In HIV-infected individuals, levels of HIV-1 RNA in blood [1,2] and semen [3,4] correlate with the risk of sexual transmission. Although HIV-1 RNA levels in blood correlate with levels in seminal plasma [3–7], local genital factors, particularly concomitant bacterial sexually transmitted infections (STIs), can increase HIV-1 shedding in semen [7–11]. Not only bacterial STI, but also chronic viral coinfections, for example herpes simplex virus type 2 (HSV-2) and cytomegalovirus (CMV), likely influence HIV-1 pathogenesis, transmission and dynamics within the genital tract [12–15].
Previous studies have demonstrated that some herpes viruses are likely important for HIV-1 transmission. For example, levels of CMV [6,15,16] and HSV-2 [17–19] DNA in semen are associated with higher HIV-1 RNA seminal levels, and HSV-2 seropositivity of source partners is associated with HIV-transmission among men who have sex with men (MSM) . However, treatment of HSV in HIV-infected potential source partners with acyclovir does not reduce HIV transmission , suggesting that other factors remain. Although levels of HIV-1, HSV-2 and CMV in the genital tract are well documented, they are not the only viruses that replicate in the male genital tract, and other members of the herpes family are sexually transmitted and are common worldwide [21,22]. These viruses often coinfect the same host and likely influence each other's virologic dynamics and host immune response [12,21–23]. One study evaluated 50 semen samples collected from HIV-infected heterosexual men in India with a median of 281 CD4+ T-cells per millilitre and found that concomitant Epstein–Barr virus (EBV) and CMV seminal shedding was associated with increased HIV-1 RNA levels in semen . Although CMV and EBV shedding was high in this cohort (70 and 56%, respectively), shedding of HSV, human herpes virus (HHV)-6, HHV-7 and HHV-8 was likely too low (8, 2, 12 and 6%, respectively) to interpret associations with HIV-1 shedding.
To further understand the role that chronic viral infections of the male genital tract play on HIV-1 dynamics and replication, this study measured viral levels of seven different herpes viruses in 236 semen samples from 115 HIV-infected antiretroviral-naive MSM and examined the relationships between levels of these viruses and the presence of HIV-1 RNA within the genital tract. In addition, we evaluated the impact of HIV-1 transmitted drug resistance mutations (DRM) on HIV-1 seminal shedding; on a subset of 36 individuals  we investigated the associations between these herpes viruses and the number and immune activation status of T-cells in semen and blood.
Materials and methods
Participants, samples and clinical laboratory tests
A total of 236 semen samples from 115 recently HIV-infected antiretroviral-naive participants from the San Diego Primary Infection Cohort  were included. Semen was collected by masturbation, as described previously [12,25,26]. At baseline, Neisseria gonorrhoeae and Chlamydia trachomatis genital tract infections were evaluated by PCR of urine samples and screening for syphilis infection was performed by rapid plasma reagin titers (RPR). In blood, CD4+ T-lymphocyte subsets were measured by flow cytometry (LabCorp) and HIV-1 RNA was quantified (Amplicor HIV Monitor Test; Roche Molecular Systems Inc. Pleasanton, California, USA). HHV-8 serology was determined using the ‘HHV-8 immunoglobulin (Ig) G Antibody ELISA Kit’ (Advanced Biotechnologies Inc. Columbia, Maryland, USA). Clinical data were collected, and estimated duration of infection (EDI) was calculated using established algorithm [24,27]; HIV-1 subtype was determined for 113 individuals using HIV-1 pol sequence data generated by Viroseq 2.0 (Applied Biosystems, Carlsbad, California, USA) using the genetic algorithm ‘Subtype Classification using Evolutionary Algorithms’ . For two participants sequences were not obtained because of blood plasma viral load less than 500 copies/ml. DRM were analyzed using the Genotypic Resistance Interpretation Algorithm on the Stanford HIV drug resistance database . The studies were conducted with appropriate written participant consent and were approved by the Human Research Protections Program at the University of California, San Diego, California, USA.
