometry [CFC]). The average viability of thawed cells was 93% (range 61–98%; 80% of samples had viability >90%).
For T cell immunophenotyping, the percent of activated (CD38+/HLA-DR+/PD1+) CD4+ and CD8+ T cells were measured; these markers of immune activation/dysfunction have been shown to be strong and independent predictors of HIV disease progression , , , , . Cells were stained with Aqua Amine Reactive Dye (AARD, Invitrogen) to discriminate dead cells, washed, and stained with fluorescently-conjugated monoclonal antibodies: CD3-Pacific Blue (BD Pharmingen), CD38-PE, HLA-DR-FITC, PD1- Alexa647 (BD Biosciences), CD4-PE Texas Red, and CD8-QDot 605 (Invitrogen). In each experiment a fluorescent-minus one control was included for CD38, HLA-DR, and PD-1. Stained cells were washed, fixed in 0.5% formaldehyde (Polyscience), and held at 4C until analysis.
For CFC, rested PBMCs were stimulated for 18–22 h at 37C with overlapping peptide pools corresponding to HIV-1 Con B Gag peptides (NIH 8117) in the presence of 0.5 ug/mL Brefeldin A and 0.5 ug/mL Monensin (Sigma-Aldrich). A control well with no stimulation was run in parallel for each sample. Cells were washed and stained with AARD, fixed, and permeabilized for intracellular staining with antibodies against CD3-Pacfic Blue, IFNγ-FITC, IL-2-PE (BD BioScience), CD4-PE Texas Red, and CD8-QDot 605 (Invitrogen). Cells were washed and stored at 4C until analysis. We focused on Gag-specific IFNγ+IL2+ T cell responses given that we have shown that these responses are associated with control of HIV replication in controllers , , .
Stained cells were run on a customized BD LSR II (BD Bioscience). 100,000 and 500,000 lymphocytes were collected for immunophenotyping and CFC samples, respectively. Data were compensated and analyzed using FlowJo (Tree Star) to determine the proportion of CD4+ and CD8+ T cells expressing each of the T cell or cytokine markers. Combinations of markers were calculated in FlowJo using the Boolean gate function. For CFC data, results from control wells with no stimulation were subtracted from stimulated results.
High sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), soluble CD14 (sCD14), and D-dimer levels were measured on stored fasting plasma samples at weeks 0, 4, and 24 at the Laboratory for Clinical Biochemistry Research at the University of Vermont. hsCRP was measured with a BN II nephelometer (Siemens Diagnostics, Deerfield, IL), IL-6 was measured with Chemiluminescent Sandwich enzyme-linked immunosorbent assay, sCD14 with a standard ELISA (both R&D Systems, Minneapolis, MN), and D-dimer was measured with an immunoturbidometric method on the Sta-R analyzer, Liatest D-DI (Diagnostica Stago, Parsippany, NJ). Interassay coefficients of variation for a number of different control materials of different values averaged ∼10% or less for all assays.
Gut-Associated Lymphoid Tissue (GALT)
Thirty colorectal biopsy specimens were obtained 10–20 cm from the anal verge using 3 mm jumbo forceps at weeks −2, 6, and 22. Eighteen to 24 biopsy pieces were placed into 10 mL RPMI-1640 media containing fetal calf serum (15%), penicillin (100 U/mL), streptomycin (100 ug/mL), and L-glutamine (2 mM). Fresh colorectal cells were isolated on the same day using a modification of a published protocol designed to optimize yield and viability of mucosal lymphocytes without compromising the detection of most surface antigens . Briefly, biopsy pieces underwent two rounds of digestion in 0.5 mg/mL collagenase type II (Sigma-Aldrich). Each digestion was followed by disruption of the tissue with a syringe bearing a 16-gauge blunt end needle and subsequent passage through a 70 µm cell strainer. Yields were 9.5–31×106 (mean 18×106) total rectal cells. 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 25C. Propidium iodide was added to stain non-viable cells and samples were run on an Accuri C6 to determine the total number of viable mononuclear cells and proportion and absolute number of viable CD45+ leukocytes and CD4+ T cells. Another aliquot of cells was frozen at −80C for subsequent nucleic acid extraction.
