Clinical and Mucosal Immune Correlates of HIV-1 Semen Levels in Antiretroviral-Naive Men

Study Participants and Design

Men who have sex with men infected with HIV for ≥1 year were recruited through the Maple Leaf Medical Clinic, Toronto between 2011 and 2013. Participants were ART naive and were screened for STIs at each study visit. All participants provided paired blood and semen samples at a single time point, and a subgroup of men who opted to defer ART provided monthly semen and blood samples for up to 1 year. All participants provided informed, written consent; ethical approval for this study was obtained through the research ethics board of the University of Toronto.

Sample Collection and Diagnostic Testing

Blood was collected into acid citrate dextran, and semen was collected by masturbation directly into a sterile container containing 10 mL of Roswell Park Memorial Institute (RPMI) medium with penicillin/streptomycin after 48 hours of abstinence [20]. Samples were processed within 2 hours of collection. Semen plasma (SP) was isolated by centrifugation at 850 × g for 10 minutes, and blood plasma (BP) was isolated after Ficoll density gradient centrifugation at 500 × g for 25 minutes. Blood plasma and SP HIV RNA VL were assayed using the Abbott RealTime HIV-1 assay using an automated m2000 system (Abbott Molecular Diagnostics). Due to occasional spillage of RPMI medium during semen collection and/or sample transport, correction for semen dilution assumed a semen volume of 2 mL, as previously validated [20]. The lower limit of detection for bVL and sVL were 50 and 300 HIV RNA copies/mL, respectively. High-level semen shedding was defined as a VL >5000 HIV RNA copes/mL [1].

Real-Time Quantification of Semen Herpesvirus Levels

Viral load quantification of herpes simplex virus (HSV)-1, HSV-2, HHV-3, EBV, CMV, HHV-7, and HHV-8 was performed by reverse transcription-polymerase chain reaction (PCR) using the genesig quantitative PCR (qPCR) detection kit (PrimerDesign Ltd); HHV-6 A-B was quantified using the RealStar PCR kit (Altona Diagnostics) [21]. In brief, deoxyribonucleic acid (DNA) was extracted from 400 μL of SP using a DNA mini extraction kit (QIAGEN, Inc., Valencia, CA). For the genesig kits, extracted SP DNA (5 μL) containing the provided internal control was combined with a PCR mix containing 10 μL 2×Precision MasterMix, 1 μL Primer/Probe mix, and 4 μL RNAse/DNAse-free water. For the RealStar PCR kit, 10 μL of extracted SP DNA was added to the mastermix containing 5 μL of master-A, 15 μL of master-B, and 1 μL of the internal control. All viruses were quantified using the Applied Biosystems 7900 HT Real-Time PCR system with the after reactions conditions: 95°C for 10 minutes followed by 50 cycles of 95°C for 10 seconds (genesig) or 15 minutes (RealStar) and 60°C for 1 minute. Results were analyzed using the Sequence Detection System, version 2.4 (Applied Biosystems). Lower limit of detection was 200 copies/mL.

Cytokine and Trappin-2/Elafin Levels in Semen Plasma

Cytokine levels in SP were measured using the Meso Scale Discovery multiplex System (Gaithersburg, MD). Fourteen cytokines/chemokines were measured (lower limit of quantification listed in parenthesis): interleukin (IL)-1α (3.6 pg/mL), IL-6 (3.6 pg/mL), IL-1β (3.6 pg/mL), IL-8 (1.8 pg/mL), monocyte chemoattractant protein (MCP)-1 (1.8 pg/mL), macrophage-derived chemokine ([MDC] 1.8 pg/mL), monokine induced by interferon-γ ([MIG] 1.8 pg/mL), macrophage inflammatory protein (MIP)-1β (29.28 pg/mL), MIP-3α (14.64 pg/mL), regulated on activation, normal T cells expressed and secreted ([RANTES] 3.6 pg/mL), IL-10 (1.8 pg/mL), IL-17 (3.6 pg/mL), interferon-inducible protein (IP)-10 (14.64), and tumor necrosis factor (TNF)-α (1.8 pg/mL). All samples were run in duplicate according to the manufacturer’s protocol. For cytokine levels below the lower limit of detection of the assay, values were reported as the lower limit of quantification based on the standard curve. Commercially available Trappin-2/Elafin enzyme-linked immunosorbent assay kits (Hycult Biotech) were used to measure SP levels of Elafin according to manufacturer’s instructions. The levels of many cytokines coassociate in semen, and so to reduce concerns around multiple comparisons we calculated a semen inflammation (SI) score: this was based on prior work in the female genital tract (FGT) [22], with slight modification to accommodate FGT-semen cytokine differences. Men who had 4 or more of 9 inflammatory cytokines (TNF-α, IL-1α, IL-1β, IL-6, IL-8, IL-17, MIP-3α, IL-1β, and MIP-1β) in the upper quartile were defined as inflamed, and the total number of cytokines in the upper quartile constituted an inflammation score (from 0 to 9).

