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Residual HIV-1 disease in seminal cells of HIV-1-infected men on suppressive HAART: latency without on-going cellular infections

Nunnari, Giuseppe; Otero, Miguel; Dornadula, Geethanjali; Vanella, Michelle; Zhang, Hui; Frank, Iana; Pomerantz, Roger J.

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Highly active antiretroviral therapy (HAART) has led to dramatic reductions in HIV-1 replication in vivo and changes in mortality and morbidity of treated patients [1–3]. Nevertheless, even in patients on virally suppressive HAART for relatively long periods of time (obtaining plasma HIV-1 RNA levels < 50 copies/ml), persistently infected resting CD4 T lymphocytes have been demonstrated in peripheral blood and lymphoid tissue [4–7]. Replication-competent virus can be recovered from these proviral-positive cells after CD8 T-lymphocyte depletion in vitro. The vast majority of these viruses are CCR5-tropic [8]. In addition, this cell reservoir is established soon after primary HIV-1 infection [9], and can be activated by pro-inflammatory cytokines in vitro and potentially in vivo [10]. Suppressive HAART initiated before primary HIV-1 seroconversion and during potential perinatal HIV-1 infection was also shown to be unable to halt the development of this replication-competent virus reservoir in CD4 T lymphocytes [11,12].

In addition to latent virus, it has been demonstrated by various complementary assays that on-going, low-level covert or cryptic HIV-1 replication occurs in the peripheral blood mononuclear cells (PBMC) and lymphoid tissue of patients on suppressive HAART [13–19]. Finally, replication-competent HIV-1 has also been demonstrated in seminal cells of certain infected men on suppressive HAART [18]. Understanding the diverse mechanisms of residual HIV-1 disease is critical for the possible development of clinical research protocols targeting long-term viral remissions and in determining the long-term durability of treatment strategies.

Sexual transmission has a major role in the spread of HIV-1. The semen of infected men may contain high levels of HIV-1, and infectious virus may be recovered from seminal cells and fluid from these men [20–23]. Seminal cells are a mixture of spermatozoa, precursors of germ cells, T lymphocytes, macrophages and epithelial cells. HIV-1 proviral DNA has been detected in several types of these cells [24–27]. The level of HIV-1 RNA in seminal fluid can also be correlated with the level in plasma, and antiretroviral therapy decreases the levels of virions not only in blood plasma, but also in seminal fluid [28,29]. Nevertheless, there are often significant differences in the viral load and viral sequences between semen and peripheral blood, suggesting that the replication of HIV-1 may be compartmentalized in vivo [30,31]. As noted above, we have recently demonstrated that replication-competent HIV-1 that is either CCR5- or dual (CXCR4 and CCR5)-tropic can be detected in the seminal cells of some men who are treated with virally suppressive HAART [18]. In addition, we and other groups have also recently shown that HIV-1 two-long terminal repeat (2-LTR) DNA circles, indicating infections of previously uninfected cells in vivo [32–36], can be detected in the PBMC of the majority of patients on virally suppressive HAART [32,37].

To determine potential compartment-specific mechanisms of HIV-1 residual disease in seminal cells, a cohort of 28 men on virally-suppressive HAART, with consistently undetectable plasma viral RNA, were analyzed for HIV-1 2-LTR DNA circles and replication-competent viral growth from their semen.

Materials and methods

Study cohort and clinical samples

We analyzed a cohort of 28 asymptomatic, HIV-1-infected men with plasma HIV-1 RNA levels < 50 copies/ml prior to and at the time of analyses (ultrasensitive reverse transcription–PCR; Roche, Branchberg, New Jersey, USA), for replication-competent virus in both seminal cells and PBMC. The patients in this study were identified from a larger group of more than 500 HIV-1-infected individuals treated in our various clinics. These patients had plasma viral RNA levels < 400 copies/ml for 5–58 months, and represent individuals who had both blood and semen analyzed at our Center over the past 3 years for both viral out-growth and HIV-1 2-LTR DNA circles. Patients were followed at the hospitals within the Jefferson Health System and collaborating health systems within the Delaware Valley. All patients were immunologically (i.e., CD4 T-lymphocyte counts) and virologically stable, with extremely stable HAART regimens (i.e., no recent changes in therapy). No active opportunistic infections or other significant medical conditions were present in any member of this cohort during the time of the study. In addition, no other sexually transmitted diseases were detected in any of the patients during this study. Some of the in vitro viral growth parameters in PBMC and semen from certain of these patients have been reported previously [18].