RNA extraction from seminal plasma, and HIV RNA quantification
HIV-1 RNA levels were measured in seminal plasma by first concentrating HIV-1 RNA from 500 μl of seminal plasma with high-speed centrifugation (23 500 × g at 4°C for 1 h), as described previously . Concentrated RNA was extracted using High-Pure Viral RNA Kit (Roche, Indianapolis, Indiana, USA) and HIV-1 cDNA was generated using the SuperScript III First-Strand Synthesis Kit (Invitrogen, Grand Island, New York, USA) with specific primer mf302 . HIV RNA in seminal plasma was quantified by real-time PCR in an 7900HT thermocycler (Applied Biosystems) with 0.005 μmol/l ROX as passive reference. A total reaction volume of 50 μl was added to each well consisting of 5 μl of cDNA, TaqMan Environmental Mastermix 2.0 (Applied Biosystems), PCR primers mf302 and mf299  (1 μmol/l), and probe mf348  (0.3 μmol/l) (supplementary Table 1, http://links.lww.com/QAD/A236). The PCR conditions were 2 min at 50°C, 10 min at 95°C, 60 cycles of 15 s at 95°C, and 60 s at 60°C. HIV RNA quantification standard was obtained from the DAIDS Virology Quality Assurance Program .
DNA extraction from seminal plasma, and herpes virus DNA quantification
Viral DNA was extracted from 200 μl of seminal plasma using QIAamp DNA Mini Kit (Qiagen, Valencia, California, USA) per manufacturer's protocol. EDTA (50 mmol/l) was added to seminal plasma to inhibit DNase activity. Levels of different herpes viruses in semen were measured by real-time PCR in an 7900HT thermocycler (Applied Biosystems) with 0.005 μmol/l ROX as passive reference. A total reaction volume of 50 μl was added to each well consisting of 10 μl DNA, TaqMan Environmental Mastermix 2.0 (Applied Biosystems), virus-specific PCR primers (1 μmol/l) and probe (0.3 μmol/l) (supplementary Table 1, http://links.lww.com/QAD/A236). Quantification standards for the different herpes viruses were obtained using plasmid preparations with known concentrations.
Multiparameter flow cytometry analysis
For a subset of 36 participants, seminal cell samples were analyzed by flow cytometry (FACS) on a dual-laser, 6-color Becton Dickinson FACS Canto using Diva (6.1) or FlowJo (9.0) software, as described previously . Cells were stained using CD45-PerCP/Cy5.5 (clone 2D1), CD45RA-PE (clone HI100), CD4-FITC (Leu 3a/3b multiclone), CD38-PE/Cy7 (clone HB7), CD3-APC (clone SK7) and CD8-APC/Cy7 (clone SK1) (BD Biosciences, San Jose, California, USA).
Statistical analyses were performed using Graph-Pad Prism 5.0 software (GraphPad Software, La Jolla, California, USA) and SAS version 9.2 (SAS Institute Inc., Cary, North Carolina, USA).
Viral load variables were transformed to the base 10 logarithm values. Nonnormal data were either dichotomized or ordinalized. Comparison of viral seminal shedding between groups was performed using Fisher exact test. Genital herpes viruses, blood HIV-1 RNA levels, CD4+ T-cell number, presence of DRM, age, or stage of HIV-infection (cutoff at 90 days after EDI) were associated with detectable seminal HIV-1 RNA shedding (as a binomial variable, i.e. with seminal levels above/below 50 copies/ml) using univariate and multivariate generalized estimating equation (GEE) to account for repeated measurements using an exchangeable correlation structure (entry criterion for multivariate analysis: P = 0.35; level of significance: P = 0.05). CMV and EBV were categorized in three groups, that is undetectable, low, and high shedding (cutoff: mean viral level among detectable samples). HIV-1 genital levels in CMV and EBV positive versus negative samples and in samples with and without DRM, as well as differences in seminal T-cell numbers and immune activation were compared using Mann–Whitney test.