Bacterial Load Quantification

For bacterial load quantification, 500 μL of thawed SP was lysed using a combination of chemical and mechanical methods and purified using AllPrep DNA/RNA Mini Kit (QIAGEN). Deoxyribonucleic acid was eluted in 100 μL of buffer EB, whereas RNA was eluted in 50 μL. Reverse transcription was performed using qScript cDNA SuperMix according to the manufacturer’s instructions (Quanta Biosciences, Gaithersburg, MD). Using the DNA fraction, bacterial load was quantfied, measured as bacterial 16S recombinant RNA (rRNA) gene copy/mL of SP using a broad-coverage qPCR assay, described previously [23].

T-Cell Populations in Blood and Semen

Mononuclear cells were stained for 30 minutes with fluorochrome-labeled monoclonal antibodies (CD3G-FITC, CD8-PETR, CD195-PerCPCy5.5, CD69-PECy7, β7integrin-APC, HLADR-APCH7, CD196-Pacific Blue, CD3-605NC, CD4-650NC) and a viability dye (Invitrogen) followed by fixation for 30 minutes in 1% paraformaldehyde and analyzed using BD LSR-II (BD Systems).

Statistical Analysis

Flow cytometry analysis was performed using FlowJo analytical software, version 9.4.11 (Tree Star). Non-parametric Mann-Whitney U and Wilcoxon signed-rank analysis were performed using IBM SPSS Statistics, version 20 (IBM Corporation). Backward stepwise linear regression analyses were performed for multiple independent covariants using SPSS, with P < .05 as the threshold for significance. Count and/or concentration variables were logarithmically transformed. For longitudinal multivariate analysis, a mixed model (MM) analysis was used to take into account multiple observations/measurements produced per individuals within the study. Variables associated with the outcome variable on univariate analysis (P < .1) were selected for the multivariable analysis.

Cross-Sectional Cohort Demographics and Viral Loads

Thirty HIV-infected, antiretroviral-naive men provided paired blood and semen samples. All participants were infected with HIV for at least 1 year (median, 3 years; range, 1–23 years). Study participants had median absolute CD4 and CD8 T-cell counts of 440 cells/mm³ (range, 190–860) and 905 cells/mm³ (range, 230–2690), respectively. The median bVL was 30544 HIV-1 RNA copies/mL (range, 824 to >500000 copies/mL), and median sVL was 5496 HIV-1 RNA copies/mL (range, 300–208152 copies/mL). All study participants were CMV seropositive, 80% (24 of 30) HSV-1 seropositive, and 33.3% (10 of 30) HSV-2 seropositive. Clinical parameters are summarized in Table 1.

Semen HIV RNA was detected in 93% (28 of 30) of participants, and 50% (15 of 30) met criteria for high-level semen shedding with an sVL ≥5000 copies of HIV-1 RNA/mL. Two individuals had asymptomatic urethral infection with Chlamydia trachomatis: both had high-level semen shedding and elevated semen cytokines and T-cell populations, and these individuals were removed from subsequent analysis. In 10.7% (3 of 28) of men, the sVL exceeded the paired bVL, meeting predefined criteria for disproportionately high HIV-1 semen shedding. At baseline, there was a weak correlation between blood and semen VL (r = 0.26; P = .160). Viral load data are summarized in Table 1.

Semen Cytokines and the Semen Viral Load

Levels of 14 cytokine/chemokines (see Methods) and of Elafin/Trappin-2 were assayed in SP (Table 2). Semen inflammation, defined as ≥4 of 9 proinflammatory cytokines in the top quartile for that cytokine (see above), was present in 6 of 28 (21%) participants, and it was strongly associated with HIV semen shedding (median sVL 4.89 log₁₀ HIV RNA copies/mL for men with SI vs 3.42 without; MW P = .008) (Figure 1). Furthermore, an inflammation score that was calculated as the number of these cytokines in the top quartile (range, 0–9) was also strongly correlated with the sVL (Spearman correlation coefficient P = .65; P < .001). Subsequent analysis demonstrated that the sVL was positively associated with semen levels of IL-1α (P = .009), IL-8 (P = .033), IL-1β (P = .005), IL-6 (P = .037), and MIP-1β (P = .031). To determine the strongest cytokine correlates of sVL, a partial correlation analysis controlling for bVL was performed. After controlling for bVL, semen levels of IL-8 (P = .002), IL-1α (P = .024), IL-1β (P = .011), IL-6 (P = .013), and MIP-1β (P = .006) remained significantly correlated with the sVL. The independent association(s) of these individual cytokines with the sVL was then assessed in a stepwise multivariate linear regression model, with IL-8 (P < .001) emerging as the strongest independent cytokine correlate of the sVL, followed by IL-1β (P = .03) (Figure 1). Trappin-2/Elafin was readily detectable in SP, but levels did not correlate with either the sVL or semen cytokine/chemokine levels.

