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Human Immunodeficiency Virus Type 1 Shedding Pattern in Semen Correlates with the Compartmentalization of Viral Quasi Species between Blood and Semen

  1. Phalguni Gupta1,a,
  2. Caroline Leroux2,a,
  3. Bruce K. Patterson4,
  4. Lawrence Kingsley1,
  5. Charles Rinaldo1,3,
  6. Ming Ding1,
  7. Yue Chen1,
  8. Kathy Kulka1,
  9. William Buchanan1,
  10. Brian McKeon2 and
  11. Ronald Montelaro2
  1. 1Department of Infectious Diseases and Microbiology (Graduate School of Public Health), University of Pittsburgh, Pittsburgh, Pennsylvania
  2. 2Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, Pennsylvania
  3. 3Department of Pathology (School of Medicine), University of Pittsburgh, Pittsburgh, Pennsylvania
  4. 4Department of Gynecology and Obstetrics, Northwestern University Medical School, Chicago, Illinois
  1. Reprints or correspondence: Dr. Phalguni Gupta, Dept. of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261 (pguptal+{at}pitt.edu).

  1. Presented in part: 7th Conference on Retroviruses and Opportunistic Infections, San Francisco, January 2000.

  1. ↵a P.G. and C.L. contributed equally to this work.

 
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Abstract

High levels of human immunodeficiency virus (HIV) type 1 have been detected in semen at all stages of disease. However, it is not clear whether HIV-1 is shed in semen continuously or intermittently. In a prospective longitudinal study, viral RNA was measured weekly for 10 weeks in semen and blood of HIV-seropositive subjects. Results showed three different patterns of HIV-1 shedding in semen: none (28%), continuous (28%), and intermittent (44%). In contrast, there was no change in blood plasma virus load during the study period. Phylogenetic analysis of the envelope sequences of HIV-1 RNA in semen and blood revealed distinct virus populations in semen and blood of intermittent shedders but similar virus populations in the semen and blood of continuous shedder. These results indicate for the first time that HIV-1 is shed primarily in an intermittent manner and that shedding patterns of HIV-1 in semen are related to compartmentalization of HIV-1 between semen and blood.

Human immunodeficiency virus (HIV) type 1 infection in adults is transmitted predominantly by sexual routes. Semen is considered to be the major vehicle for such transmission, since it contains free infectious HIV-1 and/or HIV-1-infected cells [1–6]. In situ hybridization, immunohistologic, and polymerase chain reaction (PCR) studies have demonstrated that the principal types of HIV-1-infected cells in semen are lymphocytes and macrophages [3, 7]. The concentration of HIV-1-infected cells in semen varies substantially, ranging from 0.01% to 5% of white cells [8, 9]. However, if venereal disease is present, many more inflammatory cells (and thus virus-infected cells) are present in seminal plasma [10]. Detection of high levels of cell-free HIV-1 [10–12] and relatively low levels of HIV-1-infected cells in semen at all stages of disease [2, 8, 9] suggest that HIV-1 transmission occurs primarily via cell-free virus [11]. This hypothesis is supported by the fact that cell-free simian immunodeficiency virus (SIV) is more efficient in initiating intrarectal infection than cell-associated SIV [13]. However, treatment with potent antiretroviral therapy reduces virus load in semen in parallel with that in blood [11, 14].

There is evidence that in some infected subjects HIV-1 variants in semen differ from those in blood. This evidence includes the following: a difference in envelope and protease gene sequences of HIV-1 proviral DNA in semen and blood [15–17]; lack of correlation between the seminal HIV-1 RNA concentration and plasma virus load [10, 18, 19]; detection of infectious HIV-1 in semen in the absence of plasma viremia [5, 10]; discordance of the biologic properties of HIV-1 from semen and blood with respect to syncytia-inducing (SI) properties; and resistance to reverse transcriptase (RT) inhibitory drugs [5, 10, 20]. These data indicate that in these subjects, HIV-1 in semen may not originate from blood. In contrast, in other infected subjects, identical viral variants are present in both blood and semen [16, 17], indicating a common origin of HIV-1 in these two body compartments in these subjects.