Cell-Associated HIV RNA and Total HIV DNA (GALT)
Total HIV RNA was measured from rectal cells using a published method . Three replicates of up to 500 ng RNA were assayed for total processive HIV RNA transcripts using primers (HXB2 positions 522–543, 626–643) and probe (559–584) from the LTR region . Genomic HIV RNA standards (2.5×100 to 2.5×105) were prepared from lab stocks of NL4-3 virions by extracting and quantifying HIV RNA using the Abbot Real Time assay. HIV RNA copy numbers were normalized to cellular input into the PCR, as determined by RNA mass (assuming 1 ng RNA = 1000 cells ), which has been shown to correlate with levels of GAPDH RNA . Results were further normalized by the percent of cells that were CD3+CD4+ by flow cytometry and expressed as copies/106 CD4+T cells.
Total HIV DNA was measured from rectal cells using a published method . Three replicates of up to 500 ng DNA were assayed for HIV DNA using a modification of a published TaqMan PCR assay that uses primers/probe from the LTR region (as above). External standards (105 to 1) were prepared from DNA extracted from known numbers of 8E5 cells (NIH AIDS Reagent Program), each of which contains one integrated HIV genome per cell. HIV DNA copy numbers were normalized to cellular input into the PCR, as determined by DNA mass (assuming 1 ug DNA = 160,000 cells). Results were further normalized by the percent of cells that were CD3+CD4+ by flow cytometry and expressed as copies/106 CD4+T cells.
T Cell Immunophenotyping and Cytokine Flow Cytometry (GALT)
Markers of T cell activation (CD38+/HLA-DR+) and total Gag-specific responses (Gag-specific CD4+ and CD8+ T cells expressing one or more of IFNγ, IL-2, TNFα, and/or CD107a , , ) were measured from rectal cells at weeks −2, 6, and 22. We focused on these responses given that we have shown that these mucosal T cell responses are associated with control of HIV replication in controllers .
For T cell immunophenotyping of freshly isolated rectal cells, similar methods were used as for PBMCs . For CFC, freshly isolated rectal cells were rested overnight at 37C, 5%CO2, in R15 containing 0.5 mg/mL piperacillin-tazobactam, then similar methods were used as for PBMCs . To account for the lower numbers of events and elevated baseline cytokine staining in mucosal samples, response data from peptide-stimulated wells were first compared against unstimulated controls using a published algorithm to determine statistical significance, prior to background subtraction , .
Mixed effect linear models with random slopes and intercepts were used to examine change in virologic and immunologic measurements over time. Changes in integrated HIV DNA levels were assessed by estimating the mean change and its bias-corrected and accelerated non-parametric confidence intervals, and using a paired t-test to obtain a corresponding p-value . All statistical analyses were conducted with Stata version 11.1 (Stata Corp).
Conceived and designed the experiments: HH. Performed the experiments: SAY MAM TL JKW ALF BLS EHG UO ES JMM SP RPT MPB. Analyzed the data: HH PB. Contributed reagents/materials/analysis tools: JKW BLS UO MS PWH JMM SP JNM MPB SGD. Wrote the paper: HH. Recruited and enrolled study subjects: HH KH RH. Edited the manuscript: HH SAY ALF EHG MS ES MAM TL KH RH SP PB PWH JNM JMM RPT MPB UO BLS JKW SGD.
- 1. Deeks SG, Walker BD (2007) Human immunodeficiency virus controllers: mechanisms of durable virus control in the absence of antiretroviral therapy. Immunity 27: 406–416.
- 2. Hubert JB, Burgard M, Dussaix E, Tamalet C, Deveau C, et al. (2000) Natural history of serum HIV-1 RNA levels in 330 patients with a known date of infection. The SEROCO Study Group. Aids 14: 123–131.
- 3. Madec Y, Boufassa F, Porter K, Meyer L (2005) Spontaneous control of viral load and CD4 cell count progression among HIV-1 seroconverters. Aids 19: 2001–2007.