HIV-infected participants (n = 115) were antiretroviral-naive MSM with a median age of 33 years (Table 1). All but three participants with available HIV-1 pol sequence (n = 113) were infected with HIV-1 subtype B virus (the exceptions being two B/D and one B/F1 recombinant). From these individuals, 236 single or longitudinal semen samples (median of 2 time-points for each patient, range 1–8) were collected. For 60 individuals with repeated sampling, there was a median follow-up of 65 days (IQR: 29–172). For all individuals at baseline, the median CD4 cell count was 519 cells/μl (IQR: 413–712 cells/μl), their median EDI was 97 days (IQR: 79–152), and their median blood plasma HIV-1 level was 4.83 HIV-1 RNA log10 copies/ml (IQR: 3.92–5.27). At baseline, 10 participants had positive syphilis screening tests by RPR, three were positive for urethral Chlamydia by urine PCR, another three were positive for both Chlamydia and Neisseria gonorrhoeae by urine PCR, and one participant was positive for all three (Syphilis, Chlamydia and N. gonorrhea). All participants with identified bacterial STI were treated for these infections and included in the study. Fifty-five individuals (48%) were positive for HHV-8 IgG antibody in blood plasma.
Shedding frequency and levels of HIV and herpes viruses in semen
Despite high CD4+ T-cell count (519 CD4+ T-cells/μl) and early stage disease (median 97 days EDI at baseline), 75.7% of the individuals had at least one herpes virus detected in one or more of their seminal plasma samples, and 70.3% of all samples had at least one detectable herpes virus. These included CMV (frequency 51.3%, median peak viral load 4.52 log10 DNA copies/ml), EBV (40.9%, 2.40 log10), HHV-8 (11.3%, 2.72 log10), HSV-1 or HSV-2 (10.4%, 2.83 log10), HHV-6 (7.0%, 2.82 log10), and HHV-7 (14.8%, 3.42 log10) (Table 2 and Fig. 1). Fifty of the participants (43%) were found to shed more than one herpes virus either simultaneously or over time. Although 12 participants had continuous detectable herpes virus DNA in repeated tested time-points over many months and sometimes years [median 56 days (IQR: 28–228)], most of the individuals with longitudinal sampling demonstrated intermittent herpes virus shedding, that is had seminal samples with detectable and undetectable levels of the same virus over a few weeks or months. We were able to discriminate between HSV-1 and HSV-2 by allele-specific PCR for 11 out of 12 participants with detectable HSV in their semen and found that five of these (45%) were HSV-1 and six were HSV-2.
Differences in viral levels by HIV stage and CD4+ T-cell count
To clarify other factors that may be associated with viral shedding, we investigated if there was a difference in detection of herpes viruses in semen collected before and after 90 days EDI and among those with lower and higher CD4+ T-cell counts (≥500 vs. <500 and ≥350 vs. <350 cells/μl). Overall, there was no clear relationship between EDI and seminal herpes virus shedding, but there was a trend towards increased shedding for HHV-8 (8.2 vs. 1.5% P = 0.07) and EBV (38.5 vs. 25.8% P = 0.07) in later collected samples. There was a propensity for participants with detectable seminal HIV-1 levels to be more frequently sampled within the first 90 days of their infection (P = 0.12), probably as a consequence of higher viral loads observed in the early-stage group (P < 0.01). Although HIV-1 RNA seminal shedding was associated with lower CD4+ T-cell counts (<500 cells/μl; P = 0.04), HSV seminal shedding was associated with higher CD4+ T-cell counts (>500 cells/μl; P = 0.01). At a CD4+ T-cell threshold of 350 cells/μl, only CMV showed differential shedding with CMV genital shedding being twice as frequent at lower counts (50% shedding if CD4+ T-cell count <350 cells/μl vs. 24.1% if CD4+ T-cell count >350 cells/μl). No participants had a CD4+ T-cell count less than 200.