Although HIV-1 RNA and DNA have been detected in semen of HIV-1-infected subjects, it is not clear whether the virus is shed continuously or intermittently in semen. Intermittent shedding could explain a lack of detection of HIV-1 in semen in some infected subjects at different stages of disease [11]. Furthermore, it is not known whether the shedding pattern is related to the compartmentalization of HIV-1 between semen and blood. Krieger et al. [21] reported intermittent shedding of HIV-1 in semen as detected by isolation of virus in cell culture. However, the low sensitivity of the culture technique makes it difficult to interpret these data. Here we describe a prospective longitudinal study on the dynamics of HIV-1 shedding in semen over a 10-week period and a comparative analysis of the viral variants found in semen and blood in subjects with different shedding patterns.

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Experimental Procedures

Study population, semen, and blood samples

The study population comprised 18 HIV-1-infected subjects from the Pittsburgh portion of the Multicenter AIDS Cohort Study. Participants were asymptomatic, with a median of 434 CD4 T cells (range, 117–935), and were not receiving any potent antiretroviral therapy (e.g., protease inhibitors) during the study period. Paired semen and heparinized blood samples were collected at weekly intervals for 10 weeks. Subjects did not engage in sexual activity for 48–72 h before donation of semen and did not have other sexually transmitted diseases.

Samples were processed within 4 h of collection. Blood plasma was prepared by centrifuging heparinized blood at 1200 g at room temperature. Seminal cells were separated from whole semen by centrifugation for 10 min at 800–1000 g at room temperature. Supernatant (seminal fluid) was stored frozen at −70°C. Seminal cells were washed once with Hanks' balanced salt solution (HBSS), resuspended in 5 mL of HBSS, and then subjected to ficoll-hypaque gradient centrifugation. Seminal mononuclear cells were collected at the interface, washed, and stored frozen in dimethyl sulfoxide at −130°C until use. Blood CD4 cells were counted at the first and the last visit (visit 10) by our standard flow-cytometric measurement [22], since no change in CD4 cell number was expected in a 10-week period.

Quantitation of HIV RNA in semen and blood

The levels of HIV-1 RNA in whole semen and blood plasma were quantitated by a commercial nucleic acid sequence-based amplification assay (Nuclisens) according to the manufacturer's (Organon Teknika, Durham, NC) protocol. This is a modified version of a NASBA (nucleic acid sequence-based amplification assay) [11, 23–25]. The assay had a lower limit of detection of 200–400 copies of HIV-1 RNA/mL and a linear range of detection up to 10 × 106 copies of HIV-1 RNA/mL.

In situ hybridization and immunostaining of seminal mononuclear cells

Simultaneous flow cytometric analysis of intracellular HIV-1 RNA and cellular immunophenotyping of seminal mononuclear cells was done as described elsewhere [26]. Duplicate samples containing 0.5 × 105 to 1 × 106 seminal mononuclear cells were labeled with optimal concentrations of anti-CD4-ECD (Coulter, Miami) and anti-CD 14-phycoerythrin (PE) and anti-CD45RO-PE/CY5 (PharMingen, San Diego). The cells were washed with PBS and fixed and permeablized by the addition of 50 μL of PermeaFix (Ortho Diagnostics, Raritan, NJ). The cells were permeabilized for ⩽60 min and ⩾18 h. The cells were then washed twice in PBS (pH 7.4) and once in 2× standard saline citrate. The cells were then resuspended in 50 μL of hybridization buffer containing a cocktail of 5-carboxyfluorescein-labeled oligonucleotide probes specific for HIV RNA at 43°C for 30 min. The cells were washed for 5 min with buffer I and for 30 min with buffer II at 43°C. Multiparameter four-color analysis was then performed on labeled cells by flow cytometer (model XL; Coulter).

Measurement of β-chemokine RANTES in semen

The level of RANTES was measured by ELISA (R&D Systems, Minneapolis) in 100 μL of seminal fluid per the manufacturer's protocol.