- 4. Lambotte O, Boufassa F, Madec Y, Nguyen A, Goujard C, et al. (2005) HIV controllers: a homogeneous group of HIV-1-infected patients with spontaneous control of viral replication. Clin Infect Dis 41: 1053–1056.
- 5. Betts MR, Nason MC, West SM, De Rosa SC, Migueles SA, et al. (2006) HIV nonprogressors preferentially maintain highly functional HIV-specific CD8+ T cells. Blood 107: 4781–4789.
- 6. Saez-Cirion A, Lacabaratz C, Lambotte O, Versmisse P, Urrutia A, et al. (2007) HIV controllers exhibit potent CD8 T cell capacity to suppress HIV infection ex vivo and peculiar cytotoxic T lymphocyte activation phenotype. Proc Natl Acad Sci U S A 104: 6776–6781.
- 7. Lichterfeld M, Mou D, Cung TD, Williams KL, Waring MT, et al. (2008) Telomerase activity of HIV-1-specific CD8+ T cells: constitutive up-regulation in controllers and selective increase by blockade of PD ligand 1 in progressors. Blood 112: 3679–3687.
- 8. Hersperger AR, Martin JN, Shin LY, Sheth PM, Kovacs CM, et al. (2011) Increased HIV-specific CD8+ T-cell cytotoxic potential in HIV elite controllers is associated with T-bet expression. Blood 117: 3799–3808.
- 9. Tomescu C, Duh FM, Hoh R, Viviani A, Harvill K, et al. (2012) Impact of protective killer inhibitory receptor/human leukocyte antigen genotypes on natural killer cell and T-cell function in HIV-1-infected controllers. Aids 26: 1869–1878.
- 10. Hatano H, Delwart EL, Norris PJ, Lee TH, Dunn-Williams J, et al. (2009) Evidence for persistent low-level viremia in individuals who control human immunodeficiency virus in the absence of antiretroviral therapy. J Virol 83: 329–335.
- 11. Pereyra F, Palmer S, Miura T, Block BL, Wiegand A, et al. (2009) Persistent low-level viremia in HIV-1 elite controllers and relationship to immunologic parameters. J Infect Dis 200: 984–990.
- 12. Hunt PW, Brenchley J, Sinclair E, McCune JM, Roland M, et al. (2008) 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 197: 126–133.
- 13. Hunt PW, Landay AL, Sinclair E, Martinson JA, Hatano H, et al. (2011) A low T regulatory cell response may contribute to both viral control and generalized immune activation in HIV controllers. PLoS ONE 6: e15924.
- 14. Hsue PY, Hunt PW, Schnell A, Kalapus SC, Hoh R, et al. (2009) Role of viral replication, antiretroviral therapy, and immunodeficiency in HIV-associated atherosclerosis. Aids 23: 1059–1067.
- 15. Pereyra F, Lo J, Triant VA, Wei J, Buzon MJ, et al. (2012) Increased coronary atherosclerosis and immune activation in HIV-1 elite controllers. Aids 26: 2409–2412.
- 16. Neuhaus J, Jacobs DR Jr, Baker JV, Calmy A, Duprez D, et al. (2010) Markers of inflammation, coagulation, and renal function are elevated in adults with HIV infection. J Infect Dis 201: 1788–1795.
- 17. Kuller LH, Tracy R, Belloso W, De Wit S, Drummond F, et al. (2008) Inflammatory and coagulation biomarkers and mortality in patients with HIV infection. PLoS Med 5: e203.
- 18. Boulware DR, Hullsiek KH, Puronen CE, Rupert A, Baker JV, et al. (2011) Higher levels of CRP, D-dimer, IL-6, and hyaluronic acid before initiation of antiretroviral therapy (ART) are associated with increased risk of AIDS or death. J Infect Dis 203: 1637–1646.
- 19. Duprez DA, Neuhaus J, Kuller LH, Tracy R, Belloso W, et al. (2012) Inflammation, coagulation and cardiovascular disease in HIV-infected individuals. PLoS ONE 7: e44454.