Predictors of HIV-1 RNA shedding in semen
Multiple factors likely influence seminal shedding of HIV-1 RNA. In this study we evaluated the association of seminal HIV-1 shedding to herpes viruses DNA, blood HIV-1 RNA levels, CD4+ T-cell count, presence of transmitted HIV-1 DRM, participants’ age and stage of infection (Table 3). In univariate analysis, detectable seminal HIV-1 RNA was marginally associated with higher seminal levels of CMV DNA (P = 0.06), higher HIV-1 RNA levels in blood plasma (P < 0.01), and lower numbers of CD4+ T-cells in blood (P < 0.05). There was also a weak trend toward increased HIV-1 shedding for samples with EBV at levels more than 2.7 log10 copies/ml (P = 0.17). In univariate analysis there were no associations between presence of HIV-1 RNA seminal shedding and presence of transmitted DRM, participant's age, and presence of other herpes viruses (i.e. HSV, HHV-6, HHV-7, and HHV-8). In multivariate analysis, blood HIV-1 RNA levels (P < 0.01), seminal CMV and HHV-8 levels (P < 0.05) were independent predictors of HIV-1 RNA seminal shedding. Interestingly, only high levels of CMV DNA in semen (>4.4 log10 copies/ml) were associated with detectable HIV-1 RNA in semen. In regard to HHV-8 coinfection, 87% of the semen samples with detectable HHV-8 DNA also had HIV-1 RNA shedding compared to 67% HIV-1 shedding in samples when HHV-8 was not present (Fisher's exact P = 0.16). When considering only individuals with HHV-8-positive serology, this difference was even greater with 87 vs. 56% (P = 0.04). Similarly, in our multivariable GEE model using the same covariates displayed in Table 3, but including only individuals with positive HHV-8 serology (n = 56), the association of HHV-8 shedding with HIV-1 shedding in semen remained statistically significant (P = 0.02).
As an additional endpoint, we investigated the relationships between detectable CMV or EBV DNA in semen (the most frequently detected viruses) and levels of seminal HIV-1 RNA. Seminal shedding of both herpes viruses was associated with higher levels of HIV-1 RNA within the same compartment (median 2.64 vs. 2.18 log10 copies/ml, P = 0.05 for CMV, and 2.79 vs. 2.15 log10 copies/ml, P < 0.01 for EBV, Fig. 2a and 2b). All of these observations remained true after excluding the 16 patients with concomitant bacterial STI.
Impact of transmitted HIV-1 drug resistance mutation on seminal HIV-1 levels
Because transmitted DRM in the HIV-1 pol gene can influence HIV-1 levels [32,33], we also evaluated the presence of DRM in our antiretroviral-naive cohort. Nineteen participants were found to have relevant DRM in their baseline HIV-1 pol sequences [nonnucleoside reverse transcriptase inhibitor: K101P (one), K103N (11), Y181C (one), P225H (one); nucleoside reverse transcriptase inhibitor: M41L (five), D67N/G (four), T69-insert (one), K70R (one), L74 V (one), M184 V (two), L210W (one), T215S/Y (seven), K219E/Q (three); protease inhibitor: M46I (one), I54 V (two), V82A (one), I84 V (one), L90 M (two)]. Overall, the presence of transmitted DRM was associated with a lower viral load in blood (4.31 vs. 4.62 log10 copies/ml, P = 0.05) and with a more pronounced difference in semen (median of 2.09 vs. 2.49 log10 copies/ml, P = 0.02, Fig. 2c).
Associations between seminal shedding of herpes viruses and absolute numbers and activation levels of lymphocytes in semen and blood
A recent article from our group  reported that detectable CMV levels in semen were positively correlated with the absolute numbers of seminal T-cells and with their activation status in semen and blood. On the same subset of participants, we now investigated the associations between shedding of other herpes viruses with the numbers and immune activation status (RA-CD38+) of CD4+ and CD8+ T-cells. Similar to what we previously found with CMV, the presence of seminal EBV replication was associated with higher absolute numbers of CD4+ (P < 0.01) and CD8+ (P < 0.05) T-cells in semen. None of the herpes viruses detected in the genital tract (other than CMV) significantly impacted the observed immune activation in semen or blood.
Understanding the viral and immunologic dynamics in the seminal compartment and characterizing the contribution of cofactors that increase the risk of sexual transmission could provide important information for the development of effective prevention strategies. Because HIV-1 in male genital secretions account for more than half of all HIV-transmissions among MSM in particular , we investigated a cohort of HIV-infected MSM for interactions between seminal replication of seven herpes viruses and HIV-1 shedding.
