Abstract
In resting CD4+ T lymphocytes harboring human immunodeficiency virus type 1 (HIV-1), replication-competent virus persists in patients responding to highly active antiretroviral therapy (HAART). This small latent reservoir represents between 103 and 107 cells per patient. However, the efficiency of HIV-1 DNA-positive resting CD4+ T cells in converting to HIV-1-antigen-secreting cells (HIV-1-Ag-SCs) after in vitro CD4+-T-cell polyclonal stimulation has not been satisfactorily evaluated. By using an HIV-1-antigen enzyme-linked immunospot assay, 8 HIV-1-Ag-SCs per 106 CD4+ resting T cells were quantified in 25 patients with a plasma viral load of <20 copies/ml, whereas 379 were enumerated in 10 viremic patients. In parallel, 369 and 1,238 copies of HIV-1 DNA per 106 CD4+ T cells were enumerated in the two groups of patients, respectively. Only a minority of latently HIV-1 DNA-infected CD4+ T cells could be stimulated in vitro to become HIV-1-Ag-SCs, particularly in aviremic patients. The difference between the number of HIV-1 immunospots in viremic versus aviremic patients could be explained by HIV-1 unintegrated viral DNA that gave additional HIV-1-Ag-SCs after in vitro CD4+-T-cell polyclonal stimulation. The ELISPOT approach to targeting the HIV-1-Ag-SCs could be a useful method for identifying latently HIV-1-infected CD4+ T cells carrying replication-competent HIV-1 in patients responding to HAART.
The use of highly active antiretroviral therapy (HAART) in human immunodeficiency virus type 1 (HIV-1)-infected individuals has dramatically improved the clinical outcome for many patients and has led to a substantial decline in the incidence of AIDS and in AIDS-related mortality (15). However, the presence of latently infected CD4+ T lymphocytes harboring replication-competent virus has been consistently demonstrated for the majority of HIV-1-infected individuals receiving HAART in whom plasma viremia has been successfully suppressed for prolonged periods of time (4, 8, 11, 17, 18, 29, 30, 33, 35). Two forms of HIV-1 DNA have been described, a labile unintegrated form which decays within the first 3 months after the initiation of therapy (1, 29) and a stable integrated form (1, 4, 29). This small, persistent, latent reservoir has been established to be between 103 and 107 cells per patient, and in subjects who consistently maintain undetectable plasma HIV-1 RNA levels, it seems very stable, with a mean half-life of 43.9 months (17, 31), although a half-life as short as 6 months has been reported for some patients treated during the primary infection or for patients with unusually good suppression of viral replication on HAART (30, 33). These findings render untenable the idea that HIV-1 eradication can be achieved in patients receiving HAART, and the transient detectable viremia called “blips” (30) in some patients classified as good responders to HAART suggests a residual persistent HIV-1-competent replication despite the efficiency of the treatment.
Both virologic and molecular approaches have been used to enumerate CD4+ T cells latently infected with HIV-1. One approach consists in inducing HIV-1 virus production by purified resting CD4+ T cells after their polyclonal activation by a mitogenic stimulus in the presence of allogeneic peripheral blood mononuclear cells (PBMC) previously depleted in CD8+ T cells (6, 9). The use of a limiting dilution format and detection of replication-competent forms of HIV-1 could make this method quantitative (4, 5). On the other hand, integrated forms of HIV-1 DNA can be detected in highly purified resting CD4+ T cells by the inverse PCR method, and typically less than 0.01% of resting CD4+ T cells carry integrated HIV-1 DNA (5, 7, 18, 33). Since it is necessary to quantify resting CD4+ T cells able to produce HIV-1 antigens (HIV-1-Ag), different approaches have been developed to detect HIV-1 mRNAs, such as in situ hybridization (13, 21) and a recently described reverse transcriptase PCR method, which affords possibilities of detecting unspliced and spliced HIV-1 mRNAs (20). These observations suggest that HIV-1 DNA-positive resting CD4+ T cells are able to produce viral structural proteins and even complete particles, and only 1% of the HIV-1 DNA-positive lymphocytes induce an up-regulation of HIV-1 mRNA after in vitro CD4+-T-cell polyclonal activation (20).
Our group has developed an HIV-1-Ag ELISPOT assay to enumerate HIV-1-Ag-producing cells (HIV-1-Ag-SCs) following in vitro polyclonal activation of highly purified CD4+ T lymphocytes (19). Each immunospot represents the fingerprint of one HIV-1-Ag-producing CD4+ T cell, and this assay makes it possible to identify a single cell dispersed among one million cells tested (12). Therefore, in highly purified resting CD4+ T lymphocytes from patients with detectable HIV-1 viral load in plasma or from aviremic patients receiving HAART for a long time, the number of circulating CD4+ T cells able to produce viral proteins upon polyclonal activation were enumerated by the HIV-1-Ag ELISPOT assay. We compared these results with the number of HIV-1 DNA copies in resting CD4+ T lymphocytes by using a real-time HIV-1 DNA PCR. Our results demonstrate that a minority of latently HIV-1 DNA-infected CD4+ T cells could be stimulated in vitro to become HIV-1-Ag-SCs and that the capacity of HIV-1 DNA to be transcribed and translated into HIV-1 viral proteins was weak, particularly in aviremic patients.
MATERIALS AND METHODS
Patients.