Analysis of C2-V5 sequence of HIV-1 env from semen and blood plasma

Total RNA was extracted from 100 μL of blood plasma or whole semen by the RNA extraction protocol of the Nuclisens assay as described above. RNA extracted by this procedure removed the RT-PCR inhibitor that is often associated with semen [11, 23]. In total, 5–25 μL of extracted RNA was reverse transcribed at 42°C for 50 min with 200 U of SuperScript II RNase H− RT (Gibco BRL, Grand Island, NY) using the PCR downstream primer ED1220 (20 pmol) as the primer in a reaction mixture containing 8 mM dithiothreitol (Gibco BRL), 0.4 mM each deoxynucleotide triphosphate (Pharmacia Biotech, Piscataway, NJ), 5× 1st strand buffer (Gibco BRL), and 30 U of RNAguard RNase inhibitor (Pharmacia Biotech) in a total volume of 25 μL. A nested PCR was performed with 5-fold serial dilutions of cDNA with primers ED31/BH2 and DR7/DR8 in the first and second round of PCR, respectively, as described elsewhere [18]. The conditions of the PCR assay were as follows: samples were preheated at 94°C for 2.5 min (1 cycle), denatured at 94°C for 1 min, and annealed at 55°C for 45 s, with extension at 72°C for 1 min (35 cycles) and final extension at 72°C for 10 min (1 cycle). Each PCR used 10 μL of diluted cDNA in the first round and 2 μL of first-round amplified DNA in the second-round reaction of the PCR assay. The PCR reaction mixture contained 10 pmol of each primer, 200 μM of each deoxynucleotide triphosphate, 2.5 U of AmpliTaq DNA polymerase (Perkin-Elmer, Norwalk, CT), 50 mM KCl, 1.25 mM MgCl2, 10 mM Tris-HCl (pH 8.3), and 5% glycerol in a final volume of 50 L. Amplification was monitored by visualization of an ethidium bromide-stained band of 678 bp after electrophoresis in a 2% agarose gel. Multiple HIV-1-negative controls were applied with each PCR run to detect any possible contamination.

Cloning and sequence analysis

For each sample, PCR products from ⩽3 independent PCR reactions, obtained from dilutions of the input cDNA, were cloned in the pGEM-T Easy vector (Promega, Madison, WI). Plasmid DNAs were prepared by the Wizard system (Promega) and digested with appropriate restriction enzymes to confirm the size of the insert. Subsequently, DNA from 9 to 11 clones was purified by using the DNA-pure miniprep kit (CPG, Lincoln Park, NJ) and sequenced with a Taq Dye Deoxy Terminator Cycle sequencer kit (Applied Biosystems, Foster City, CA) in an ABI Prism 377 DNA sequencer (Applied Biosystems).

Sequences from each cloned viral DNA were aligned by the clustal W multiple sequence alignment program [27]. Genetic distances were calculated with DNADIST software from the PH YLIP package [28] by the maximum likelihood method. Neighbor-joining trees were constructed with the clustal W package, and a bootstrap analysis using 100 bootstrap replicates was conducted to assess the support at each internal nodes.

Nucleotide accession numbers

Sequences were deposited in GenBank under accession numbers AF256230-AF256286 (patient 1), AF256287-AF256305 (patient 2), AF256306-AF256322 (patient 4), AF256323-AF256404 (patient 6), and AF256405-AF256465 (patient 8).

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Results

Virus load in semen and blood

On the basis of the prevalence of HIV-1 RNA in semen at all visits, three patterns of HIV-1 shedding emerged. Representative seminal and blood virus load data of 4 subjects from each of these patterns are shown in figure 1. Of the 18 subjects studied, HIV-1 RNA was detected in semen at all 10 visits in 5 (28%), with levels varying among subjects (figure 1B); in 5 other subjects (28%), HIV-1 RNA was not detected in semen at any of the 10 visits (figure 1C; and in the remaining 8 subjects (44%), HIV-1 RNA was detected intermittently in semen during 15%–70% of all visits (figure 1A). The difference in the level of seminal virus load between a positive and a negative visit in this intermittent shedding group ranged between 100- and 10,000-fold. In contrast to the observed variations in seminal virus levels, blood plasma virus load did not change significantly during this 10-week study period, even in the group with intermittent shedding of HIV-1 in semen (figure 1).