Peripheral blood (80–100 ml) was obtained from each patient by phlebotomy. Seminal samples were obtained by self-masturbation to ejaculation into a sterile container. By using an ultrasensitive reverse transcription–PCR for seminal fluid [19], HIV RNA was shown to be < 20 copies/ml in all of these patients (data not shown). All samples were processed within 2 h of collection. Screening of these patients for clinical study protocols was approved by the Thomas Jefferson Institutional Review Board, and each patient signed an informed consent form.

Viral growth co-culture assays

CD8 T lymphocytes were depleted from the isolated PBMC by binding to magnetic beads conjugated to anti-CD8 antibody (Biosource, Camarillo, California, USA). This process decreases the fraction of CD8 T lymphocytes in the PBMC from approximately 20–30% to 3–5%, as analyzed by flow cytometry [18]. Depletion of CD8 T lymphocytes significantly increased in vitro growth of HIV-1 from the PBMC because CD8 T lymphocytes secrete chemokines and other factors that inhibit the replication of the virus [38]. Macrophages and their precursors were depleted from PBMC by incubating the samples overnight to allow these cells to attach to the plastic plates [39]. The remaining cells were then stimulated with 5 μg/ml phytohemagglutinin (PHA) (Sigma, St. Louis, Missouri, USA ) and 50 U/ml interleukin-2 (IL-2) (Gibco-BRL, Grand Island, New York, USA). PBMC were isolated from blood samples obtained from HIV-1-seronegative subjects by the same procedure. The PBMC from the patients were then mixed in a 1 : 1 ratio with those from HIV-1-seronegative subjects (2 × 106 cells each) and cultured in RPMI-1640 medium with 10% fetal calf serum (FCS) and penicillin plus streptomycin at 37°C for 6 weeks. Twice a week, half the medium was replaced with fresh medium, and once a week, half the cells were replaced with 2 × 106 fresh PBMC (after stimulation with PHA and IL-2 and depletion of CD8 T-lymphocytes) from HIV-1-seronegative subjects.

The seminal cell pellets, isolated from semen by low speed centrifugation for 10 min, were washed twice with cold phosphate-buffered saline (PBS), and then 3 × 106 seminal cells were mixed with 2 × 106 PBMC (after depletion of CD8 T lymphocytes and stimulation with PHA and IL-2) from HIV-1-seronegative subjects. After 24 h, the cells were washed three times with PBS, and the cultures were maintained in the presence of IL-2 (10 U/ml) for 6 weeks. Twice a week, half of the medium was replaced with fresh medium, and once a week the cells were replenished with 2 × 106 fresh, PHA-stimulated PBMC from HIV-1-seronegative subjects. HIV-1 p24 antigen was measured in the supernatants by an enzyme-linked immunosorbent assay (Dupont, Wilmington, Delaware, USA). Positive cultures were those demonstrating ≥ 30 pg/ml p24 antigen in the culture supernatant. All procedures were performed under level P3 biosafety conditions to minimize the possibility of cross-contamination.

HIV-1 2-LTR circular DNA PCR

Seminal mononuclear cells and PBMC separated from seminal fluid and blood plasma, respectively, by discontinuous Ficoll centrifugation, without CD8-positive T-lymphocyte depletion or in vitro stimulation, were used directly for HIV-1 2-LTR circular DNA analysis. After washing the PBMC and seminal mononuclear cells with PBS the cells were cryopreserved. The seminal mononuclear cells and PBMC used for 2-LTR circular DNA analysis were obtained from each patient on the same date as the cells used for in vitro viral growth assays. DNA was extracted from these cells by using a standard ‘quick lysis’ methodology [39]. As noted previously by our group [18], relatively low seminal cell aliquots in comparison to PBMC always make studies of HIV-1 infection of seminal mononuclear cells somewhat technically difficult.