Ten patients with a detectable HIV-1 viral load in plasma and 25 patients highly adherent to HAART on the basis of long-term undetectable plasma viral RNA were recruited for this study, which obtained the approval of the local ethics committee (Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale de l'Hôpital de Montpellier, no. 98-7564). All patients gave written informed consent. The characteristics of the patients are presented in Table 1. HIV-1 RNA in plasma was measured by an HIV-1 RNA PCR assay (Amplicor HIV-1 monitor test; Roche Diagnostics Systems, Meylan, France). Samples undetectable by the standard assay (threshold, 200 copies/ml) were reanalyzed using an ultrasensitive procedure (threshold, 20 copies/ml; Roche Diagnostic System). The number of CD4+ T cells was determined by flow cytometry (XL-apparatus; Beckman-Coulter, Villepinte, France) after cell staining with fluorescein isiothiocyanate-conjugated antibodies (Ab) directed against the CD4 receptor (Beckman).
TABLE 1.
Profiles of the HIV-1-infected patients studied
| Patient no. | Drug regimen at time of studya | Duration of virologic supression (mo) | Plasma HIV-1 RNA level (no. of copies/ml) | CD4+-T-cell count (cells/μl) |
|---|---|---|---|---|
| 1 | ABC+TNV+3TC+ABT | 0 | 57 | 492 |
| 2 | ABC+TNV+ABT | 0 | 81 | 253 |
| 3 | AZT+3TC+DDI+ABT | 0 | 1,210 | 635 |
| 4 | AZT+3TC+ABC+TNV | 0 | 1,300 | 692 |
| 5 | None | 0 | 3,120 | 322 |
| 6 | AZT+3TC+EFV | 0 | 7,490 | 440 |
| 7 | None | 0 | 1,200 | 442 |
| 8 | None | 0 | 14,400 | 614 |
| 9 | None | 0 | 152,000 | 320 |
| 10 | None | 0 | 390,000 | 292 |
| 11 | AZT+3TC+ABC | 3 | <20 | 325 |
| 12 | AZT+3TC+NVP | 6 | <20 | 878 |
| 13 | AZT+3TC+DDI | 6 | <20 | 372 |
| 14 | AZT+3TC+ABC+NVP | 11 | <20 | 386 |
| 15 | AZT+3TC+EFV | 24 | <20 | 469 |
| 16 | AZT+3TC+ABC+EFV | 21 | <20 | 798 |
| 17 | 3TC+TNV+ABT | 26 | <20 | 302 |
| 18 | DDI+FTC+EFV | 30 | <20 | 828 |
| 19 | DDI+FTC+EFV | 38 | <20 | 308 |
| 20 | AZT+3TC+TNV | 50 | <20 | 745 |
| 21 | AZT+3TC+DDI+NVP | 30 | <20 | 980 |
| 22 | AZT+3TC+NVP | 57 | <20 | 573 |
| 23 | 3TC+TNV+NVP | 60 | <20 | 599 |
| 24 | AZT+3TC+NVP | 36 | <20 | 850 |
| 25 | AZT+3TC+ABC | 60 | <20 | 1,300 |
| 26 | AZT+3TC+EFV | 22 | <20 | 332 |
| 27 | ABC+3TC+TNV+NFV | 3 | <20 | 333 |
| 28 | AZT+3TC+ABC+ABT | 30 | <20 | 351 |
| 29 | 3TC+ABC+TNV+ABT | 3 | <20 | 440 |
| 30 | AZT+3TC+ABC | 5 | <20 | 636 |
| 31 | ABC+3TC+NVP | 2 | <20 | 230 |
| 32 | 3TC+EFV+TNV | 20 | <20 | 739 |
| 33 | ABC+TNV+ABT | 22 | <20 | 1,466 |
| 34 | ADI+FTC+EFV | 35 | <20 | 422 |
| 35 | AZT+3TC+EFV | 1 | <20 | 209 |
Abbreviations for antiretroviral drugs: AZT, zidovudine; 3TC, lamivudine; ABC, abacavir; NVP, nevirapine; EFV, efavirenz; NFV, nelfinavir; ABT, lopinavir plus ritonavir; TNV, tenofovir; DDI, didanosine; FTC, emtricitabine.
Isolation of resting CD4+ T cells.
CD4+ T cells from HIV-1-infected patients were purified from EDTA-treated blood samples using a Rosette sep CD4 cell enrichment cocktail, according to the manufacturer's instructions (Stemcell Technologies, Meylan, France). Ex vivo-activated CD4+ T cells were eliminated using magnetic beads coupled with anti-HLA-DR monoclonal Ab (Dynabeads M-450; Dynal, Oslo, Norway). Briefly, cells and beads (bead-to-cell ratio of 4:1) were shaken for 30 min at 4°C, and the unbound cell fractions were removed and washed twice in phosphate-buffered saline (PBS) supplemented with 2% fetal calf serum. The enriched CD4+-T-cell population contained more than 98% CD4+ T cells, and less than 1% of enriched resting CD4+ T cells were spontaneously activated (Fig. 1). Aliquots of 106 CD4+ T cells were stored in liquid nitrogen until tested in the HIV-1-Ag ELISPOT and the HIV-1 DNA real-time PCR assays.
FIG. 1.