Figure 1
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Figure 1

Human immunodeficiency virus type 1 RNA measurements in semen and blood plasma in 3 groups of subjects: A, those who shed virus intermittently; B, those who shed virus persistently; and C, nonshedders.

Relationship between seminal virus load and semen volume, seminal mononuclear cells, blood CD4 cells, and sex partners

We examined whether the different shedding patterns of HIV-1 in semen were due to a difference in semen volume or seminal mononuclear cells. There was no relationship between the pattern of virus load as a group or at a particular visit and the semen volume or the number of mononuclear cells per milliliter of semen in that group or at that particular visit (data not shown). Furthermore, the patterns of seminal virus load did not correlate with blood CD4 T cell number. These results indicate that intermittent shedding of HIV-1 in semen was not due to fluctuation of semen volume, semen mononuclear cells, or blood CD4 T cell number.

To investigate whether chronic infection or inflammation of the genitourinary tract was linked to patterns of HIV-1 shedding in semen, several covariates were examined from the last visit prior to the baseline evaluation of this study. No differences were observed among the study groups for physical examination and self-reported findings of herpes zoster, facial herpes, genital or anal herpes, syphilis, gonorrhea, nonspecific urethritis, anal warts, molluscum contagiosum, or abdominal parasite diseases. In fact, only 1 or 2 persons in the entire study group reported such findings. Since we did not perform microbiologic assays to detect common bacterial urethral pathogens or collect urethral smears for Gram's stain microscopy, it is possible that we missed an association between subclinical urethritis and intermittent shedding of HIV-1 in semen, as reported by Winter et al. [29]. This seems unlikely, however, because of the very low (8%) prevalence of subclinical urethritis reported by Winter et al. [29]. This low prevalence of urethritis would weaken any association between shedding of HIV-1 in semen and urethritis. However, we found an association between a greater number of sex partners (>7) and shedding of HIV-1 in semen, since 4 of 5 subjects with continuous HIV-1 shedding in semen reported multiple sex partners versus only 1 of 5 study participants with multiple sex partners who never shed HIV-1 in semen.

Immunophenotyping and expression of HIV-1 transcripts in seminal mononuclear cells

We considered the possibility that seminal HIV-1 was derived from mononuclear cells present in semen and that therefore intermittent shedding of HIV-1 in semen was due to the fluctuation of the viral RNA expression in seminal mononuclear cells. To test this hypothesis, we examined the relationship between seminal virus load and the number of activated CD4+, HIV-1-expressing seminal mononuclear cells. Figure 2 shows a representative flow cytometric detection of HIV-1 gag-pol RNA in seminal mononuclear cells from 1 intermittent shedder (subject 1) at visits 1, 3, 5, and 9. Table 1 summarizes the flow cytometry data of the percentage of activated CD4+, HIV-1 gag-pol RNA-expressing seminal mononuclear cells and the activated CD4+ (CD4+, CD45RO+) seminal mononuclear cells from 3 subjects who intermittently shed HIV-1 in semen. These results indicate that the percentage of activated CD4+ cells and HIV-1 gag-pol-positive CD4 cells remained at similar levels at all visits regardless of virus load in semen. These results indicate that the intermittent shedding of HIV-1 in semen was not due to fluctuations in levels of viral RNA expression in seminal mononuclear cells and was not related to the number of activated CD4+ cells in seminal mononuclear cells.

Figure 2
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Figure 2

Flow cytometry analysis of CD4+ human immunodeficiency virus (HIV) gag-pol RNA-positive cells in seminal mononuclear cells from subject 1 who shed HIV-1 intermittently. Data are from visits 2, 4, 7, and 10 (V2-V10). ECD, energy-coupled dye; FITC, fluorescein isothiocyanate.

Virus load and β-chemokine level in semen

β-chemokines such as RANTES, macrophage inflammatory protein (MIP)-1β, and MIP-1α inhibit viral infection and thereby control viral replication [30–33]. To determine whether the intermittent shedding of HIV-1 was due to fluctuation in the level of β-chemokines, we measured the level of RANTES in semen samples at all visits from 4 subjects who shed HIV-1 intermittently in semen. As shown in figure 3, there was no change in RANTES levels in semen during the 10-week study. Thus, intermittent shedding of HIV-1 in semen was not correlated with the level of β-chemokines in semen.