HIV-1 2-LTR circular DNA was then analyzed using a semi-quantitative DNA PCR technique [32], with modifications in the amplification parameters to optimize sensitivity and specificity. The amplification parameters were an initial denaturation step at 95°C for 10 min, amplification for 35 cycles at 95°C for 1 min, 60°C for 1 min, 72°C for 1 min, and a final extension at 72°C for 5 min. The sense and anti-sense primers used were 5′-GTAACTAGAGATACCCTCAAC-3′ and 5′-CAGATCTGGTCTAACCAGAGA-3′, respectively. PBMC DNA from HIV-1-seronegative individuals was used as the negative control. The amplified PCR products were transferred to Gene Screen + nylon membrane (Dupont) for Southern blotting utilizing a specific probe, which binds to the 2-LTR circle junction amplicon: 5′-AGTGGCGAG CCCTCAGATGCTGC-3′, labeled with 32P. After washing the membranes, a PhosphorImager (Molecular Dynamics, Sunnyvale, California, USA) was used to visualize and quantify the specific bands of the amplicons. To normalize the input cell number used for detection of 2-LTR circular forms in these DNA PCR procedures, the human β-globin gene was evaluated quantitatively from seminal mononuclear cells and PBMC, with a specific primer pair and a radiolabeled probe, as described previously [39,40]. Of note, spermatozoa were not a portion of seminal cell aliquots (i.e., seminal mononuclear cells only) and so were not included in calculations of copies of viral DNA/1 × 106 cells.

To prepare the standard curves for this assay, H9 T cells acutely infected with HIV-1 strain NL4-3 were used. DNA extracted from these cells served as a template to amplify 2-LTR circular DNA. The band isolated from the positive control was 531 base pairs in length.

The copy numbers of 2-LTR circle amplicons were calculated based upon the quantity of amplified DNA, as measured spectrophotometrically. This positive control amplicon DNA was serially diluted and analyzed by Southern blotting. Copy numbers of 2-LTR DNA circular forms in the study samples could be quantitated using this standard curve.

The 2-LTR circular DNA was expressed as copies/1 × 106 cells; variability was < 15%. Reproducible quantification of 2-LTR DNA circles was obtained to a level as low as 10 copies/1 × 106 cells, with detection (but not quantitation) below this level. All PCR procedures were conducted in designated and separate hoods and thermocyclers devoted solely to low viral copy experiments. Negative controls were included in all batched experimental runs.


In these analyses, CD8 T lymphocyte-depleted PBMC of 16 out of 28 HIV-1-infected men on virally-suppressive HAART (57%) grew virus in vitro; this is similar to data reported from other laboratories [41]. In addition, for five patients (18%) virus was grown from seminal cells in vitro, in co-culture with CD8 T lymphocyte-depleted PBMC from HIV-1-seronegative-individuals. Again these data are similar to those of previous studies carried out in our laboratories [18] (Table 1). Nonetheless, pure resting T-lymphocytes from peripheral blood may yield higher numbers of patients demonstrating in vitro viral growth [6].

Table 1:
Residual HIV-1 in the semen of men on suppressive HAART.

Eighteen patients were positive for 2-LTR circular DNA in PBMC (64%), while 10 were negative (Table 1). Two patients with virus-positive PBMC cultures had no detectable HIV-1 2-LTR circular DNA in PBMC, and four patients with detectable PBMC-associated 2-LTR DNA circles had virus-negative PBMC cultures. Of importance, none of the patients’ seminal mononuclear cells (n = 28) were shown to have detectable 2-LTR circular DNA. This included all five patients with viral growth in vitro (Fig. 1). Of interest, each of the patients with viral growth from their seminal cells also showed viral growth from their PBMC. Only one patient (#28) out of five demonstrated no 2-LTR DNA circles in PBMC while having PBMC and seminal mononuclear cells that were both positive for viral growth. All of these patients had positive, low-levels of HIV-1 gag DNA by PCR in seminal mononuclear cells (data not shown), which is demonstrable also in the PBMC from the patients on long-term HAART with clinically undetectable plasma viral RNA on suppressive HAART, as reported previously [18]. Finally, to determine whether semen-specific inhibitors of 2-LTR circle DNA PCR were present, selected semen samples were mixed with 2-LTR DNA circle-positive PBMC from other patients. In these mixing studies, the 2-LTR circle DNA PCR always yielded a specific amplicon (Fig. 1), demonstrating no obvious inhibitors of these reactions.