Efficiency of ex vivo spontaneously activated CD4+-T-cell elimination. Enrichments of CD4+ T cells (I and II) were characterized according to membrane HLA-DR receptor expression (Ib and IIb). Aliquots of CD4+ T cells were incubated with a mixture of phycoerythrin-cyanin 5 (PC5)-conjugated anti-CD4 and fluorescein isiothiocyanate (FITC)-conjugated anti-HLA-DR antibodies (Beckman-Coulter). (I) Purified CD4+ T cells. (II) Ex vivo-activated CD4+ T cells were eliminated using magnetic beads coupled with anti-HLA-DR monoclonal Ab (Dynal). The horizontal line separating the HLA-DR negative and positive subpopulations was defined using an isotypic control (Ia and IIa).
Culture conditions.
Resting CD4+-T-cell samples were cultured in flasks at 106 cells/ml with monoclonal human Ab directed against CD3 and CD28 receptors plus irradiated PBMC without CD8+ T cells from HIV-1-seronegative individuals (9). Briefly, 24-well culture plates (Falcon, Meylan, France) were coated overnight with anti-CD3 Ab (200 ng/106 cells). PBMC from healthy controls were depleted of CD8+ T cells by using beads coupled with anti-CD8 Ab (Dynal) and then gamma irradiated (25 Gy). After washings with PBS, enriched CD4+ T cells were cultured with soluble anti-CD28 Ab (200 ng/106 cells) plus 3 × 106 irradiated PBMC. Then, the cells were cultured at 37°C in a 5% CO2-humidified atmosphere and tested at day 5 using the HIV-1 Ag ELISPOT assay. In addition, unstimulated CD4+ T cells, not exposed to either anti-CD3 Ab, anti-CD28 Ab, or allogeneic cells, were also cultured under the same conditions. For five patients (no. 11, 15, 20, 25, and 30), aliquots of resting CD4+ T cells were cultured with or without 1 μg of synthetic peptide (enfuvirtide; Roche Pharma, Nutley, N.J.)/ml that corresponds to a linear 36-amino-acid sequence within HR2 of gp41 (14, 24). In four preliminary experiments, 106 resting CD4+ T cells were cultured with different concentrations of enfuvirtide (0.01 to 5 μg/ml), and at day 5 of culture, the viable cells were counted by the trypan blue exclusion test. At 1 and 5 μg of enfuvirtide/ml, the viability of the cells was 93.4% (± 1.7) and 92.5% (± 2.1), respectively.
HIV-1-Ag ELISPOT assay.
This assay was performed as previously described (12, 19). Briefly, Immobilon-P membrane 96-well plates (MAIPN 4550; Millipore Corporation, Bedford, Mass.) were coated overnight at 4°C with a mixture of anti-HIV-1 polyclonal Ab prepared as previously described (12). Sera from 10 HIV-1 patients with a complete serologic pattern in Western blots were pooled, adsorbed on CEM cells at a concentration of 5 × 106 cells/ml for 60 min at 37°C under agitation, and then used at a 1:250 dilution. After washings with PBS, 105 CD4+ T cells from HIV-1 patients were added to each well. Plates were then incubated for 24 h at 37°C in a 5% CO2-humidified atmosphere and washed with PBS, PBS-0.05% Tween 20, and again PBS. One hundred microliters of biotinylated anti-p24 monoclonal Ab at 1:1,000 dilution (R4, genetics systems HIV-1 Ag EIA; Bio-Rad, Marnes la Coquette, France) was added and incubated for 6 h at 37°C. After washing with PBS, a solution of alkaline phosphatase-labeled streptavidin diluted at 1:500 in PBS was added, and the plates were incubated for 45 min at 37°C. The plates were washed three times in PBS and developed with a chromogenic substrate (a mixture of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium; Sigma, St. Louis, Mo.). Spots appeared as purple precipitates after 10 min, and the reaction was stopped by washing the plates with distilled water. HIV-1 immunospots were counted by video camera imaging and computer-assisted analysis (KS ELISPOT; Carl Zeiss Vision, Hallbermoos, Germany). The frequency of HIV-1-Ag-SCs was equal to the frequency of the total number of cells producing HIV-1-Ag in vitro minus the frequency of HIV-1-producing cells obtained with unstimulated CD4+ T cells at day 5 of culture. Unstimulated cells from untreated HIV-1-infected patients generated a frequency of HIV-1-producing cells of between 3 to 10 cells per million.
HIV-1 DNA real-time PCR assay.
A real-time PCR assay was used to quantify proviral HIV-1 DNA according to the instructions of the ANRS, the French agency for AIDS research, for HIV-1 quantification (M. Burgard, M. L. Chaix, N. Ngo, N. Leruez, and C. Rouzioux, Proc. 8th Conf. Retrovir. Opportun. Infect., abstr. 243, 2001). Briefly, total DNA was extracted from 106 purified resting CD4+ T cells or 8E5 cells, using the QIAamp DNA blood Midikit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions, and stored at −80°C. HIV-1 DNA was quantified by real-time PCR using primers that target the long terminal repeat (LTR) gene. The reaction was performed with the LightCycler (Roche Diagnostics Systems). Briefly, 2 μg of DNA was amplified with the sense primer NEC 152 (5′-GCCTCAATAAAGCTTGCCTTGA-3′) and the reverse primer NEC 131 (5′-GGCGCCACTGCTAGAGATTTT-3′) in the presence of a dually (FAM and TAMRA) labeled NEC LTR probe (5′-AAGTAGTGTGTGCCCGTCTTRTKTGACT-3′) (K = G+T; R = G+A). The first PCR cycle allowed us to quantify the HIV-1 DNA by reference to a standard curve (fivefold dilutions of 8E5 cell DNA). To determine precisely the amount of DNA in purified resting CD4+ T cells, all samples were tested using LightCycler-Control kit DNA (Roche), which quantifies the human β-globin gene. All samples from each patient were tested in the same assay, and the results were expressed as the number of DNA copies/106 CD4+ T cells tested.
