Human Immunodeficiency Virus Type 1 in the Semen of Men Receiving Highly Active Antiretroviral Therapy
Hui Zhang, M.D., Ph.D., Geethanjali Dornadula, Ph.D., Maria Beumont, M.D., Lawrence Livornese, M.D., Bonnie Van Uitert, M.D., Kelly Henning, M.D., and Roger J. Pomerantz, M.D.
Background Highly active antiretroviral therapy can effectivelydecrease the levels of human immunodeficiency virus type 1 (HIV-1)virions in peripheral plasma and seminal fluid of infected men.Whether the genital tract of HIV-1–infected men who arereceiving highly active antiretroviral therapy and who haveno detectable virus in the peripheral plasma harbors replication-competentvirus is not known.
Methods We collected peripheral-blood and semen samples fromseven men with HIV-1 infection who were receiving highly activeantiretroviral therapy and who had no detectable viral RNA (fewerthan 50 copies per milliliter) in plasma and analyzed the samplesfor cell-associated proviral DNA using a quantitative polymerase-chain-reactionassay. Replication-competent viruses were evaluated by cell-cocultureassays. Proviral DNA and replication-competent virus obtainedfrom peripheral-blood and seminal cells were also analyzed bysequencing relevant viral genes.
Results Despite the long-term suppression of HIV-1 RNA in theplasma of the seven men, proviral DNA was detected in seminalcells in four. Replication-competent viruses were recoveredfrom peripheral-blood cells in three men and from the seminalcells in two of these three men. The viruses recovered fromthe seminal cells had no genotypic mutations suggestive of resistanceto antiretroviral drugs and were macrophage-tropic, a featurethat is characteristic of HIV-1 strains that are capable ofbeing sexually transmitted.
Conclusions In HIV-1–infected men who are receiving highlyactive antiretroviral therapy and who have no detectable levelsof viral RNA in plasma, the virus may be present in seminalcells and therefore may be capable of being transmitted sexually.
Sexual transmission has a major role in the spread of humanimmunodeficiency virus type 1 (HIV-1). The semen of infectedmen may contain high levels of HIV-1, and infectious virusescan be recovered from seminal cells or seminal fluid from thesemen.1,2,3,4 Seminal cells are mixtures of spermatozoa, precursorsof germ cells, T lymphocytes, macrophages, and epithelial cells,and HIV-1 proviral DNA has been detected in several types ofthese cells.4,5,6,7,8 The level of HIV-1 RNA in seminal fluidcan also be correlated with the level in plasma, and antiretroviraltherapy decreases the levels not only in plasma but also inseminal fluid.9,10 There are often significant differences inthe viral load and the viral sequences between semen and peripheralblood, suggesting that the replication of HIV-1 may be compartmentalizedin vivo.11,12
Recently, therapy with combinations of antiretroviral drugs,referred to as highly active antiretroviral therapy, has resultedin the suppression of HIV-1 RNA in plasma to below the limitsof detection of many assay systems and increased the survivalof many HIV-1–infected patients.13,14 In these patients,the viral load in lymphoid tissues is dramatically decreased,15,16but proviral DNA can still be detected in resting CD4 T lymphocytesin the peripheral blood, and replication-competent virus canbe recovered from these cells, indicating that the cells couldbe a reservoir for viral replication.17,18,19 HIV-1 can replicatein many types of cells and many locations in the body, but whethertissues and cells other than resting CD4 T lymphocytes in peripheralblood harbor replication-competent virus in patients who arereceiving long-term highly active antiretroviral therapy isnot known. We examined whether proviral DNA and replication-competentvirus were present in the seminal cells of HIV-1–infectedmen who were receiving highly active antiretroviral therapyand in whom no viral RNA could be detected in plasma.
Methods
Study Subjects
We studied seven men with HIV-1 infection who had plasma levelsof HIV-1 RNA below 400 copies per milliliter when measured bya reverse-transcriptase–polymerase-chain-reaction (RT-PCR)assay on three occasions at least one month apart. The men hadpretreatment plasma HIV-1 RNA levels of at least 1000 copiesper milliliter and had been taking highly active antiretroviraltherapy for 5 to 41 months. They were identified from amonga cohort of more than 400 men with HIV-1 infection who weretreated in our clinics. Approximately four other men who metthe eligibility criterion declined to participate in the study.On the days on which samples of peripheral blood and semen wereobtained for the study, the plasma HIV-1 RNA levels in all sevenmen were below 50 copies per milliliter, as measured with asensitive RT-PCR technique described previously.20,21,22 Thesemen samples were collected by masturbation into clean containers.All samples were processed within two hours after collection.The study protocol was approved by the university's institutionalreview board, and all the men gave written informed consent.