Despite relatively preserved immunity and early stage disease, a high proportion of participants (75.7%) had at least one genital sample with shedding of herpes viruses. Similar to previous reports [16,35], CMV and EBV were frequently detected in about half of all analyzed seminal samples, whereas HSV and HHV-6, HHV-7, and HHV-8 were found at lower frequencies, between 7–14.8%. Interestingly, 45% of HSV-DNA found in the semen was HSV-1 rather than HSV-2, suggesting that future studies involving genital HSV replication should discern each type.
Although individuals with detectable HIV-1 levels in semen were more often identified earlier during HIV-infection, there were trends toward increased shedding more than 90 days EDI for HHV-8 (P = 0.07) and for EBV (P = 0.07), whereas there was no difference for the other herpes viruses. This suggests that individuals within the first 90 days of infection are more likely to shed seminal HIV-1 independently of herpes viruses coinfections, probably as a consequence of higher blood HIV-1 viral load. On the contrary, seminal shedding of herpes viruses may have a greater impact on HIV-1 shedding after acute HIV-infection has passed, that is more than 90 days EDI. Further, immune status, as measured by CD4+ T-cell count, also influenced HIV-1 and herpes virus shedding. Although HIV-1 (P = 0.04) and CMV (P < 0.01) seminal shedding were associated with lower CD4+ T-cell counts, consistent with previous reports [15,21], there was strong evidence that HSV shedding occurred at CD4+ cell counts more than 500 cells/μl (P = 0.01). These results contrast with a recent report , documenting borderline increased HSV-shedding in individuals with CD4+ cell counts less than 500 cells/μl (P = 0.08). On the contrary, the participants in our study had relatively intact immune systems and did not include severely immunocompromised persons in whom the association between herpes viruses and HIV-1 shedding may be stronger. Also, as our cohort included only asymptomatic HIV-infected individuals, it is possible that HSV-shedders with low CD4+ cells and ulcerative genital disease were not included in this study, but this should be pursued in future investigations.
Similar to previous studies [3,4], the strongest predictor of HIV-1 seminal shedding in our cohort was the level of HIV-1 RNA in blood; however, high levels of CMV DNA (>4.4 log10 copies/ml) in semen were also independently associated with HIV-1 shedding . The lack of association between seminal CMV and HIV-1 shedding when CMV was present at lower levels could suggest that a certain threshold of CMV replication is needed to influence HIV-1 RNA shedding or that an unidentified factor influencing both HIV-1 and CMV shedding could be present, like localized immune activation [12,35,37,38]. This study also found that presence of seminal EBV replication was strongly associated with higher levels of HIV-1 RNA in semen and also with higher CD4+ T-cell counts in semen (both P < 0.01) suggesting that also EBV plays a role in HIV-1 dynamics. Perhaps most interesting, this study identified that the presence of HHV-8 was also an independent predictor for HIV-1 RNA seminal shedding. Unlike the previous Indian study of heterosexual individuals , the ability to detect this effect is likely a consequence of the higher rate of HHV-8 seminal shedders in our cohort (11.3 vs. 6%) [21,35]. Seminal shedding of HHV-8 has been reported more frequently in MSM [39–41], but a limitation is that we did not evaluate oral shedding [42–45], and this should be pursued given these results. With the historical connections between Kaposi sarcoma and the male-to-male sex for HIV-1 and HHV-8 infections, the associations between HIV-1 and HHV-8 genital tract shedding should be explored further and might be especially important among African populations in which HHV-8 and Kaposi sarcoma prevalence is higher [46–48].
We observed significantly lower HIV-1 RNA levels in blood plasma of participants in which the HIV population had a DRM (P = 0.05), and this difference was even more pronounced in semen (P = 0.02). Previous reports have demonstrated the effect of DRM on blood plasma viral load [32,33], but while intuitive, this is the first report of this effect on seminal viral load. The fitness cost of DRM seems to have a higher impact on replication capacity of HIV-1 in the genital compartment compared to wild-type virus. Because all of our cohort participants were antiretroviral-naive, all of the observed DRM were likely transmitted, so no conclusions can be made on how failing antiretroviral therapy selecting for DRM can influence seminal shedding of HIV-1.