Statistical analysis.
The correlations between variables were analyzed by Spearman's rank test. Results were compared using the Mann-Whitney U test. P values of <0.05 were considered statistically significant.
RESULTS
The number of CD4+ T cells latently infected with HIV-1 was assessed with the following: (i) HIV-1-infected patients highly adherent to HAART with a viral plasma RNA load below 20 copies/ml for at least 6 months (n = 25), (ii) patients incompletely responding to HAART with a persistent residual HIV-1 plasma viral load (n = 5), and (iii) untreated patients (n = 5) (Table 1). Circulating CD4+ T cells were highly purified (>95%), and the preparations contained less than 1% of ex vivo-activated CD4+ T cells. Then, the enriched CD4+ T cells were stimulated by strong in vitro CD4+ T-cell polyclonal activation induced by Ab directed against the CD3 and CD28 receptors plus allogeneic CD8+-depleted and irradiated PBMC from healthy controls. Finally, CD4+ T lymphocytes latently infected with HIV-1 were assessed by the HIV-1-Ag ELISPOT assay for their capacity to produce HIV-1 viral proteins. In parallel, the proviral HIV-1 DNA copies were quantified in inactivated CD4+ T cells using a real-time PCR assay.
HIV-1-Ag-SCs were detected in all patients tested, with a median frequency of 11.2 cells per million CD4+ T lymphocytes. For the patients who responded to HAART (Table 2), the median number of HIV-1-specific spots was 8 per 106 resting CD4+ T lymphocytes tested (25th percentile, 3.7; 75th percentile, 20). In contrast, the number of HIV-1-Ag-SCs (median, 379; 25th percentile, 15; 75th percentile, 806) was found to be significantly higher (P = 0.04) for 10 patients who showed continuously detectable levels of plasma HIV-1 RNA copies (Table 3). After having established the frequency of latently HIV-1-infected CD4+ T lymphocytes able to synthesize HIV-1-Ag following in vitro CD4+-T-cell polyclonal activation, we analyzed in parallel the proviral HIV-1 DNA burden in the enriched unstimulated CD4+-T-cell preparations. The HIV-1 DNA copies were quantified for all the HIV-1-infected patients, with a median of 589 copies per 106 CD4+ T cells tested. For responder patients receiving HAART over a long term (Table 2), the median number of HIV-1 DNA copies was 369/106 resting CD4+ T lymphocytes (25th percentile, 148; 75th percentile, 1,072), whereas the number of HIV-1 DNA copies (median, 1,238; 25th percentile, 617; 75th percentile, 3,006) was found to be significantly higher (P = 0.03) for patients (five untreated and five treated) with detectable plasma HIV-1 RNA. For seven aviremic patients, higher levels of HIV-1 DNA (>1,000 copies/106 CD4+ T lymphocytes) were found, and the number of HIV-1-Ag-SCs was also higher compared to those for the other responder patients (Table 3). These results could be explained by a transient emergence of viral replication despite HAART (30, 32). Such “blips” could allow the infection of other CD4+ T cells, thus contributing to the increase of the frequency of inducible HIV-1-Ag-SCs.
TABLE 2.
Determination of HIV-1 DNA-positive resting CD4+ T cells secreting HIV-1 viral proteins in HAART patients
| Patient no. | HIV-1-DNA level (no. of copies/106 resting CD4+ T cells) | Frequency of HIV-1- antigen-secreting cells/106 resting CD4+ T cells |
|---|---|---|
| 11 | 192 | 1.1 |
| 12 | 546 | 3.6 |
| 13 | 301 | 2.5 |
| 14 | 725 | 4.0 |
| 15 | 128 | 3.7 |
| 16 | 1,344 | 4.5 |
| 17 | 369 | 2.0 |
| 18 | 34 | 7.3 |
| 19 | 193 | 3.7 |
| 20 | 23 | 1.0 |
| 21 | 1,941 | 1.1 |
| 22 | 174 | 9.0 |
| 23 | 453 | 8.0 |
| 24 | 101 | 2.7 |
| 25 | 132 | 0.4 |
| 26 | 1,231 | 4.6 |
| 27 | 1,072 | 1.9 |
| 28 | 227 | 7.7 |
| 29 | 468 | 4.5 |
| 30 | 26 | 3.0 |
| 31 | 4,013 | 3.1 |
| 32 | 2,934 | 8.3 |
| 33 | 1,598 | 2.0 |
| 34 | 1,480 | 7.5 |
| 35 | 827 | 43.6 |
| Median | 369 | 8.0 |
TABLE 3.