- 20. Amanna IJ, Carlson NE, Slifka MK (2007) Duration of humoral immunity to common viral and vaccine antigens. N Engl J Med 357: 1903–1915.
- 21. Cimerman S, Sucupira MC, Lewi DS, Diaz RS (2007) Less sensitive HIV-1 enzyme immunoassay as an adjuvant method for monitoring patients receiving antiretroviral therapy. AIDS Patient Care STDS 21: 100–105.
- 22. Yukl SA, Boritz E, Busch M, Bentsen C, Chun T-W, et al. (2013) Challenges in Detecting HIV Persistence during Potentially Curative Interventions: A Study of the Berlin Patient. PLoS Pathog 9: e1003347.
- 23. Henrich TJ, Hu Z, Li JZ, Sciaranghella G, Busch MP, et al. (2013) Long-Term Reduction in Peripheral Blood HIV Type 1 Reservoirs Following Reduced-Intensity Conditioning Allogeneic Stem Cell Transplantation. Journal of Infectious Diseases 207: 1694–1702.
- 24. Blankson JN, Bailey JR, Thayil S, Yang HC, Lassen K, et al. (2007) Isolation and Characterization of Replication-Competent HIV-1 from a Subset of Elite Suppressors. J Virol 81: 2508–2518.
- 25. Lamine A, Caumont-Sarcos A, Chaix ML, Saez-Cirion A, Rouzioux C, et al. (2007) Replication-competent HIV strains infect HIV controllers despite undetectable viremia (ANRS EP36 study). Aids 21: 1043–1045.
- 26. Mens H, Kearney M, Wiegand A, Shao W, Schonning K, et al. (2010) HIV-1 continues to replicate and evolve in patients with natural control of HIV infection. J Virol 84: 12971–12981.
- 27. Graf EH, Mexas AM, Yu JJ, Shaheen F, Liszewski MK, et al. (2011) 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 7: e1001300.
- 28. Morse CG, Dodd LE, Nghiem K, Costello R, Csako G, et al. (2013) Elevations in D-dimer and C-reactive protein are associated with the development of osteonecrosis of the hip in HIV-infected adults. Aids 27: 591–595.
- 29. Buzon MJ, Massanella M, Llibre JM, Esteve A, Dahl V, et al. (2010) HIV-1 replication and immune dynamics are affected by raltegravir intensification of HAART-suppressed subjects. Nat Med 16: 460–465.
- 30. Yukl SA, Shergill AK, McQuaid K, Gianella S, Lampiris H, et al. (2010) Effect of raltegravir-containing intensification on HIV burden and T-cell activation in multiple gut sites of HIV-positive adults on suppressive antiretroviral therapy. Aids 24: 2451–2460.
- 31. Llibre JM, Buzon MJ, Massanella M, Esteve A, Dahl V, et al. (2012) Treatment intensification with raltegravir in subjects with sustained HIV-1 viraemia suppression: a randomized 48-week study. Antivir Ther 17: 355–364.
- 32. Massanella M, Negredo E, Puig J, Puertas MC, Buzon MJ, et al. (2012) Raltegravir intensification shows differing effects on CD8 and CD4 T cells in HIV-infected HAART-suppressed individuals with poor CD4 T-cell recovery. Aids 26: 2285–2293.
- 33. Hatano H, Strain MC, Scherzer R, Bacchetti P, Wentworth D, et al. (2013) Increase in 2-LTR Circles and Decrease in D-dimer After Raltegravir Intensification in Treated HIV-Infected Patients: A Randomized, Placebo-Controlled Trial. J Infect Dis [Epub ahead of print].
- 34. Autran B, Carcelain G, Li TS, Blanc C, Mathez D, et al. (1997) Positive effects of combined antiretroviral therapy on CD4+ T cell homeostasis and function in advanced HIV disease. Science 277: 112–116.
- 35. Emu B, Sinclair E, Favre D, Moretto WJ, Hsue P, et al. (2005) 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 79: 14169–14178.