This study has a number of limitations. Because this was a retrospective observational study, we cannot establish a causal relationship between the described correlations. Morover, screening for STI was performed only at baseline and an unrecognized incurred bacterial STI could confound the observations. Further, although this is the largest study of its kind, the sample size still limited power in discerning a significant effect for some viruses, for example HSV or HHV-6. In any case, the effect of these relatively rarer herpes viruses on HIV-1 seminal shedding, and thus on the potential for HIV-1 transmission in MSM risk group, is likely relatively small. Lastly, as this study included only MSM from a relatively small geographic area, it is necessary to determine if the results can be generalized for other risk groups or populations.
To our knowledge, this is the largest study of this kind (n = 236 samples, 115 different participants) investigating the simultaneous interactions between HIV-1 and seven different herpes viruses in a cohort of MSM followed since primary HIV-infection. This large dataset permitted the description of the shedding of herpes viruses and HIV-1 in different subsets of participants, and a multivariate analysis taking in account a number of potentially confounding variables (age, stage of infections, blood HIV-1 viral load, and CD4+ T-cell counts). This study is also unique in that it evaluated the possible influence of DRM and the associations between herpes viruses and lymphocytes subsets and immune activation in semen in a subgroup of participants. Consistent with previous studies [15,21], blood HIV-1 levels, seminal CMV and EBV were the most significant associations for HIV-1 shedding in semen. Uniquely interesting, HHV-8 was associated with increased seminal HIV-1 shedding. Moreover, we described for the first time how presence of transmitted DRM impacts HIV-1 replication in the genital tract. Taken together, these results provide a clearer picture of how the highly prevalent herpes viruses influence the dynamics of HIV-1 in the male genital tract and how they likely influence the risk of sexual transmission of HIV-1. Future studies should determine if these correlations hold true in patients on ART, and if this is the case, adding CMV suppressive therapy to standard ART in high-risk individuals or in discordant couples may be clinically relevant.
We are grateful to all the participants in the San Diego Primary Infection Cohort, the CFAR Genomic and Sequencing and Translational Virology Cores, and Nadir Weibel for his support and inspiring discussions. We also thank and commemorate our dear friend and outstanding research colleague Marek Fischer for all his contributions and support to our research over many years. HIV RNA quantification standard was obtained through the NIH AIDS Research and Reference Reagent Program, DAIDS, NIAID: HIVVQA RNA Quantification Standard from the DAIDS Virology Quality Assurance (VQA) Program; Primer and Probe for quantification of herpes viruses as well as the plasmids and quantification standards were kindly provided by Fred Lakeman.
S.G. participated in the study design, performed the laboratory experiments, participated in the data analyses for this study and wrote the primary version of the manuscript; S.M. participated in study design, performed statistical analysis and wrote the primary version of the manuscript; C.A., J.A.Y., C.A.S. participated in study design, participated in the data analyses and revised the manuscript, M.V.V. performed the laboratory experiments; S.J.L. and D.M.S. enrolled participants, D.D.R., S.J.L., and D.M.S. designed the present study, participated in data analysis and revised the manuscript. All authors read and approved the final manuscript.
This work was supported by the Department of Veterans Affairs, the James Pendleton Charitable Trust; the US National Institutes of Health (NIH) awards AI69432, AI043638, MH62512, MH083552, AI100665, AI077304, AI36214, AI047745, AI74621, GM093939 and AI080353, AI306214 (CFAR); Swiss National Science Foundation grant PASMP3_136983; the California HIV Research Program RN07-SD-702; and the National Institute of General Medical Sciences grant GM093939. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
Conflicts of interest
S.G. and M.V.V. do not have any commercial or other associations that might pose a conflict of interest. D.D.R. has served as a consultant for Bristol-Myers Squibb, Gilead Sciences, Merck & Co, Monogram Biosciences, Biota, Chimerix, Tobira, and Gen-Probe. D.M.S. has received grant support from ViiV Pharmaceuticals and consultant fees from Gen-Probe.
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