Quantitative HIV-1 DNA and RNA Analyses
Peripheral-blood cells were separated from plasma by discontinuousFicoll centrifugation at 1000xg for 20 minutes at 4°C. Seminalcells were separated from seminal fluid by centrifugation at500xg for 10 minutes at 4°C. The cell-associated HIV-1 proviralDNA levels and virion-associated RNA levels were measured asdescribed previously.20,21,22 To separate the virions from plasmaand seminal fluid, the samples were centrifuged at 150,000xgfor one hour. The RNA was isolated from the pellets by acidguanidinium thiocyanate–phenol–chloroform extraction.23DNA was extracted from peripheral-blood mononuclear cells andseminal cells with use of a standard method.20 The HIV-1 gagDNA and RNA sequences were detected with the primer–probeset SK38, SK39, and SK19.20,21,22 The -globin gene was quantitatedin the DNA from peripheral-blood mononuclear cells and seminalcells by a PCR assay with PCO3 and PCO4 as the primer pair,as described previously,20,21,22 as a loading control, to confirmthat each sample contained similar quantities of DNA beforethe initiation of PCR. The phosphorus-32–labeled Southernblots of these PCR mixtures were quantitated with a PhosphorImager(Molecular Dynamics, Sunnyvale, Calif.). The results of Southernblotting of the patients' samples were compared with those forextracts of ACH-2 cells, a T-cell line that contains one integratedHIV-1 genome per cell for the DNA PCR assay,24 for the quantitationof proviral DNA and with an HIV-1 gag RNA construct transcribedin vitro for the RT-PCR assay22 for the quantitation of RNAvirions. Two healthy men were studied as negative controls torule out cross-contamination of the samples.
Coculture Assays
CD8 T lymphocytes were depleted from the isolated peripheral-bloodmononuclear cells by binding to magnetic beads conjugated withanti-CD8 antibody (Biosource, Camarillo, Calif.). This processdecreases the fraction of CD8 T lymphocytes in the peripheral-bloodmononuclear cells from approximately 20 to 30 percent to 3 to5 percent, as analyzed by flow cytometry. Depletion of CD8 Tlymphocytes significantly increases in vitro outgrowth of HIV-1from the peripheral-blood mononuclear cells17,18,19 becauseCD8 cells secrete chemokines and other factors that inhibitthe replication of the virus.25 Macrophages and their precursorswere depleted from peripheral-blood mononuclear cells by incubatingthe samples overnight to allow these cells to attach to theplastic plates. The remaining peripheral-blood lymphocytes werethen stimulated with 5 µg of phytohemagglutinin per milliliter(Sigma, St. Louis) and 50 U of interleukin-2 per milliliter(GIBCO-BRL, Grand Island, N.Y.). Peripheral-blood lymphocyteswere isolated from blood samples obtained from normal subjectswith the same procedure. The peripheral-blood lymphocytes fromthe patients were then mixed in a 1-to-1 ratio with those fromnormal subjects (2 million cells each) and cultured in RPMI-1640medium with 10 percent fetal-calf serum and penicillin plusstreptomycin at 37°C for six weeks. Twice a week, half themedium was replaced with fresh medium, and once a week, halfthe cells were replaced with 2 million fresh peripheral-bloodlymphocytes from normal subjects after stimulation with phytohemagglutininand interleukin 2 and depletion of CD8 T lymphocytes.
The seminal-cell pellet was washed twice with cold phosphate-bufferedsaline, and 3 million cells were mixed with 2 million peripheral-bloodlymphocytes from normal subjects after the depletion of CD8T lymphocytes and stimulation with phytohemagglutinin and interleukin-2.After 24 hours, the cells were washed three times with phosphate-bufferedsaline, and the cultures were maintained in the presence ofinterleukin-2 (10 U per milliliter) for six weeks. Twice a week,half of the medium was replaced with fresh medium, and oncea week, the cells were replenished with 2 million fresh, treatedperipheral-blood lymphocytes from normal subjects. HIV-1 p24antigen was measured in the supernatants by an enzyme-linkedimmunosorbent assay (ELISA) (Dupont, Wilmington, Del.). Allprocedures were performed under level P3 biosafety conditionsto minimize the possibility of cross-contamination.