Figure 3
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Figure 3

Measurement of RANTES in semen from 4 subjects who shed human immunodeficiency virus (HIV) type 1 intermittently

Sequence analysis of HIV-1 from semen and blood plasma

A 650-bp region corresponding to the C2-V5 region of HIV-1 gp120 from HIV-1 RNA isolated from semen and blood plasma was sequenced to determine the viral genetic diversity in 2 subjects (6 and 8) with continuous shedding and in 3 subjects (1, 2, and 4) with intermittent shedding of HIV-1 in semen. According to the deduced amino acid sequences of the V3 loop, the sequences derived from these 5 subjects belonged to the subtype B clade, and each set clustered as monophyletic groups compared with those from other patients in this study. Hence, these data indicate that there was no evidence of cross-contamination among patient samples.

We initially determined whether intermittent shedding was due to any rapid genetic variation of HIV-1 between visits. For this purpose, we analyzed sequences of virus population from semen and blood plasma samples at multiple visits from 1 intermittent shedder (subject 1: visits 3, 8, and 10) where we had sufficient viral RNA for amplification. From each sample, at least 10 clones were sequenced at multiple dilutions of the amplified DNA, to avoid sampling error. Phylogenetic analysis (figure 4A) revealed that both blood plasma- and semen-derived variants formed a tightly clustered group of sequences between visits (mean percentages of nucleotide divergences, 0.7 ± 0.4 for semen and 5 ± 2.8 for blood plasma), showing no indication of significant genetic diversity among visits. We extended this observation by analyzing HIV-1 variants from semen and blood plasma of 1 continuous shedder (subject 6) at visits 1,3,6, and 10. As shown in figure 4B, no major difference in genetic diversity was observed between visits (mean percentages of nucleotide divergences, 3.2 ±1.8 for blood plasma and 2.3 ± 1.5 for semen).

Figure 4
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Figure 4

Phylogenetic tree of human immunodeficiency virus type 1 envelope (C2-V5) sequence from HIV-1 RNA isolated from semen and blood plasma of subject 1, who shed virus intermittently (A), and from subject 6, who shed virus continuously (B), at visits indicated.

Next, we compared sequences of HIV-1 variants in blood with those in semen of 3 intermittent shedders (subject 1 : visits 3, 8, and 10; subject 2: visit 5; subject 4: visit 8) and 2 continuous shedders (subject 6: visits 1, 3, 6, and 10; subject 8: visits 1,6, 7, and 10) at visits with sufficient amplifiable HIV-1 RNA (figure 5). The percentage of divergence at the nucleotide level among the plasma-derived clones was 3.2 ± 1.8, 3.4 ± 1.8, 3.4 ± 1.8, 5 ± 2.8, 2.1 ± 1.1, and 3.8 ± 1.6 from subjects 6, 8, 1, 4, and 2, respectively. Overall, such diversity among plasmaderived clones was higher than the diversity among semenderived clones with 2.3 ± 1.5, 2.9 ± 1.3, 0.7 ± 0.4, 1.8 ± 0.3, and 0.6 ± 0.6 for subjects 6, 8, 1, 4, and 2, respectively. However, as shown in figure 5, in all 3 intermittent shedders (subjects 1, 2, and 4), semen-derived variants in general formed a tightly clustered group of sequences distinct from that in their blood plasma. In general, semen-derived variants were more clustered than their blood counterparts, as shown by the greater mean percentage of divergence of HIV-1 variants in plasma (mean, 3.6; range, 2.1–5.0) compared with those in semen (mean, 1.03; range, 0.6–1.8). In addition, on the basis of the charge of the amino acids at positions 11, 13, 19, 23, 24, 25, and 32 of the V3 loop [34–36], all semen and plasma clones from subjects 2 and 4 had the consensus sequence associated with non-SI-inducing (NSI) phenotype. However, in subject 1, HIV-1 variants with SI genotypes were obtained only in blood plasma and not in semen.