- 36. Ferre AL, Hunt PW, Critchfield JW, Young DH, Morris MM, et al. (2009) Mucosal immune responses to HIV-1 in elite controllers: a potential correlate of immune control. Blood 113: 3978–3989.
- 37. Ferre AL, Hunt PW, McConnell DH, Morris MM, Garcia JC, et al. (2010) HIV controllers with HLA-DRB1*13 and HLA-DQB1*06 alleles have strong, polyfunctional mucosal CD4+ T-cell responses. Journal of virology 84: 11020–11029.
- 38. Ferre AL, Lemongello D, Hunt PW, Morris MM, Garcia JC, et al. (2010) 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 84: 10354–10365.
- 39. Hunt PW, Hatano H, Sinclair E, Lee TH, Busch MP, et al. (2011) HIV-specific CD4+ T cells may contribute to viral persistence in HIV controllers. Clin Infect Dis 52: 681–687.
- 40. Hatano H, Somsouk M, Sinclair E, Harvill K, Gilman L, et al. (2013) Comparison of HIV DNA and RNA in Gut-associated lymphoid tissue of HIV-Infected Controllers and Non-Controllers. Aids (in press)..
- 41. Hunt PW, Martin JN, Sinclair E, Bredt B, Hagos E, et al. (2003) T cell activation is associated with lower CD4+ T cell gains in human immunodeficiency virus-infected patients with sustained viral suppression during antiretroviral therapy. J Infect Dis 187: 1534–1543.
- 42. Stramer SL, Glynn SA, Kleinman SH, Strong DM, Caglioti S, et al. (2004) Detection of HIV-1 and HCV infections among antibody-negative blood donors by nucleic acid-amplification testing. N Engl J Med 351: 760–768.
- 43. Stekler J, Swenson PD, Wood RW, Handsfield HH, Golden MR (2005) Targeted screening for primary HIV infection through pooled HIV-RNA testing in men who have sex with men. Aids 19: 1323–1325.
- 44. Nugent CT, Dockter J, Bernardin F, Hecht R, Smith D, et al. (2009) Detection of HIV-1 in alternative specimen types using the APTIMA HIV-1 RNA Qualitative Assay. J Virol Methods 159: 10–14.
- 45. Lelie PN, van Drimmelen HA, Cuypers HT, Best SJ, Stramer SL, et al. (2002) Sensitivity of HCV RNA and HIV RNA blood screening assays. Transfusion 42: 527–536.
- 46. Busch MP, Hecht FM (2005) Nucleic acid amplification testing for diagnosis of acute HIV infection: has the time come? Aids 19: 1317–1319.
- 47. Palmer S, Wiegand AP, Maldarelli F, Bazmi H, Mican JM, et al. (2003) New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma. J Clin Microbiol 41: 4531–4536.
- 48. Keating SM, Hanson D, Lebedeva M, Laeyendecker O, Ali-Napo NL, et al. (2012) Lower-sensitivity and avidity modifications of the vitros anti-HIV 1+2 assay for detection of recent HIV infections and incidence estimation. J Clin Microbiol 50: 3968–3976.
- 49. Bernardin F, Tobler L, Walsh I, Williams JD, Busch M, et al. (2008) Clearance of hepatitis C virus RNA from the peripheral blood mononuclear cells of blood donors who spontaneously or therapeutically control their plasma viremia. Hepatology 47: 1446–1452.
- 50. Lee TH, el-Amad Z, Reis M, Adams M, Donegan EA, et al. (1991) Absence of HIV-1 DNA in high-risk seronegative individuals using high-input polymerase chain reaction. Aids 5: 1201–1207.
- 51. Lee TH, Paglieroni T, Utter GH, Chafets D, Gosselin RC, et al. (2005) High-level long-term white blood cell microchimerism after transfusion of leukoreduced blood components to patients resuscitated after severe traumatic injury. Transfusion 45: 1280–1290.
