Several controls were included for the nested PCR as previously described.35 First, a separate reaction mix was included for the first-round PCR identical to that described above, except only a single primer to the LTR was included and the primer to the Alu repeat element was not included. This control identified what component of the second-round PCR was a consequence of linear amplification from the LTR in the first round PCR (referred to as “LTR primer alone”). Second, a separate reaction mix was included for the first round identical to that described previously, except Taq DNA polymerase was not added. This control identified what component of the second-round PCR was a consequence of PCR amplification from the LTR alone and not integrated HIV-1 DNA. This control measured amplification from only the second-round LTR-LTR PCR or total HIV-1 DNA (summarized in Table 1 and Fig. 2).
All samples for the first- and second-round PCR assays were run in replicates of 5, given the varying length of the Alu-LTR amplicon, leading to high intra-assay variability of the Alu-LTR first-round reaction. A mean value of the 5 replicates was used to calculate the integrated copy number. To normalize for cell equivalents in the input DNA, we performed a separate real-time PCR with primers and beacons to detect the number of CCR5 copies as previously described.36
Given that DNA from the first round PCR was diluted 10-fold in the second round PCR, a difference of more than 3 cycles (which approximates a 10-fold change in template) between the threshold cycle (Ct) from the Alu-LTR/LTR-LTR reaction and the Ct from the LTR primer alone control reaction was required to demonstrate true amplification from the Alu-LTR reaction, and therefore to confirm that integrated HIV-1 DNA was present and quantifiable (see Fig. 2). The assay detected a minimum of 16 copies per reaction. Therefore, using a target number of cell equivalents of between 50,000 and 100,000 cells per reaction, the assay had a lower limit of detection of 16 to 32 copies per 100,000 cells.
Generation of Standards for Quantification of Integrated HIV-1
A standard that contained integrated HIV-1 DNA at random locations, and therefore at varying distances from the Alu repeat elements, was generated as previously described.34 Briefly the 293T-cell line was infected with a vesicular stomatitis virus G (VSV-G) pseudotyped HIV-1 that contained a deletion in envelope (VSV-G-HIVΔenv). Pseudotyped VSV-G-HIVΔenv was titrated using a reporter cell line, CEM-T-cell line, stably transfected with a green fluorescent protein (GFP) construct linked to the HIV-1 LTR promoter (CEM-GFP) to achieve a high multiplicity of infection (MOI = 1). Virus stock was filtered though a 0.45-μm filter (Sartorius, Goettingen, Germany) and treated with DNase 1 (Promega, Madison, WI) and added to 293T cells. Cells were passaged for 4 weeks to ensure that all the extracellular chromosomal forms of viral DNA were cleared as previously described.34 We therefore assumed that all HIV-1 genomes in the culture were now in an integrated form and quantified the amount of HIV-1 using an alternative real-time PCR assay that had both primers within the LTR.19,34,37
Quantification of Total HIV-1 DNA
To measure total HIV-1 DNA, a separate real-time PCR was performed. Briefly, each 50-μL reaction contained 5 μL of DNA, and the final concentration of each component was as follows: 1 × Taqman buffer II (Perkin Elmer, Waltham, MA), 3.5 mM of MgCl2, 0.8 mM of deoxynucleoside triphosphate, 1.25 U of AmpliTaq Gold DNA polymerase, 0.4 μM of each primer (sense: 5′-TCTCTAGCAGTGGCGCCCGAACA-3′ and antisense 5′-TCTCCTTCTAGCCTCCGCTAGTC-3′, and 0.2 μM of molecular beacon 5′ FAM (6-carboxyfluorescein)-CGGGAG TACTCACCAGTCGCCGCCCC CTCCCG (4-dimethylaminophenlazobenzoic acid, DABCYL) 3′. The PCR was performed in a spectrofluorometric thermal cycler (iCycler), and the cycling conditions were 95°C for 10 minutes, followed by 45 cycles of 94°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds.
HIV-1 RNA: Cell-Associated and Plasma
HIV-1 US RNA was quantified from PBMCs as previously described.25 HIV-1 plasma RNA was quantified using the Roche Amplicor assay (Roche Diagnostics, Basel, Switzerland). For samples with an HIV-1 viral load >700,000 copies/mL, serial dilutions were performed to achieve an accurate value.