The Semen Viral Load and Local Herpesvirus Reactivation

Herpesvirus qPCR was performed on all semen samples; 63% (19 of 30) passed internal control standards for CMV and 67% (20 of 30) for EBV. Internal control/run failure for the remaining samples may be due to the presence of PCR inhibitors, a common issue with qPCR assays in SP [24]. Cytomegalovirus was detected in the SP of 11 of 19 (58%), and EBV was detected in 6 of 20 (30%) participants; median CMV and EBV VL were 486 copies CMV DNA/mL (range, 200–266761) and 200 copies EBV DNA/mL (range, 200–409157), respectively. Semen HSV-1, HSV-2, HHV-3, HHV-7, and HHV-8 were undetectable in all patients, and HHV6 was only detected at very low levels in the semen of 2 participants. Therefore, subsequent analysis was focused on CMV and EBV.

Semen levels of CMV but not EBV correlated with the HIV sVL (P = .014 and P = .511, respectively (Figure 2). Semen CMV levels tended to be higher in men with SI (median 4.40 vs 2.38 log₁₀ DNA copies/mL; MW P = .063) and were directly correlated with the inflammation score (Spearman’s rho = 0.64; P = .006), whereas EBV levels were not related with either the presence or degree of inflammation. In particular, CMV levels correlated with semen cytokines IL-10 (0.048), IL-1β (<0.001), IL-6 (0.009), MIP-1β (0.004), and TNF-α (0.006). The independent association of these cytokines with semen CMV VL was assessed in a stepwise multivariate linear regression model with semen IL-1β level (P < .001) correlating most strongly with CMV VL.

Semen T-Cell Immune Parameters and the Semen Viral Load

Semen mononuclear cells (SMCs) were analyzed by flow cytometry in the subset of men (13 of 30; 43.3%) who consented to be observed longitudinally, to evaluate the prospective associations of T-cell phenotype and activation status with sVL. Ninety-three semen samples (approximately 7 samples per participant) were collected, and for practical reasons flow cytometry was performed on 76 of these samples (82%): adequate semen T-cell numbers (above our cutoff of ≥50 cell events) were present for CD4⁺ T-cell analysis in 69 of 76 (91%) and for CD8⁺ T-cell analysis in 73 of 76 (96%). Semen T cells were immunologically much more activated than those in the blood of the same participant. In particular, coexpression of CD38 and HLA-DR was higher on both semen CD4⁺ T cells (median, 16.25% vs 0.89%; P < .001) and CD8⁺ T cells (median, 13.75% vs 3.91%; P = .039), as was the early activation/T_RM marker CD69 on both CD4⁺ (32.80% vs 3.14%; P < .001) and CD8⁺ (57.0 vs 5.17; P < .001) T cells. Semen CD4⁺ T cells also expressed higher levels of the HIV coreceptor CCR5 (24.15% vs 4.3%; P < .001) and of the Th17 marker CCR6 (28.8 vs 4.3; P = .003), whereas expression of β7, a proxy for the mucosal homing integrin α₄β₇⁺, was lower on semen CD4⁺ T cells (0.9% vs 2.1%; P = .013). The proportion of CD3⁺ T cells expressing the gamma-delta receptor (CD3G) tended to be higher in peripheral blood mononuclear cells than SMCs (2.84 vs 0.94; P = .180), although semen CD3G⁺ T cells expressed higher levels of CCR6 (83.7 vs 0.43; P < .001).

Overall, CD4⁺ and CD8⁺ T-cell numbers in the semen were strongly correlated with a number of immune and virologic semen parameters, including the CMV VL (Spearman rho = 0.78 and 0.89, both P < .01) and the SI score (Spearman rho = 0.77 and 0.85, both P < .001), and less strongly with the semen HIV VL (Spearman rho = 0.48 and 0.64, P = .11 and P = .02, respectively). No T-cell associations were seen with semen levels of EBV or with the semen bacterial load (see below).