DNA-Sequence Analyses
The sequences of the V3 loop of the gp120 region of the viralenvelope (env), protease, and RT genes of HIV-1 were determinedby a nested PCR assay of both proviral DNA (directly from peripheral-bloodand seminal-cell samples) and virion-encapsidated RNA (fromreplicating virus in the coculture). The viral RNA was reverse-transcribedwith an antisense external primer, and the complementary DNA(cDNA) was amplified with the PCR. A second PCR was performedwith a primer pair internal to the primer pair used in the firstPCR, and the amplified DNA was isolated from agarose gels andanalyzed by sequencing with an automated sequencer (Prism model377, with XL upgrade, Perkin–Elmer Applied Biosystems,Foster City, Calif.). If there were variations in the sequences,the PCR fragments were cloned into the pGEM-T vector (Promega,Madison, Wis.). Multiple clones were selected from DNA of seminalcells and peripheral-blood mononuclear cells as well as fromcDNA from replicating viral RNA, and the sequences were analyzed.The outer primer pair for the V3 loop of the gp120 region ofthe viral envelope was KK30 and KK40, and the inner primer pairwas KK10 and KK20, as described previously.26 (The sequenceshave been deposited in the GenBank data base under accessionnumbers AF098718 through AF098734.)
Determinations of Viral Phenotype
To determine the viral phenotypes, viral isolates from the cocultureswere cultured on MT-2 T lymphocytes and human macrophages (containing250 pg of HIV-1 p24 antigen equivalents per milliliter). Themacrophages were isolated from peripheral-blood mononuclearcells from normal subjects, as described previously.22 Viralgrowth in the MT-2 cells was determined on the basis of theformation of syncytium and the production of p24 antigen inculture. The growth of HIV-1 in macrophages was determined onthe basis of the detection of p24 antigen in the culture supernatantsby ELISA (Dupont).
Results
Detection of Cell-Associated HIV-1 Proviral DNA
The characteristics of the seven men are shown in Table 1. Thelevels of HIV-1 RNA in the plasma and seminal fluid of thesemen were below 50 copies per milliliter and thus were much lowerthan the levels in men with untreated HIV-1 infections who wereevaluated by the same assay (1500 to 75,000 copies per milliliterof seminal fluid).22 These results suggest that in our subjects,highly active antiretroviral therapy inhibited viral replicationnot only in the bloodstream but also in the genital tract. However,cell-associated viral DNA was detected in peripheral-blood mononuclearcells from all the men (Figure 1). The number of copies of HIV-1gag DNA ranged from 5 to 40 per million peripheral-blood mononuclearcells.
Figure 1. Detection of Proviral DNA in Seminal Cells and Peripheral-Blood Mononuclear Cells from Seven Men with HIV-1 Infection.
Cellular DNA was extracted from seminal cells and peripheral-blood mononuclear cells (PBMCs), and amplified by the PCR, with gag SK38 and SK39 as the primer pair. As a means of confirming that each sample contained similar quantities of DNA before PCR, -globin DNA was also amplified in each sample, with PCO3 and PCO4 as the primer pair.20,21,22 The HIV-1 DNA standards were prepared from ACH-2 cells (a T-cell line with one integrated HIV-1 genome per cell)24 and amplified by the PCR at the same time as were the samples from the patients; values are given as the number of copies per million cells. Blood and semen samples from two healthy men served as negative controls. A second set of samples was obtained from Patients 3 and 4 two to three months after the first set.
Cell-associated proviral DNA was also detected in the seminalcells from four men (Figure 1 and Table 1). The number of copiesof HIV-1 gag DNA ranged from less than 5 to 90 per million seminalcells. The sequence analyses of the V3-loop region of gp120indicated that the proviral DNA was not the result of contaminationwith common laboratory strains, as analyzed by a search of theGenBank data base (Table 2) (and data not shown). In two men(Patients 3 and 4), the levels of proviral DNA in seminal cellsand peripheral-blood mononuclear cells were examined again twoto three months after the first analysis. There was a smalldecrease in the level of proviral DNA in seminal cells in Patient4. Patient 3 had no detectable proviral DNA in seminal cellson either occasion.
Table 2. Genotypes and Phenotypes of HIV-1 from Peripheral-Blood Mononuclear Cells and Seminal Cells.
Recovery of Replication-Competent HIV-1
Replication-competent HIV-1 was recovered from peripheral-bloodlymphocytes from three men and seminal cells from two of thethree (Table 1 and Figure 2). In Patient 4, replication-competentHIV-1 was recovered from both peripheral-blood lymphocytes andseminal cells on two occasions. The analyses of the sequencesin the V3 loop of the gp120 region of the viral envelope withthe use of the GenBank data base indicated that these viruseswere primary isolates and not laboratory strains causing contamination(Table 2) (and data not shown). The viral sequences differedamong these three men, thus demonstrating that there was nocross-contamination of the samples. When CD8 T lymphocytes werenot depleted from the peripheral-blood cells used in the cocultures,we could not isolate viruses from either peripheral-blood lymphocytesor seminal cells from any of the men (data not shown).