HIV-1 RNA copies/mL less than the detection limit of 50 copies/mL were included in the analysis as 50 copies/mL. Total HIV-1 DNA and US HIV-1 RNA copies less than their detection limits of 20 and 200 copies per 106 PBMCs, respectively, were included for the analysis as 20 and 200 copies per 106 PBMCs. Integrated HIV-1 DNA levels <16 copies per input cellular DNA were included as 16 copies and adjusted for the amount of DNA added to the assay (15,000 to 150,000 cell equivalents) to copies per 106 PBMCs. HIV-1 DNA, integrated HIV-1 DNA, and US HIV-1 RNA per 106 PBMCs were then converted to copies per 106 CD4 T cells using the proportion of CD4 T cells to total PBMCs at each time point.
Comparisons of continuous variables between groups were performed with the Wilcoxon rank sum test, whereas the Fisher exact test was used for contingency analysis. Correlations were performed with Spearman rank correlation. P values <0.05 were considered significant.
Kinetics of Cell-Associated HIV-1 DNA and RNA After STI
The change in all virologic parameters over time during treatment and after STI is summarized in Figure 3. As expected, plasma HIV-1 RNA decreased during ARV treatment and was >5000 copies/mL at the time of reintroduction of ARVs (as per the treatment protocol). The change in HIV-1 DNA and US HIV-1 RNA was similar to that in plasma HIV-1 RNA. The proportions of individuals with detectable HIV-1 DNA and US HIV-1 RNA at the end of the first ARV interruption were 78% (15 of 19 individuals) and 21% (3 of 14 individuals), respectively; at the end of the second interruption, the proportions were 81% (9 of 11 individuals) and 22% (2 of 9 individuals), respectively; and at the end of the third interruption, the proportions were 90% (9 of 10 individuals) and 20% (2 of 10 individuals), respectively. There were no significant differences in the proportions of individuals with detectable HIV-1 DNA or HIV-1 US RNA or in the copy number of HIV-1 DNA or HIV-1 US RNA at each interruption.
Virologic Determinants of Treatment Success
Treatment success, or a “good response,” was achieved in 7 individuals (5 after the first interruption and 2 after the third interruption). The clinical and virologic differences between good (n = 7) and poor (n = 12) responders are summarized in Table 2. In brief, there was no significant difference in baseline HIV-1 plasma RNA, CD4 T-cell count, HIV-1 integrated DNA, or HU use between good and poor responders. The plasma HIV-1 RNA 12 weeks after the initiation of ARV was significantly lower in good responders compared with poor responders (median [interquartile range (IQR)] in good responders was 50 [50 to 50] copies/mL and was 430 [100 to 1260] copies/mL in poor responders; P = 0.005), and plasma HIV-1 RNA was significantly more likely to be undetectable in good responders (<50 copies/mL; P = 0.017). HIV-1 DNA and HIV-1 US RNA were only quantified after the commencement of ARVs, and there were no significant differences between good and poor responders at any time in the proportion of individuals with detectable HIV-1 DNA and HIV-1 US RNA (Fisher exact test; P > 0.1 for all time points) or in the copy number of HIV-1 DNA or HIV-1 US RNA.
Because week 12 plasma HIV-1 RNA was the best predictor of good response of all virologic components, we then investigated whether its predictive capability was also valid in the total cohort (n = 59 [18 good responders and 41 poor responders]). As observed in the subjects enrolled in the virology substudy, there was significantly lower plasma HIV-1 RNA at week 12 after initiation of ARVs in good responders compared with poor responders in the total cohort (P = 0.006). Finally, the proportion of individuals with plasma HIV-1 RNA lower than detection at week 12 for the total cohort was significantly greater for good responders compared with poor responders (odds ratio [OR] = 4.1; P = 0.02). Baseline plasma HIV-1 RNA was significantly lower in good responders compared with poor responders in the total cohort (P = 0.01), however, which was not observed in the smaller cohort enrolled in the virology substudy. In a multivariate analysis on the total cohort, baseline and week 12 plasma HIV-1 RNA were significant independent predictors of a good response (P = 0.035 and P = 0.047, respectively).