Determination of HIV-1 DNA-positive resting CD4+ T cells secreting HIV-1 viral proteins in HIV-1 patients with a detectable plasma viral load
| Patient no. | HIV-1 DNA level (no. of copies/106 resting CD4+ T cells) | Frequency of HIV-1- antigen-secreting cells/106 resting CD4+ T cells |
|---|---|---|
| 1 | 10,000 | 857 |
| 2 | 1,667 | 723 |
| 3 | 589 | 4.7 |
| 4 | 655 | 15 |
| 5 | 353 | 7.7 |
| 6 | 617 | 18 |
| 7 | 1,573 | 666 |
| 8 | 903 | 92 |
| 9 | 3,006 | 1,028 |
| 10 | 15,831 | 806 |
| Median | 1,238 | 379 |
The question arises as to whether during the activation of CD4+ T lymphocytes, de novo-sensitized HIV-1 could infect other cultured T lymphocytes and cause overestimation of the ELISPOT results. To answer this question, HIV-1 DNA was measured in highly purified CD4+ T cells recovered from five patients with undetectable plasma viremia, before and after 5 days of CD4+-T-cell stimulation. The numbers of HIV-1 DNA copies per million CD4+ T cells tested were not significantly different (P > 0.05) before and at the term of the culture (data not shown). Moreover, since integrated proviral DNA is replicated like all other genomic DNA when cells proliferate, we measured the numbers of HIV-1 DNA copies and HIV-1-Ag-SCs in resting CD4+ T cells from five patients, cultured with or without 1 μg of an HIV-1 fusion inhibitor (enfuvirtide; Roche Pharma)/ml. As shown in Table 4, similar numbers of HIV-1 DNA copies and HIV-1-Ag-SCs were found in the resting CD4+ T cells cultured with or without enfuvirtide. These results strongly suggest that de novo-sensitized HIV-1 did not infect other T lymphocytes in the culture. In addition, previously published data indicate that the average HIV-1 generation time is 2.6 days, defined as the time from release of virion until it infects another cell and causes the release of a new generation of HIV-1 virions (28). The number of HIV-1-Ag-SCs measured at the term of the CD4+ T-cell polyclonal activation was not overestimated by a potential de novo HIV-1 infection.
TABLE 4.
Effect of the addition of enfuvirtide on de novo HIV-1 infection in resting CD4+-T-cell culture
| Patient no. | HIV-1 DNA level (no. of copies/106 resting CD4+ T cells)
|
Frequencies of HIV-1-antigen-secreting cells/106 resting CD4+ T cells
|
||
|---|---|---|---|---|
| +a | −b | +a | −b | |
| 11 | 125 | 200 | 7 | 12 |
| 15 | 76 | 110 | 2 | 2 |
| 20 | 26 | 30 | 2 | 2 |
| 25 | 125 | 140 | 2 | 2 |
| 30 | 35 | 37 | 4 | 3 |
CD4+ T cells (106/ml) were cultured with monoclonal human antibodies directed against CD3 and CD28 receptors plus irradiated PBMC without CD8+ T cells from controls and 1 μg of enfuvirtide/ml.
CD4+ T cells were cultured in the absence of enfuvirtide.
DISCUSSION
Enumeration of the peripheral resting CD4+ T cells latently infected with HIV-1 is crucial in order to quantify this cellular HIV-1 reservoir. Considering the very small number of these cells (11, 18, 33), the development of a specific, sensitive, and reproducible assay to determine their frequency is needed. Several methods have been proposed based on the following: (i) the detection of HIV-1 proviral DNA-positive cells by PCR assays (4, 5, 8, 10, 18, 29, 33, 35) and (ii) the synthesis of p24 antigen and the production of infectious virus following in vitro polyclonal activation of CD4+ T cells (6). Moreover, it is important to quantify the rare HIV-1 DNA-infected cells which are able to sustain a viral cycle leading to the production of HIV-1 viral proteins and virions. To this end, the enumeration of these cells was recently estimated by the detection of spliced and multispliced HIV-1 mRNAs (13, 20) and by an HIV-1-Ag ELISPOT assay, which is based on the detection of HIV-1-Ag-SCs induced by strong in vitro CD4+-T-cell polyclonal activation (19). The sensitivity and the specificity of this ELISPOT assay were found to be sufficient to detect a very small number of CD4+ T cells which could synthesize HIV-1-Ag among a few million cells (12).
Assuming that one cell latently infected with HIV-1 carries only one HIV-1 integrated provirus, Hermankova et al. (20) assessed the capacity of the HIV-1 DNA transcription to be only 1%. When we calculated the capacity of the HIV-1 viral DNA to be transcribed and that of the cells to be converted to HIV-1-Ag-SCs, we observed similar results (2.2%). However, recently published data show that latently infected resting CD4+ T lymphocytes harbor a mean of 3.2 integrated HIV-1 DNA copies (23). Thus, considering that in aviremic patients a mean of 3.2 HIV-1 DNA copies are integrated in each HIV-1-infected CD4+ T cell, the capacity of HIV-1-DNA-positive cells to induce HIV-1-Ag-SCs can be established at 7%. It remains that since one HIV-1 immunospot is the fingerprint of one HIV-1-Ag-SC and represents one cell carrying competent integrated HIV-1 DNA, the results obtained by using the HIV-1-Ag ELISPOT assay are independent of the number of integrated HIV-1 DNA genes, as substantiated by the weak statistical correlation between the results of the PCR and ELISPOT assays in aviremic and viremic patients (r = 0.64 and r = 0.58, respectively).