Figure 5
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Figure 5

Phylogenetic tree of human immunodeficiency virus (HIV) type 1 envelope (C2-V5) sequence from HIV-1 RNA isolated from semen and blood (A) of 3 intermittent shedders (subject 1: visits 3, 8, 10; subject 2: visit 5; subject 4: visit 8) and (B) from 2 continuous shedders (subject 6: visits 1, 3, 6, 10; subject 8: visits 1, 6, 7, 10).

Table 1
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Table 1

Correlation of seminal virus load with human immunodeficiency virus (HIV) transcription in seminal mononuclear cells.

Phylogenetic analysis of HIV-1 sequences from continuous shedders (subjects 6 and 8) revealed a distributed pattern of viral variants between semen and blood plasma, with no evidence of clustering in either body compartment (figure 5). The mean percentage of divergence among viral variants in blood plasma (3.2 ±1.8 for subject 6 and 3.4 ±1.8 for subject 8) was very similar to that in semen (2.5 ± 1.5 and 2.9 ± 1.3 for subjects 6 and 8, respectively). All HIV-1 variants in both body compartments of these 2 continuous shedders had NSI genotypes.

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Discussion

In a cross-sectional study, we previously found that HIV-1 can be detected in semen in 66% of seropositive subjects at all stages of disease [10]. It was not clear whether the observed lack of detection of HIV-1 in semen in that study was due to the absence of shedding of HIV-1 or the intermittent shedding of HIV-1 in semen. In the present study, we found three different HIV-1 shedding patterns in semen: none, continuous, and intermittent. Of the subjects who shed HIV-1 in semen, the intermittent shedding pattern was predominant (61%).

In the present study, we analyzed various potential factors as determinants of the different HIV-1 shedding patterns in semen. We found that the pattern was not related to semen volume, number of seminal mononuclear cells, or to percentage of CD4 T cells in blood. We also demonstrated that the intermittent shedding of HIV-1 in semen was not due to fluctuation in the level of expression of HIV-1 RNA in seminal mononuclear cells. This was further supported by the lack of correlation between the number of activated CD4+ (CD4+, CD45RO+) seminal mononuclear cells and the intermittency of HIV-1 in semen. Furthermore, our data indicate that the intermittent shedding of HIV-1 is not due to the fluctuation of the β-chemokine RANTES level, which suppresses HIV-1 infection by a competitive interaction with the viral coreceptor [31]. This finding is in contrast with the recent report of Iversen et al. [37], who found a positive correlation of cervical HIV-1 shedding with genital chemokine secretion. Sequence analysis of HIV-1 at multiple visits in subjects with intermittent and continuous shedding of HIV in semen also did not indicate any rapid genetic variation between visits as the reason for the intermittent shedding of HIV-1.

Our data indicate that in subjects with intermittent shedding of HIV-1 in semen, the virus population in semen was distinct from that in blood and there was no correlation between the level of virus in semen and blood. At certain visits, HIV-1 was not detected in semen but there was a significant amount of virus in blood. Sequence analysis of the C2-V5 region of the viral envelope gene of HIV-1 variants obtained from semen and blood plasma RNA at multiple visits from the subjects with intermittent shedding indicated that HIV-1 variants in semen differed from those in blood. These results from viral RNA support our previous data [16] and those of Zhu et al. [17], which were obtained by sequence analysis of proviral DNA from semen and peripheral blood mononuclear cells. Moreover, in 1 intermittent shedder, the SI genotype was detected only in blood and not in semen. These results suggest that, in intermittent shedders, HIV-1 in semen was not recently derived from peripheral blood. Furthermore, in these subjects, the seminal mononuclear cells were not the origin of the HIV-1 in semen, as evidenced by the lack of correlation between the seminal virus load and the percentage of HIV-1-expressing seminal mononuclear cells. In contrast, in continuous shedders, the HIV-1 RNA population in semen was similar to that in blood, indicating that HIV-1 in semen in this group of subjects may come from blood. This was supported by the concordance of HIV-1 RNA level between semen and blood in continuous shedders at all visits. This observation of different patterns of compartmentalization with different shedding patterns of HIV in semen could explain our previous findings [16] and those of Zhu et al. [17], in which differential representations of integrated proviral sequences were found between blood and semen in 3 subjects but not in 2 others. On the basis of our current findings, it is conceivable that these 2 subjects with similar virus populations in semen and blood could belong to the group of continuous shedders. In contrast, the other 3 subjects with distinct HIV-1 species in semen and blood could be grouped with intermittent shedders. The 60% of subjects with distinct HIV-1 genotypes in the previous study matches well with 61% of subjects with intermittent shedding in this study. Of interest, nonshedders also had very low level of HIV RNA in their blood, indicating a concordanance of HIV replication between these two compartments in this group of subjects.