HIV-1 Integrated DNA Was Associated With an Early Response to Treatment
To gain further insight into potential mechanisms of virologic rebound, we then determined correlations between each of the viral quantification methods during this first year of ARVs. Baseline integrated HIV-1 DNA was assessed relative to other components at all available time points, whereas plasma HIV-1 RNA, total HIV-1 DNA, and HIV-1 US RNA were assessed relative to each other at corresponding time points. After Bonferroni adjustment for multiple testing, only 2 comparisons were significantly correlated. Integrated HIV-1 DNA at baseline was strongly correlated with plasma HIV-1 RNA at week 12 (P = 0.006, r = 0.81; n = 15; Fig. 4A) but not with plasma HIV-1 RNA at baseline. The association remained significant even when we only considered individuals with detectable integrated HIV-1 DNA (P = 0.03). Total HIV-1 DNA and HIV-1 US RNA (on ARVs) were not significantly associated with plasma HIV-1 RNA at baseline or week 12 (Fig. 4B-C). Total HIV-1 DNA and US HIV-1 RNA at week 12 were significantly correlated (P = 0.03, r = 0.68; n = 19; see Fig. 4D), however. When we only included individuals in whom HIV-1 US RNA was detectable (n = 4), the association was no longer significant.
We demonstrate here that the viral load measured 12 weeks after initiation of ARVs in primary infection was strongly associated with successful virologic control after interruption of ARVs. Although the concentration of integrated HIV-1 DNA before initiation of treatment was strongly correlated with HIV-1 RNA after 12 weeks of ARVs, quantification of integrated HIV-1 DNA itself before initiation of ARVs or that of HIV-1 DNA or US HIV-1 RNA did not predict a good or poor response after interruption of ARVs.
The only significant virologic factor that was correlated with clinical outcome, defined as the extent of rebound of viral replication, was the plasma HIV-1 RNA level at week 12 of treatment. This remained significant when we looked at all subjects in the study (n = 59). In addition, the plasma HIV-1 RNA level before initiation of ARVs was significantly associated with clinical outcome, as previously reported by our group and others.13,38,39 It was interesting that there was a correlation between integrated HIV-1 DNA before therapy and the week 12 plasma HIV-1 RNA level, suggesting that the total number of infected cells before ARVs may determine the pattern of viral kinetics after the initiation of ARVs, specifically the second phase decay, as predicted in earlier mathematic models of HIV-1 viral kinetics.40,41 In this study, we were unable to address the relation between integrated DNA prior to treatment and HIV RNA on treatment because detailed viral kinetic analyses were not performed, but this relation should be explored in future studies after treatment of primary infection.
We were unable to demonstrate that a more sensitive marker of the number of infected cells (HIV-1 DNA or integrated HIV-1 DNA) or residual viral replication (eg, HIV-1 US RNA) may predict clinical outcome after cessation of ARVs. Another study of treatment interruption after acute HIV-1 infection also showed that the concentration of HIV-1 DNA did not predict the likelihood of viral rebound14 in contrast to studies of treatment interruption in chronic infection, where the baseline concentration of HIV-1 DNA was lower in individuals with a successful outcome after treatment interruption.11,42 We were unable to measure total HIV-1 DNA at baseline; however, the level of HIV-1 DNA before each treatment interruption or at any time on treatment was not associated with the likelihood of viral rebound.
No previous studies have evaluated the role of cell-associated HIV-1 RNA in predicting viral rebound after cessation of ARV. In this study, few individuals had detectable cell-associated HIV-1 US RNA before (20%) or after (∼50%) treatment interruption. This is consistent with previous studies, which showed that HIV-1 US RNA is found in some but not all individuals receiving ARVs and that the amount of HIV-1 US RNA increases soon after treatment interruption.25,27-29 We chose to measure HIV-1 US RNA in preference to MS RNA because we and others have demonstrated that HIV-1 US RNA is more commonly detected in individuals receiving ARV than MS RNA encoding for tat-rev.25,33 Another group that studied HIV-1 US RNA and MS RNA during STI in chronic infection found that MS RNA encoding nef transcripts was more frequently detected than MS RNA encoding tat/rev transcripts and cell-associated US RNA in individuals receiving ARVs, however. In addition, the level of MS RNA encoding nef predicted rebound viremia after treatment interruption.27 Measurement of transcripts other than cell-associated US RNA may be a more sensitive marker to predict viral rebound.