Figure 2. Growth Kinetics of Replication-Competent HIV-1 from Seminal Cells.
Approximately 3 million fresh seminal cells from HIV-1–infected men were incubated for 24 hours with 2 million peripheral-blood lymphocytes from normal subjects after the depletion of CD8 T lymphocytes and stimulation with phytohemagglutinin and interleukin-2. The cell mixtures were then washed and cultured in the presence of interleukin-2 (10 U per milliliter) for six weeks. Every week, half the cells were replaced with 2 million treated peripheral-blood lymphocytes from normal subjects, and the supernatants were analyzed for HIV-1 p24 antigen by ELISA. Two samples from each culture were analyzed.
Genotypic and Phenotypic Characterization of Replication-Competent HIV-1
To investigate whether the replication-competent viruses recoveredfrom seminal cells could be sexually transmitted, the sequencesin the V3-loop region and the phenotype of the viruses wereexamined (Table 2). According to the net amino acid chargesin the V3 loop (a charge of +4 or less indicates a macrophage-tropicvirus, and a charge of +5 or more usually indicates a T-cell-line–tropicvirus) and the growth patterns in cell culture,27,28 the virusisolated from the seminal cells of Patient 6 was a typical macrophage-tropicstrain, whereas the virus isolated from the seminal cells ofPatient 4 had dual tropism. Most HIV-1 strains transmitted sexuallyare macrophage-tropic or have dual tropism11,29,30; therefore,the replication-competent viruses isolated from the seminalcells of these men are potentially capable of initiating a primaryinfection in a sexual partner, even though these men were receivinghighly active antiretroviral therapy and had undetectable levelsof viral RNA in plasma.
The differences in the sequences of the V3-loop region betweenthe viruses recovered from peripheral-blood lymphocytes andthose recovered from seminal cells from Patients 4 and 6 suggestthat at least some viral replication is compartmentalized withinthe male genital tract and the peripheral blood, as describedpreviously.11 The sequences also indicate that the recoverable,replication-competent viruses from seminal cells and peripheral-bloodlymphocytes are probably derived from the proviral DNA in thesecells (Table 2). In Patient 6, the replication-competent virusfrom seminal cells and the DNA of a provirus from seminal cellshad the same V3-loop sequence, whereas in Patient 4, the sequencesdiffered slightly. In Patient 6, however, the V3 sequence ofreplication-competent virus from the peripheral-blood lymphocytesdiffered substantially from that of the proviral DNA from peripheral-bloodmononuclear cells.
Strains of HIV-1 that are resistant to RT inhibitors as wellas protease inhibitors can be transmitted sexually.31 To determinethe drug sensitivity of the replication-competent HIV-1 andproviral DNA isolated from seminal cells and peripheral-bloodmononuclear cells of our patients, we sequenced the RT and proteaseregions.32 We found a single mutation coding for drug resistancein the protease gene (isoleucine was substituted for leucineat position 10) of the proviral DNA from peripheral-blood mononuclearcells from Patient 4. No drug-resistance mutations were detectedin either the replication-competent virus recovered from theseminal cells or the seminal-cell–associated proviralDNA from Patient 4 or Patient 6. Similarly, no drug-resistancemutations were detected in seminal-cell–associated proviralDNA from Patients 2 and 7. Finally, as previously reported,18no other drug-resistance mutations were detected in the proviralDNA from peripheral-blood mononuclear cells from Patient 2,Patient 6, or Patient 7 (data not shown).
Discussion
We isolated replication-competent virus from the seminal cellsof HIV-1–infected men who were receiving highly activeantiretroviral therapy and who had no detectable levels of viralRNA in plasma. This finding suggests that the genital tractcan be a reservoir for HIV-1 replication in men. This phenomenoncould be due to a very slow turnover of some cells harboringproviral DNA. Theoretically, if no drug-resistant mutants developedduring highly active antiretroviral therapy, there would beno reinfection of cells in microenvironments in which therewere inhibitory concentrations of the drugs. However, the blood–testesbarrier may prevent antiviral drugs from entering testiculartissue in high concentrations, therefore creating a viral sanctuary.Other sites of tissue–blood endothelial barriers, includingthe brain and the retina,33,34 have also been shown to harborHIV-1–infected cells. Although levels of HIV-1 RNA werebelow the level of detection in the seminal fluid of the menin the present study, there could still be covert viral replicationin the genital tract, as may occur in lymphoid tissue.19
We did not identify the types of cells in the genital tractthat contained replication-competent HIV-1. In untreated HIV-1–infectedmen, the virus is found in macrophages and CD4 T lymphocytesin the semen18; germ cells such as spermatogonia and their progenymay also contain provirus, although the sequences in germ cellsmay be defective or incomplete.6,7,35 Germ cells do not haveCD4 molecules on their surfaces,36 and therefore, HIV-1 shouldnot be able to enter them, unless there is a CD4-independentmechanism of entry.