- 52. Lee TH, Chafets DM, Reed W, Wen L, Yang Y, et al. (2006) Enhanced ascertainment of microchimerism with real-time quantitative polymerase chain reaction amplification of insertion-deletion polymorphisms. Transfusion 46: 1870–1878.
- 53. Graf EH, O'Doherty U (2013) Quantitation of integrated proviral DNA in viral reservoirs. Curr Opin HIV AIDS 8: 100–105.
- 54. Sinclair E, Tan QX, Sharp M, Girling V, Poon C, et al. (2006) Protective immunity to cytomegalovirus (CMV) retinitis in AIDS is associated with CMV-specific T cells that express interferon- gamma and interleukin-2 and have a CD8+ cell early maturational phenotype. J Infect Dis 194: 1537–1546.
- 55. Giorgi JV, Hultin LE, McKeating JA, Johnson TD, Owens B, et al. (1999) Shorter survival in advanced human immunodeficiency virus type 1 infection is more closely associated with T lymphocyte activation than with plasma virus burden or virus chemokine coreceptor usage. J Infect Dis 179: 859–870.
- 56. Hunt PW, Cao HL, Muzoora C, Ssewanyana I, Bennett J, et al. (2011) Impact of CD8+ T-cell activation on CD4+ T-cell recovery and mortality in HIV-infected Ugandans initiating antiretroviral therapy. Aids 25: 2123–2131.
- 57. Hatano H, Jain V, Hunt PW, Lee T-H, Sinclair E, et al. (2013) Cell-Based Measures of Viral Persistence Are Associated With Immune Activation and Programmed Cell Death Protein 1 (PD-1)–Expressing CD4+ T cells. Journal of Infectious Diseases 208: 50–56.
- 58. Pereyra F, Addo MM, Kaufmann DE, Liu Y, Miura T, et al. (2008) Genetic and immunologic heterogeneity among persons who control HIV infection in the absence of therapy. J Infect Dis 197: 563–571.
- 59. Shacklett BL, Yang O, Hausner MA, Elliott J, Hultin L, et al. (2003) Optimization of methods to assess human mucosal T-cell responses to HIV infection. J Immunol Methods 279: 17–31.
- 60. Kumar AM, Borodowsky I, Fernandez B, Gonzalez L, Kumar M (2007) Human immunodeficiency virus type 1 RNA Levels in different regions of human brain: quantification using real-time reverse transcriptase-polymerase chain reaction. J Neurovirol 13: 210–224.
- 61. Fischer M, Huber W, Kallivroussis A, Ott P, Opravil M, et al. (1999) Highly sensitive methods for quantitation of human immunodeficiency virus type 1 RNA from plasma, cells, and tissues. J Clin Microbiol 37: 1260–1264.
- 62. Yukl SA, Gianella S, Sinclair E, Epling L, Li Q, et al. (2010) Differences in HIV burden and immune activation within the gut of HIV-positive patients receiving suppressive antiretroviral therapy. J Infect Dis 202: 1553–1561.
- 63. Critchfield JW, Lemongello D, Walker DH, Garcia JC, Asmuth DM, et al. (2007) Multifunctional human immunodeficiency virus (HIV) gag-specific CD8+ T-cell responses in rectal mucosa and peripheral blood mononuclear cells during chronic HIV type 1 infection. J Virol 81: 5460–5471.
- 64. Betts MR, Brenchley JM, Price DA, De Rosa SC, Douek DC, et al. (2003) Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods 281: 65–78.
- 65. Hatano H, Hayes TL, Dahl V, Sinclair E, Lee TH, et al. (2011) A randomized, controlled trial of raltegravir intensification in antiretroviral-treated, HIV-infected patients with a suboptimal CD4+ T cell response. J Infect Dis 203: 960–968.
- 66. Critchfield JW, Young DH, Hayes TL, Braun JV, Garcia JC, et al. (2008) Magnitude and complexity of rectal mucosa HIV-1-specific CD8+ T-cell responses during chronic infection reflect clinical status. PLoS One 3: e3577.
- 67. Efron B, Tibshirani RJ (1993) An Introduction to the Bootstrap. London: Chapman and Hall.