This is the first study to determine if quantification of integrated HIV-1 DNA may predict viral rebound after interruption of ARVs. There have been 4 previous methods described to quantify integrated HIV-1 DNA. The methods include 1-step Alu-LTR amplification;34 nested PCR using linker primers;21,22 real-time nested PCR using Alu-specific primers and a virus specific primer without a tag sequence;24,35 and, more recently, real-time nested PCR using Alu-specific primers and a virus-specific primer with a tag sequence.43,44 By the addition of a tag sequence to the virus-specific primer, the specificity of amplification is substantially improved, because amplification of unintegrated forms of HIV-1 is reduced. We did not use a tag sequence in this study but accounted for the potential contribution of unintegrated HIV-1 DNA to the detection of integrated HIV-1 DNA by including “no polymerase” and LTR alone controls. We only considered true detection of integrated HIV-1 DNA, if there was at least a 3-threshold cycle shift between the LTR alone control and the Alu-LTR/LTR-LTR reaction. This stringent approach may have potentially underestimated the amount of integrated HIV-1 DNA present and reduced the sensitivity of the assay in vivo. The frequency of detection of integrated HIV-1 DNA in untreated individuals was approximately 40%, however, which is similar to that recently reported using a similar technique.24
There were several limitations to this study. First, we were unable to measure all parameters on baseline specimens, given the large number of cells required to quantify integrated HIV-1 DNA. We did, however, measure residual HIV-1 DNA and HIV-1 US messenger RNA (mRNA) before each interruption. We hypothesized that this was the best time to quantify the size of the residual reservoir. Second, the sensitivity of each of the assays used was limited by the total amount of DNA added to each assay. This was dependent on sample availability and inhibition of PCR in the presence of >1 μg of cellular DNA. Therefore, we were unable to detect and quantify integrated HIV-1 DNA and HIV-1 US mRNA in a significant number of specimens, although the frequency of detection was consistent with previous reports with different assay techniques.24,27 Given the detailed virologic analyses required for each patient, the sample size was small; therefore, these findings need to be confirmed in a larger study. This study only addressed virologic parameters associated with viral rebound when other factors may clearly be relevant, such as the CD8 T-cell, immune activation, and neutralizing antibody response.38,45-47 Finally, in this study, we only evaluated potential viral reservoirs in blood, and it is possible that the source of rebound virus may be from other anatomic sites that have a high burden of HIV-1 infection, such as the gastrointestinal tract or lymphoid tissue.48-50
In summary, we identified that HIV-1 RNA measured 12 weeks after initiation of ARVs was the only virologic variable associated with viral rebound after treatment interruption. The use of more complex assays to measure cell-associated virus in blood, including quantification of integrated HIV-1 DNA, did not help to predict the likelihood of viral rebound after STI in the setting of PHI.
The authors thank all the participants and investigators of the PULSE study and Ming Lee Goh and Jane Howard for development of the integrated HIV cell line.