Fig. 1.:
HIV-1 2-LTR DNA circles analyzed by PCR in seminal cells of infected men on suppressive HAART. (a) Seminal cell DNA was evaluated by quantitative PCR for HIV-1 2-LTR DNA circles. PBMC DNA samples positive for HIV-1 2-LTR circles that were obtained from representative patients, are also illustrated alone as positive controls (patients #6 and #22) and in mixing experiments with seminal cells (SC) (see right side of top right autoradiograph: patients #17 and #18). ‘Negative PBMC’ are PBMC from HIV-1-seronegative men used as negative controls. (b) β-Globin DNA PCR was used as seminal cell loading control. PBMC samples were used as positive β-globin controls.

In summary, this study indicates that HIV-1-specific 2-LTR circular DNA that represents cryptic viral replication in vivo, is common in PBMC of patients on viral suppressive HAART but is not found in the seminal cells of these patients.


In this study, we demonstrate that HIV-1 2-LTR circular DNA, suggestive of cryptic viral growth replication in vivo, is not detectable in seminal cells of men on suppressive HARRT, even when seminal cells yield replication-competent virus by in vitro viral growth. This is contrary to the findings in the PBMC of HIV-1-infected individuals on suppressive HAART, in which a significant majority of patients maintain detectable 2-LTR DNA circles in PBMC [32,37].

These data suggest that there may be different mechanisms which may be compartment-specific, in patients on virally-suppressive HAART, for residual HIV-1 disease. In PBMC and lymphoid tissue it appears that both cryptic replication as well as proviral latency are molecular mechanisms yielding residual HIV-1 disease on suppressive HAART [4–18,42,43]. In contrast, proviral latency alone appears to be the major mechanism of viral persistence in the semen of infected men on virally-suppressive HAART.

HIV-1 2-LTR circular DNA is the end product of non-integrating ‘pre-integration’ complexes, after transport from the cytoplasm to the nucleus of newly infected cells. 2-LTR DNA circles have been reported by one group to have a relatively short half-life [32], although another laboratory has reported a longer half-life [33]. Further studies are necessary to confirm the in vivo stability of HIV-1 2-LTR circular DNA. Nonetheless, 2-LTR DNA circles represent infections of previously uninfected cells. As such, they can be considered the ‘foot-prints’ of cryptic HIV-1 replication in vivo. The 2-LTR DNA circles are formed after nuclear ligases join the 5′ and 3′ LTR of retroviral DNA into a circular episomal moiety. Measurements of these viral DNA moieties are potentially more relevant than measuring plasma or PBMC-associated viral RNA, as HIV-1-specific RNA may be defective [19,42], whereas 2-LTR DNA circles occur only after active viral infection of a previously uninfected cell [34–36].

The present study suggests that there may be differences in the mechanisms of HIV-1 persistence in various compartments within the body, with some regions having more cryptic viral replication than others. One could speculate that this lack of on-going cryptic replication in semen may be based on lower levels of local transport of cells with new viral infections into seminal fluid, but certainly further studies are necessary. This and future studies will be critical in determining the diverse molecular mechanisms of HIV-1 persistence during virally-suppressive HAART [44]. Therefore, these studies may assist in the rational development of new therapeutic approaches to induce either long-term HIV-1 remission by improving the durability of antiretroviral therapy, or significantly less likely viral eradication, in patients on virally-suppressive HAART.


The authors thank C. Coates and C. Dascenzo for clinical assistance with the patients in this study, R. M. Victor and B. O. Gordon for their excellent secretarial support, and especially the patients who volunteered for these studies.


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HAART; HIV-1; latency; reservoirs; semen

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