Bacterial Load in Semen and the Semen Viral Load

To broadly assess whether the endogenous microbiome of the semen compartment [25] impacted semen immunology and sVL, total bacterial load was quantified by 16S rRNA. In men free of bacterial STIs, the total bacterial load was positively correlated with both the HIV sVL (Spearman rho = 0.51; P = .016) (Figure 3) and the SI score (Spearman rho = 0.48; P = .024), and it was higher in participants meeting criteria for SI (median log₁₀ bacterial load 7.03 vs 5.40; P = .003). Furthermore, the bacterial load was significantly correlated with semen levels of the individual cytokines IL-8 (P = .004), MCP-1 (P = .022), MDC (P = .017), MIP-3α (P = .008), IL-6 (P = .012), IP-10 (P = .007), and TNF-α (0.046). However, no association of the bacterial load was observed with baseline clinical parameters or the local reactivation of CMV or EBV.

Longitudinal Cohort Demographics

A subgroup of participants (n = 13) provided monthly, paired semen and blood samples for up to 12 months, or until initiation of ART, to permit assessment of sVL and semen immunology changes within an individual. Herpes simplex virus-1 and HSV-2 seroprevalence was 92% (12 of 13) and 46% (6 of 13), respectively, and the median duration of follow-up was 7 months (range, 2–12 months) with a total of 93 sample visits (an average of just over 7 visits per participant).

Patterns and Correlates of Human Immunodeficiency Virus Shedding in Semen

High-level semen shedding (>5000 copies/mL) had been detected at enrollment in 45.5% (5 of 11) of the STI-free prospective participants: during follow up, high-level shedding was detected at 1 study visit or more in 69% (9 of 13) and overall at 51.6% (48 of 93) of study visits. The median sVL was 0.95 log₁₀ (0.79-fold) lower than the bVL (median bVL 4.45 log₁₀ copies/mL, range 2.92–5.18 log₁₀ copies/mL vs median sVL 3.50 log₁₀ copies/mL, range 2.48–5.00 log₁₀ copies/mL), but there was substantial inter-individual variability. In approximately half of the participants, the sVL was consistently lower than the blood (6 of 13 participants; representative example) (Figure 4A). Although only 23% (3 of 13) of study men had an undetectable sVL at any time point, 1 individual maintained an undetectable sVL at each of 9 study visits despite a median bVL of 3.69 log₁₀ copies/mL (range, 3.06–3.87 log₁₀ copies/mL) (Figure 4B). One participant (1 of 13) consistently demonstrated a disproportionately high sVL (Figure 4C), whereas in the remaining 6 of 13 participants, the sVL was sometimes lower than blood and sometimes disproportionately high (Figure 4D). In addition, the variability in semen versus blood HIV-1 RNA levels was assessed by calculating the ratio and variance of log₁₀ semen/log₁₀ blood RNA levels (Table 3). Although the median semen/blood VL ratio was 0.87, it exceeded 1 in 2 participants (15%), and there was considerable heterogeneity in the variance of this ratio, ranging from 0.002 to 0.149.

Longitudinal Correlates of Human Immunodeficiency Virus Shedding in Semen

Semen parameters that had been associated with the sVL in the cross-sectional analysis also varied over time. Cytomegalovirus reactivation was more common than EBV in semen, with detection in 9 of 13 vs 4 of 13 participants at any time point, and at 29.0% (27 of 93) vs 7.5% (7 of 93) of total study visits, respectively. The median semen CMV VL was 241 copies/mL (range, 200–266761 copies/mL) and exceeded 5000 copies/mL during 29.6% (8 of 27) reactivations; the median semen EBV VL was also 200 copies/mL (range, 200–353125 copies/mL) and exceeded 5000 copies/mL during 28.6% (2 of 7) reactivations. Semen flow cytometry assays were performed for 76 of 93 samples collected, and sufficient (>50 cells per sample) semen CD4⁺ T cells for analysis were present in 69 of 76 (90.1%) samples and CD8⁺ T cells in 73 of 76 (96.1%) samples. In general, the semen bacterial load, semen cytokine levels, and T-cell parameters were relatively stable over time (data not shown).

Finally, a multivariable MM that incorporated the longitudinal viral, immune, and bacterial load data was then used to assess independent associations of the HIV sVL (see Methods). In this prospective analysis, the only independent associations of HIV sVL were the bVL (P < .001) and semen levels of the proinflammatory chemokine IL-8 (P < .001). For every log₁₀ unit increase in bVL, the sVL increased by 0.40 log₁₀, and for every log₁₀ increase in the IL-8, the sVL increased by 0.75 log₁₀. In this prospective analysis, the semen to blood HIV VL ratio, which was calculated to assess the contribution of individual semen parameters to the local HIV VL, was most strongly associated with semen IL-8 levels (P < .001) (Figure 5).