1. Palella FJ Jr, Delaney KM, Moorman AC, et al. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. N Engl J Med
2. Chen LF, Hoy J, Lewin SR. Ten years of highly active antiretroviral therapy for HIV infection. Med J Aust
3. Ananworanich J, Hirschel B. Interrupting highly active antiretroviral therapy in patients with HIV. Expert Review of Anti Infective Therapy
4. Julg B, Goebel FD. Treatment interruption in HIV therapy: a SMART strategy? Infection
5. Ananworanich J, Gayet-Ageron A, Le Braz M, et al. CD4-guided scheduled treatment interruptions compared with continuous therapy for patients infected with HIV-1: results of the Staccato randomised trial. Lancet
6. El-Sadr WM, Lundgren JD, Neaton JD, et al. CD4+ count-guided interruption of antiretroviral treatment. N Engl J Med
7. Markowitz M, Jin X, Hurley A, et al. Discontinuation of antiretroviral therapy commenced early during the course of human immunodeficiency virus type 1 infection, with or without adjunctive vaccination. J Infect Dis
8. Kaufmann DE, Lichterfeld M, Altfeld M, et al. Limited durability of viral control following treated acute HIV infection. PLoS Med
9. Rosenberg ES, Billingsley JM, Caliendo AM, et al. Vigorous HIV-1-specific CD4+ T cell responses associated with control of viremia [see comments]. Science
10. Fagard C, Oxenius A, Gunthard H, et al. A prospective trial of structured treatment interruptions in human immunodeficiency virus infection. Arch Intern Med
11. Yerly S, Gunthard HF, Fagard C, et al. Proviral HIV-DNA predicts viral rebound and viral setpoint after structured treatment interruptions. AIDS
12. Joos B, Trkola A, Fischer M, et al. Low human immunodeficiency virus envelope diversity correlates with low in vitro replication capacity and predicts spontaneous control of plasma viremia after treatment interruptions. J Virol
13. Bloch MT, Smith DE, Quan D, et al. The role of hydroxyurea in enhancing the virologic control achieved through structured treatment interruption in primary HIV infection: final results from a randomized clinical trial (PULSE). J Acquir Immune Defic Syndr
14. Hoen B, Fournier I, Lacabaratz C, et al. Structured treatment interruptions in primary HIV-1 infection: the ANRS 100 PRIMSTOP trial. J Acquir Immune Defic Syndr
15. Rosenberg ES, Altfeld M, Poon SH, et al. Immune control of HIV-1 after early treatment of acute infection. Nature
16. Zhang L, Chung C, Hu BS, et al. Genetic characterization of rebounding HIV-1 after cessation of highly active antiretroviral therapy. J Clin Invest
17. Chun TW, Davey RT Jr, Engel D, et al. Re-emergence of HIV after stopping therapy. Nature
18. Shaunak S, Teo I. Monitoring HIV disease with new and clinically useful surrogate markers. Curr Opin Infect Dis
19. Zhang L, Ramratnam B, Tenner-Racz K, et al. Quantifying residual HIV-1 replication and decay of the latent reservoir in patients on seemingly effective antiretroviral therapy. N Engl J Med
20. Chun T-W, Stuyer L, Mizell S, et al. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc Natl Acad Sci USA
21. Kumar R, Vandegraaff N, Mundy L, et al. Evaluation of PCR-based methods for the quantitation of integrated HIV-1 DNA. J Virol Methods
22. Vandegraaff N, Kumar R, Burrell CJ, et al. Kinetics of human immunodeficiency virus type 1 (HIV) DNA integration in acutely infected cells as determined using a novel assay for detection of integrated HIV DNA. J Virol
23. Sonza S, Maerz A, Deacon N, et al. Human immunodeficiency virus type 1 replication is blocked prior to reverse transcription and integration in freshly isolated peripheral blood monocytes. J Virol
24. Carr JM, Cheney KM, Coolen C, et al. Development of methods for coordinate measurement of total cell-associated and integrated human immunodeficiency virus type 1 (HIV-1) DNA forms in routine clinical samples: levels are not associated with clinical parameters, but low levels of integrated HIV-1 DNA may be prognostic for continued successful therapy. J Clin Microbiol
25. Lewin S, Vesanen M, Kostrikis L, et al. The use of real-time PCR and molecular beacons to detect virus-replication in HIV-1-infected individuals on prolonged effective antiretroviral therapy. J Virol
26. Vesanen M, Markowitz M, Cao Y, et al. Human immunodeficiency virus type-1 mRNA splicing pattern in infected persons is determined by the proportion of newly infected cells. Virology