Reinfection of peripheral-blood mononuclear cells and seminalcells should not occur in patients who are receiving highlyactive antiretroviral therapy and who have undetectable levelsof HIV-1 RNA in plasma and seminal fluid. Resting CD4 T lymphocytesfrom local lymphoid tissue, which may have a relatively longlife,37 could be reservoirs for the virus. The sequences ofHIV-1 isolated from the seminal cells differed from those obtainedfrom peripheral-blood lymphocytes in some men; therefore, someof the infected seminal cells may not have come directly fromthe peripheral blood. These T lymphocytes or macrophages couldhave been infected by blood-borne viruses before therapy wasinitiated, but they would have had to survive for very longperiods. In preliminary studies of seminal cells separated bymagnetic beads conjugated with anti–CD3 antibody, proviralDNA was detected in both the T-lymphocyte–replete (CD3antigen–positive) and T-lymphocyte–depleted cellularfractions (data not shown).
Seminal cells harboring proviral DNA could be vehicles for thesexual transmission of HIV-1. These infected seminal cells couldcome in direct contact with the target cells (i.e., CD4 T lymphocytes,macrophages, and dendritic cells) in the mucosa of the sexualpartners, resulting in the transmission of the virus. The virionsproduced from seminal cells after transfer from a man who isreceiving highly active antiretroviral therapy to a sexual partnershould be infectious because the concentrations of antiviraldrugs in the semen would be diluted to low levels. Our genotypicand phenotypic analyses indicate that the replication-competentviruses recovered from the seminal cells of our study subjectsare macrophage-tropic or have dual tropism, suggesting thatthey have the potential to initiate and establish a primaryinfection in the sexual partners of these men.10,29,30 Our studiesalso demonstrate that replication-competent viruses from theseminal cells remain sensitive to antiretroviral drugs. Thisfinding further suggests that proviral DNA in seminal cells,from which these viruses were most likely derived, may representarchival or fossil viral sequences derived by replication soonafter primary infection.
In summary, replication-competent viruses can be recovered fromseminal cells of HIV-1–infected men who are receivinghighly active antiretroviral therapy and who have undetectablelevels of viral RNA in plasma, suggesting that sexual transmissionof HIV-1 is possible despite the use of seemingly effectivetherapy.
Supported in part by funds from Thomas Jefferson University(to Dr. Zhang) and by grants from the Public Health Service(AI33810 and AI38666, to Dr. Pomerantz).
We are indebted to Ms. Mindy Klein-Dahan, R.N., for her diligentassistance in the clinics, to Mr. Richard Kedanis and Mr. SunYong for technical assistance, to Dr. Muhammad Mukhtar for assistancewith sequence alignments, to the patients of the Jefferson HealthSystem who generously volunteered for these studies, and toMs. Rita M. Victor and Ms. Brenda O. Gordon for their assistancein the preparation of the manuscript.
Source Information
From the Dorrance H. Hamilton Laboratories, Center for Human Virology, Division of Infectious Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, 1020 Locust St., Suite 329, Philadelphia, PA 19107, where reprint requests should be addressed to Dr. Pomerantz.
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-globin gene was quantitated in the DNA from peripheral-blood mononuclear cells and seminal cells by a PCR assay with PCO3 and PCO4 as the primer pair, as described previously,20,21,22 as a loading control, to confirm that each sample contained similar quantities of DNA before the initiation of PCR. The phosphorus-32–labeled Southern blots of these PCR mixtures were quantitated with a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.). The results of Southern blotting of the patients' samples were compared with those for extracts of ACH-2 cells, a T-cell line that contains one integrated HIV-1 genome per cell for the DNA PCR assay,24 for the quantitation of proviral DNA and with an HIV-1 gag RNA construct transcribed in vitro for the RT-PCR assay22 for the quantitation of RNA virions. Two healthy men were studied as negative controls to rule out cross-contamination of the samples. 
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