In this study, we used both a genomic and a protein-directed approach to analyze HIV-1 gene expression in latently infected resting CD4+ T lymphocytes from the peripheral blood of HIV-1-infected patients on HAART with a detectable or undetectable HIV-1 viral load. The frequency of resting CD4+ T cells carrying HIV-1 DNA and/or secreting HIV-1-Ag after in vitro CD4+-T-cell activation from viremic versus aviremic patients was not statistically different. These results indicate the following for viremic patients compared with aviremic ones: (i) the number of CD4+ T cells carrying HIV-1 DNA was greater, with long-term HAART responders showing a significant reduction in the number of HIV-1 proviral DNA-positive cells; and (ii) the CD4+-T-cell activation induced a higher number of HIV-1-Ag-SCs. The difference between the numbers of HIV-1 immunospots found in viremic patients and in HAART responders could be explained in part by the presence of virions attached to the cell surface, which could increase the number of HIV-1 DNA-positive cells (data not shown), and unintegrated viral genomes in the cytoplasm of infected cells that could be integrated into the cell genome and then transcribed into viral mRNA. This hypothesis is sustained by the fact that the number of latently HIV-1-infected resting CD4+ T cells during the follow-up of acute seroconverters treated early with HAART showed a biphasic decay (1, 27). This reservoir persists for a short time (1 to 6 days) before becoming nonfunctional, and only stable, integrated forms of HIV-1 genomes persist in aviremic patients (3, 34). These observations for resting CD4+ T cells are the consequence of the blockage of the virus life cycle at the level of nuclear import of the preintegration complex containing the viral genome (2).
The data we obtained for 10 HIV-1 patients with a detectable viral load are in agreement with data from two other studies: Kostrikis et al. detected 1,017 copies of HIV-1 DNA/106 PBMC using real-time PCR (25), and Blankson et al. detected 205 infectious units per million resting CD4+ T cells by limiting dilution coculture (1). Our data suggest that the majority of HIV-1 proviral DNA in resting CD4+ T lymphocytes cannot be readily activated for the HIV-1-Ag production and were in agreement with the low level of HIV-1 mRNAs observed in resting CD4+ T cells (20, 26). Indeed, some of the integrated HIV-1 DNA detected in resting CD4+ T lymphocytes may be intrinsically defective; this may be due to mutations in the LTR or in Tat (16) or under strong epigenetic regulation that cannot be readily reversed by a short-term cellular activation (22).
The results reported here, which are mainly based on an HIV-1 protein-directed approach with the latently HIV-1-infected proviral cells, confirm the fact that there is an HIV-1 cellular reservoir in viremic and aviremic patients despite a good response to antiretroviral therapy. Despite strong CD4+-T-cell polyclonal activation, numerous lymphocytes carrying HIV-1 DNA did not become HIV-1-Ag-SCs. Reporting that HIV-1 transcription in latently HIV-1-infected competent CD4+ T cells produced only low levels of HIV-1 mRNA, Hermankova et al. suggested that the targeting of this reservoir would be difficult by a protein-directed approach (20). Our results provide direct in vitro evidence that HIV-1 latency in resting CD4+ T cells operates at the level of a single HIV-1-Ag-SC and that the HIV-1-Ag approach is well founded. We were able to directly count HIV-1-Ag-SC, from viremic and aviremic HIV-1-infected individuals, despite the very low frequency of these cells. The results we obtained by using this method are consistent with the results obtained by the detection of HIV-1 mRNAs using PCR or in situ hybridization. These data indicate that the targeting of this HIV-1 CD4+-T-cell reservoir by a protein-directed approach is now available.
Since the reservoir represents a serious impediment to the long-term goal of the eradication of HIV-1, this new analytical approach, based on the measurement of the inducible viral translation of the latently infected peripheral CD4+ T cells, may provide a useful assay for assessing the effectiveness of future candidates for antiretroviral therapies.
Acknowledgments
We specially thank Nicolas Gauthier (Roche Diagnostics Systems) for technical assistance.
This work was supported by grants from the Delegation de la Recherche Clinique du Centre Hospitalier Universitaire de Montpellier, the Agence Nationale pour la Recherche sur le SIDA (ANRS), the Ensemble contre le SIDA (SIDACTION), and Beckman Coulter France.
REFERENCES
- 1.Blankson, J. N., D. Finzi, T. C. Pierson, B. P. Sabundayo, K. Chadwick, J. B. Margolick, T. C. Quinn, and R. F. Siliciano. 2000. Biphasic decay of latently infected CD4+ T cells in acute human immunodeficiency virus type 1 infection. J. Infect. Dis. 182:1636-1642. [DOI] [PubMed] [Google Scholar]