27. Fischer M, Joos B, Hirschel B, et al. Cellular viral rebound after cessation of potent antiretroviral therapy predicted by levels of multiply spliced HIV-1 RNA encoding nef. J Infect Dis
28. Fischer M, Gunthard HF, Opravil M, et al. Residual HIV-RNA levels persist for up to 2.5 years in peripheral blood mononuclear cells of patients on potent antiretroviral therapy. AIDS Res Hum Retroviruses
29. Fischer M, Trkola A, Joos B, et al. Shifts in cell-associated HIV-1 RNA but not in episomal HIV-1 DNA correlate with new cycles of HIV-1 infection in vivo. Antivir Ther
30. Zhang L, Ramratnam B, Tenner-Racz K, et al. Quantifying residual HIV-1 replication in patients receiving combination antiretroviral therapy. N Engl J Med
31. Palmer S, Wiegand AP, Maldarelli F, et al. New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma. J Clin Microbiol
32. Maldarelli F, Palmer S, King MS, et al. ART suppresses plasma HIV-1 RNA to a stable set point predicted by pretherapy viremia. PLoS Pathog
33. Furtado M, Callaway D, Phair J, et al. Persistence of HIV-1 transcription in peripheral-blood mononuclear cells in patients receiving potent antiretroviral therapy. N Engl J Med
34. Butler SL, Hansen MS, Bushman FD. A quantitative assay for HIV DNA integration in vivo. Nat Med
35. O'Doherty U, Swiggard WJ, Jeyakumar D, et al. A sensitive, quantitative assay for human immunodeficiency virus type 1 integration. J Virol
36. Zhang L, Lewin S, Markowitz M, et al. Measuring recent thymic emigrants in blood of normal persons and HIV-1-infected patients before and after effective therapy. J Exp Med
37. Solomon A, Lane N, Wightman F, et al. Enhanced replicative capacity and pathogenicity of HIV-1 isolated from individuals infected with drug-resistant virus and declining CD4+
T-cell counts. J Acquir Immune Defic Syndr
38. Oxenius A, Price DA, Gunthard HF, et al. Stimulation of HIV-specific cellular immunity by structured treatment interruption fails to enhance viral control in chronic HIV infection. Proc Natl Acad Sci USA
39. Lafeuillade A, Poggi C, Hittinger G, et al. Predictors of plasma human immunodeficiency virus type 1 RNA control after discontinuation of highly active antiretroviral therapy initiated at acute infection combined with structured treatment interruptions and immune-based therapies. J Infect Dis
40. Ho D, Neumann A, Perelson A, et al. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature
41. Perelson A, Essunger P, Cao Y, et al. Decay characteristics of HIV-1-infected compartments during combination therapy. Nature
42. Kabamba-Mukadi B, Henrivaux P, Ruelle J, et al. Human immunodeficiency virus type 1 (HIV-1) proviral DNA load in purified CD4+ cells by LightCycler real-time PCR. BMC Infect Dis
43. Brussel A, Sonigo P. Analysis of early human immunodeficiency virus type 1 DNA synthesis by use of a new sensitive assay for quantifying integrated provirus. J Virol
44. Yamamoto N, Tanaka C, Wu Y, et al. Analysis of human immunodeficiency virus type 1 integration by using a specific, sensitive and quantitative assay based on real-time polymerase chain reaction. Virus Genes
45. Trkola A, Kuster H, Rusert P, et al. Delay of HIV-1 rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies. Nat Med
46. Huber M, Fischer M, Misselwitz B, et al. Complement lysis activity in autologous plasma is associated with lower viral loads during the acute phase of HIV-1 infection. PLoS Med
47. Benito JM, Lopez M, Ballesteros C, et al. Immunological and virological effects of structured treatment interruptions following exposure to hydroxyurea plus didanosine. AIDS Res Hum Retroviruses
48. Brenchley JM, Schacker TW, Ruff LE, et al. CD4+ T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract. J Exp Med
49. Mehandru S, Poles MA, Tenner-Racz K, et al. Lack of mucosal immune reconstitution during prolonged treatment of acute and early HIV-1 infection. PLoS Med
50. Fischer M, Joos B, Wong JK, et al. Attenuated and nonproductive viral transcription in the lymphatic tissue of HIV-1-infected patients receiving potent antiretroviral therapy. J Infect Dis
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Keywords:© 2008 Lippincott Williams & Wilkins, Inc.
HIV-1 DNA; integrated HIV-1 DNA; primary HIV-1 infection; treatment interruption; unspliced HIV-1 RNA