This investigation raises an important question about the origin of HIV-1 in semen. It is thought that lymphocytes enter the seminal compartment through the epididymis [38], because the number of leukocytes in semen of vasectomized men is greatly reduced [39]. The prostate and seminal vesicles are the major contributors of seminal fluid, which contains high levels of HIV-1 [11]. Vasectomized subjects can transmit HIV-1, and vasectomization usually occurs at the proximal end of the these two glands in the male reproductive duct. Therefore, it is possible that prostate and seminal vesicles could be the major source of seminal HIV-1. In addition, since other types of microbial infection have been reported in the prostate [40–42], this organ appears to be the most likely candidate for a reservoir for seminal virus.

On the basis of the data reported here, we propose the following model to explain the origin and pattern of HIV-1 shedding in semen. In nonshedders, because of a low virus load in the blood, very low levels of virus and/or virus-infected cells enter the male genital organ. Like its counterpart in blood, viral replication in the genital organ is probably controlled by the local immune system. In intermittent shedders, at some point during the course of HIV-1 infection (possibly during acute infection), HIV-1-infected lymphocytes enter the male genital organ through the testis or epididymis because of local inflammation and subsequently reside in specific genital tissue (e.g., the prostate). In this group of infected subjects, the barrier at the blood-testis junction would not allow passage of lymphocytes or high-molecular-weight macromolecules (e.g., viruses) after initial introduction of virus or virus-infected cells. In addition, the migration of blood lymphocytes through the epididymis is restricted, giving rise to a sequestered compartment in which HIV-1 could continue to replicate in specific genital tissue. Since the immune environment in the male genital organ is thought to differ than that in the blood [3, 38, 43, 44], the level of HIV-1 replication in the face of immune selection in the male genital organ would be expected to differ from than that in blood, giving rise to the diversity of HIV-1 populations in these two body compartments. Since other types of bacterial infections are found in the prostate [40–42] and the prostate contains lymphoid cells [40–42], this organ may be a source of seminal HIV-1 in intermittent shedders. Thus, intermittent shedding of HIV-1 in semen would be due to a fluctuation of viral transcription from infected cells in the prostate. This notion is supported by a study by Kiessling et al. [45], who showed that, during an episode of asymptomatic prostatitis, the seminal virus load increased 100-fold over that in peripheral blood and that protease-resistant HIV-1 variants were found in semen, but not in blood.

In continuous shedders, inflammation at the testis-blood junction or at the epididymis would allow continuous passage of lymphocytes, virus particles, or both to enter from blood to the male genital organ and thereby contribute to similar viral representations between semen and blood. The fact that the continuously shedding subjects in this study had significantly more sex partners than the nonshedders supports such a possibility of inflammation in the male genital organ.

In summary, our data indicate that the source of HIV-1 in semen is complex and is related to pattern of shedding in semen. Direct examination of viral variants in prostate, semen, and blood could confirm the prostate origin of HIV-1.

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Acknowledgments

We thank Judy Malenka for secretarial assistance and the participants and staff of the Pitt Men's Study for dedication and support.

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Footnotes

  • Informed consent was obtained from patients in accordance with University of Pittsburgh institutional review board human experimentation guidelines.

  • Grant support: NIH (AI-35041, HD-32256, AI-46271).

  • Received January 13, 2000.
  • Revision received March 30, 2000.
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  1. J Infect Dis. (2000) 182 (1): 79-87. doi: 10.1086/315644
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