- 2.Bukrinsky, M. I., N. Sharova, M. P. Dempsey, T. L. Stanwick, A. G. Bukrinskaya, S. Haggerty, and M. Stevenson. 1992. Active nuclear import of human immunodeficiency virus type 1 preintegration complexes. Proc. Natl. Acad. Sci. USA 89:6580-6584. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Bukrinsky, M. I., T. L. Stanwick, M. P. Dempsey, and M. Stevenson. 1991. Quiescent T lymphocytes as an inducible virus reservoir in HIV-1 infection. Science 254:423-427. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Chun, T. W., L. Carruth, D. Finzi, X. Shen, J. A. DiGiuseppe, H. Taylor, M. Hermankova, K. Chadwick, J. Margolick, T. C. Quinn, Y. H. Kuo, R. Brookmeyer, M. A. Zeiger, P. Barditch-Crovo, and R. F. Siliciano. 1997. Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature 387:183-188. [DOI] [PubMed] [Google Scholar]
- 5.Chun, T. W., D. Engel, M. M. Berrey, T. Shea, L. Corey, and A. S. Fauci. 1998. Early establishment of a pool of latently infected, resting CD4(+) T cells during primary HIV-1 infection. Proc. Natl. Acad. Sci. USA 95:8869-8873. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Chun, T. W., D. Engel, S. B. Mizell, L. A. Ehler, and A. S. Fauci. 1998. Induction of HIV-1 replication in latently infected CD4+ T cells using a combination of cytokines. J. Exp. Med. 188:83-91. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Chun, T. W., D. Engel, S. B. Mizell, C. W. Hallahan, M. Fischette, S. Park, R. T. Davey, Jr., M. Dybul, J. A. Kovacs, J. A. Metcalf, J. M. Mican, M. M. Berrey, L. Corey, H. C. Lane, and A. S. Fauci. 1999. Effect of interleukin-2 on the pool of latently infected, resting CD4+ T cells in HIV-1-infected patients receiving highly active anti-retroviral therapy. Nat. Med. 5:651-655. [DOI] [PubMed] [Google Scholar]
- 8.Chun, T. W., D. Finzi, J. Margolick, K. Chadwick, D. Schwartz, and R. F. Siliciano. 1995. In vivo fate of HIV-1-infected T cells: quantitative analysis of the transition to stable latency. Nat. Med. 1:1284-1290. [DOI] [PubMed] [Google Scholar]
- 9.Chun, T. W., J. S. Justement, S. Moir, C. W. Hallahan, L. A. Ehler, S. Liu, M. McLaughlin, M. Dybul, J. M. Mican, and A. S. Fauci. 2001. Suppression of HIV replication in the resting CD4+ T cell reservoir by autologous CD8+ T cells: implications for the development of therapeutic strategies. Proc. Natl. Acad. Sci. USA 98:253-258. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Chun, T. W., J. S. Justement, R. A. Lempicki, J. Yang, G. Dennis, Jr., C. W. Hallahan, C. Sanford, P. Pandya, S. Liu, M. McLaughlin, L. A. Ehler, S. Moir, and S. Fauci. 2003. Gene expression and viral production in latently infected, resting CD4+ T cells in viremic versus aviremic HIV-infected individuals. Proc. Natl. Acad. Sci. USA 100:1908-1913. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Chun, T. W., L. Stuyver, S. B. Mizell, L. A. Ehler, J. A. Mican, M. Baseler, A. L. Lloyd, M. A. Nowak, and A. S. Fauci. 1997. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc. Natl. Acad. Sci. USA 94:13193-13197. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Corne, P., M. F. Huguet, L. Briant, M. Segondy, J. Reynes, and J. P. Vendrell. 1999. Detection and enumeration of HIV-1-producing cells by ELISPOT (enzyme-linked immunospot) assay. J. Acquir. Immune Defic. Syndr. Hum. Retrovir. 20:442-447. [DOI] [PubMed] [Google Scholar]
- 13.Derdeyn, C. A., J. M. Kilby, G. D. Miralles, L.-F. Li, G. Sfakianos, M. S. Saag, R. D. Hockett, and R. P. Bucy. 1999. Evaluation of distinct blood lymphocyte populations in human immunodeficiency virus type 1-infected subjects in the absence or presence of effective therapy. J. Infect. Dis. 180:1851-1862. [DOI] [PubMed] [Google Scholar]
- 14.Derdeyn, C. A., J. M. Decker, J. N. Sfakianos, Z. Zhang, W. A. O'Brien, L. Ratner, G. M. Shaw, and E. Hunter. 2001. Sensitivity of human immunodeficiency virus type 1 to fusion inhibitors targeted to the gp41 first heptad repeat involves distinct regions of gp41 and is consistently modulated by gp120 interactions with the coreceptor. J. Virol. 75:8605-8614. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Detels, R., A. Munoz, G. McFarlane, L. A. Kingsley, J. B. Margolick, J. Giorgi, L. K. Schrager, J. P. Phair, et al. 1998. Effectiveness of potent antiretroviral therapy on time to AIDS and death in men with known HIV infection duration. JAMA 280:1497-1503. [DOI] [PubMed] [Google Scholar]
- 16.Emiliani, S., W. Fischle, M. Ott, C. Van Lint, C. A. Amella, and E. Verdin. 1998. Mutations in the tat gene are responsible for human immunodeficiency virus type 1 postintegration latency in the U1 cell line. J. Virol. 72:1666-1670. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Finzi, D., J. Blankson, J. D. Siliciano, J. B. Margolick, K. Chadwick, T. Pierson, K. Smith, J. Lisziewicz, F. Lori, C. Flexner, T. C. Quinn, R. E. Chaisson, E. Rosenberg, B. Walker, S. Gange, J. Gallant, and R. F. Siliciano. 1999. Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat. Med. 5:512-517. [DOI] [PubMed] [Google Scholar]
- 18.Finzi, D., M. Hermankova, T. Pierson, L. M. Carruth, C. Buck, R. E. Chaisson, T. C. Quinn, K. Chadwick, J. Margolick, R. Brookmeyer, J. Gallant, M. Markowitz, D. D. Ho, D. D. Richman, and R. F. Siliciano. 1997. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 278:1295-1300. [DOI] [PubMed] [Google Scholar]
- 19.Fondere, J. M., J. F. Planas, M. F. Huguet, V. Baillat, F. Bolos, J. Reynes, and J. P. Vendrell. 2004. Enumeration of latently infected CD4+ T cells from HIV-1-infected patients using an HIV-1 antigen ELISPOT assay. J. Clin. Virol. 29:33-38. [DOI] [PubMed] [Google Scholar]
- 20.Hermankova, M., J. D. Siliciano, Y. Zhou, D. Monie, K. Chadwick, J. B. Margolick, T. C. Quinn, and R. F. Siliciano. 2003. Analysis of human immunodeficiency virus type 1 gene expression in latently infected resting CD4+ T lymphocytes in vivo. J. Virol. 77:7383-7392. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Hockett, R. D., J. M. Kilby, C. A. Derdeyn, M. S. Saag, M. Sillers, K. Squires, S. Chiz, M. A. Nowak, G. M. Shaw, and R. P. Bucy. 1999. Constant mean viral copy number per infected cell in tissues regardless of high, low, or undetectable plasma HIV RNA. J. Exp. Med. 189:1545-1554. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Jordan, A., P. Defechereux, and E. Verdin. 2001. The site of HIV-1 integration in the human genome determines basal transcriptional activity and response to Tat transactivation. EMBO J. 20:1726-1738. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Jung, A., R. Maier, J. P. Vartanian, G. Bocharov, V. Jung, U. Fischer, E. Meese, S. Wain-Hobson, and A. Meyerhans. 2002. Multiply infected spleen cells in HIV patients. Nature 418:144. [DOI] [PubMed] [Google Scholar]
- 24.Ketas, T. J., I. Frank, P. J. Klasse, B. M. Sullivan, J. P. Gardner, C. Spenlehauer, M. Nesin, W. C. Olson, J. P. Moore, and M. Pope. 2003. Human immunodeficiency virus type I attachment, coreceptor, and fusion inhibitors are active against both direct and trans infection of primary cells. J. Virol. 77:2762-2767. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Kostrikis, L. G., G. Touloumi, R. Karanicolas, N. Pantazis, C. Anastassopoulou, A. Karafoulidou, J. J. Goedert, and A. Hatzakis. 2002. Quantitation of human immunodeficiency virus type 1 DNA forms with the second template switch in peripheral blood cells predicts disease progression independently of plasma RNA load. J. Virol. 76:10099-10108. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Lewin, S. R., M. Vesanen, L. Kostrikis, A. Hurley, M. Duran, L. Zhang, D. D. Ho, and M. Markowitz. 1999. Use of real-time PCR and molecular beacons to detect virus replication in human immunodeficiency virus type 1-infected individuals on prolonged effective antiretroviral therapy. J. Virol. 73:6099-6103. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Ngo-Giang-Huong, N., C. Deveau, I. Da Silva, I. Pellegrin, A. Venet, M. Harzic, M. Sinet, J. F. Delfraissy, L. Meyer, C. Goujard, and C. Rouzioux. 2001. Proviral HIV-1 DNA in subjects followed since primary HIV-1 infection who suppress plasma viral load after one year of highly active antiretroviral therapy. AIDS 15:665-673. [DOI] [PubMed] [Google Scholar]
- 28.Perelson, A. S., A. U. Neumann, M. Markowitz, J. M. Leonard, and D. D. Ho. 1996. HIV-1 dynamics in vivo: virion clearance rate, infected cell life-span, and viral generation time. Science 271:1582-1586. [DOI] [PubMed] [Google Scholar]
- 29.Persaud, D., T. Pierson, C. Ruff, D. Finzi, K. R. Chadwick, J. B. Margolick, A. Ruff, N. Hutton, S. Ray, and R. F. Siliciano. 2000. A stable latent reservoir for HIV-1 in resting CD4(+) T lymphocytes in infected children. J. Clin. Investig. 105:995-1003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Ramratnam, B., J. E. Mittler, L. Zhang, D. Boden, A. Hurley, F. Fang, C. A. Macken, A. S. Perelson, M. Markowitz, and D. D. Ho. 2000. The decay of the latent reservoir of replication-competent HIV-1 is inversely correlated with the extent of residual viral replication during prolonged anti-retroviral therapy. Nat. Med. 6:82-85. [DOI] [PubMed] [Google Scholar]
- 31.Siliciano, J. D., J. Kajdas, D. Finzi, T. C. Quinn, K. Chadwick, J. B. Margolick, C. Kovacs, S. J. Gange, and R. F. Siliciano. 2003. Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells. Nat. Med. 9:727-728. [DOI] [PubMed] [Google Scholar]
- 32.Tamalet, C., A. Lafeuillade, J. Fantini, C. Poggi, and N. Yahi. 1997. Quantification of HIV-1 viral load in lymphoid and blood cells: assessment during four-drug combination therapy. AIDS 11:895-901. [DOI] [PubMed] [Google Scholar]
- 33.Wong, J. K., M. Hezareh, H. F. Günthard, D. V. Havlir, C. C. Ignacio, C. A. Spina, and D. D. Richman. 1997. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science 278:1291-1295. [DOI] [PubMed] [Google Scholar]
- 34.Zack, J. A., S. J. Arrigo, S. R. Weitsman, A. S. Go, A. Haislip, and I. S. Chen. 1990. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell 61:213-222. [DOI] [PubMed] [Google Scholar]
- 35.Zhang, L., B. Ramratnam, K. Tenner-Racz, Y. He, M. Vesanen, S. Lewin, A. Talal, P. Racz, A. S. Perelson, B. T. Korber, M. Markowitz, and D. D. Ho. 1999. Quantifying residual HIV-1 replication in patients receiving combination antiretroviral therapy. N. Engl. J. Med. 340:1605-1613. [DOI] [PubMed] [Google Scholar]