The risk of HIV-1 sexual transmission is independently associated with the HIV RNA viral load (VL) in a person's genital tract and blood.1 Although the genital and blood VLs are moderately correlated, localized factors such as genital coinfections can increase HIV levels in the genital compartment.2–4 The semen HIV VL is usually lower than that in the blood but is sometimes disproportionately high.5,6 Importantly, both this phenomenon and the absolute level of HIV RNA in semen have been associated with the compartmentalized reactivation of cytomegalovirus (CMV).5,7
CMV is a common human herpesvirus (HHV) with a very high prevalence in both the general population (∼65%) and in HIV-infected individuals (∼95%)8,9 and has been hypothesized to be an important cofactor in HIV transmission.10 CMV is generally asymptomatic after primary infection in the context of normal T-cell immune function,11 although asymptomatic reactivation and viral DNA shedding are common in the saliva, vaginal secretions, and semen of immunocompetent individuals. CMV reactivation may cause severe eye and gut disease in people with advanced HIV infection and associated T-cell dysfunction, and reactivation in this context constitutes an AIDS-defining illness.12,13 However, even during the early HIV stages, localized genital CMV reactivation is seen more commonly than in HIV-uninfected individuals, and this compartmentalized genital reactivation of CMV has been associated with mucosal immune activation14–16 and a 10-fold increase in semen HIV levels.5 A 10-fold increase in the HIV genital VL would be expected to approximately double the probability of HIV sexual transmission.1 In keeping with this, CMV levels were higher in the semen of men who had transmitted HIV to their partner.17 However, the impact of CMV therapy on HIV genital levels has not been assessed.
HIV-infected individuals have a high prevalence of coinfection by herpes simplex virus type 2 (HSV-2),18,19 which is associated with genital immune activation,20,21 increased genital HIV levels,22,23 and an increased probability of HIV transmission.24,25 HSV-2–suppressive therapy with the antiviral drugs acyclovir/valacyclovir reduces HIV RNA levels in both blood plasma and genital secretions, although it remains unclear whether this reduction represents a direct inhibition of HIV replication by acyclovir26,27 or is mediated through a reduced HSV-2–associated inflammation.2,28 Importantly, although acyclovir decreased the frequency of genital ulcers and HIV blood VL, this did not reduce the risk of HIV transmission to a person's sexual partner.29
Valganciclovir is a biologically inactive prodrug of ganciclovir.30 It is the first-line option for CMV prophylaxis in immunosuppressed individuals31 and is as effective as intravenous ganciclovir in efficacy, safety, and bioavailability.32,33 A dose of 900 mg/d is commonly used for CMV prophylaxis or as maintenance therapy in individuals treated for CMV disease, particularly in those with suppressed T-cell immune function.34,35 Furthermore, valganciclovir therapy in HIV-infected, antiretroviral (ART)-treated men has been shown to reduce T-cell activation in blood.14 We hypothesized that CMV suppression with valganciclovir in HIV-infected men would reduce the HIV semen VL and that this would be mediated through a decrease in CMV-associated mucosal/systemic immune activation. To test this hypothesis, we evaluated the impact of valganciclovir in HIV-infected, ART-naive men in an open-label observational study.
All participants provided written informed consent. The study protocol was approved by the Research Ethics Board at the University of Toronto, Canada. Participants were identified by study number, and data were analyzed anonymously.
All participants were 18 years or older, HIV-CMV–coinfected, and ART-naive men with a recent CD4+ T-cell count >400/mm3. Participants were screened based on a recent sample analysis demonstrating detectable HIV VLs.
Participants with urethral leukocytes, gonorrhea, chlamydia, or a history of clinical anal or genital herpes were excluded, as were those with abnormal hemoglobin (<100 g/L), neutrophil count (<750 cells/μL), or platelet count (<150 × 103/μL), ganciclovir or valacyclovir allergies, or a history of low compliance.
Study Design and Sampling
Participants were recruited through the Maple Leaf Medical Clinic (Toronto, Canada) from 2007 to 2008. Participants were provided with 900 mg of oral valganciclovir daily for 2 weeks in an open-label fashion. Participants completed questionnaires and provided clinical samples at each of 3 study visits: baseline (pretreatment), after 2 weeks of valganciclovir (2 weeks postenrollment), and 6 weeks after therapy completion (8 weeks postenrollment). First-void urine was screened for leukocytes by urine dipstick (Bayer Diagnostics, Mayfield, MA) and for infection with Neisseria gonorrhoeae or Chlamydia trachomatis by Amplicor CT/NG assay (Roche Diagnostics, Indianapolis, IN).
This observational clinical study began screening in 2007. At this time, the open-label observational format that we used did not meet the criteria for a clinical trial, as defined by the International Committee of Medical Journal Editors policy on clinical trial registration.36 Therefore, the study was not formally registered as a clinical trial, although criteria for what constitutes a clinical trial have evolved since that time.37
Paired blood and semen specimens were collected at each visit. Blood was collected into 3 acid citrate dextran tubes, and semen was collected by masturbation into 10 mL sterile RPMI supplemented with 100 U/mL penicillin and 0.1 mg/mL streptomycin and processed within 2 hours of sample collection. Seminal plasma and seminal mononuclear cells were isolated by density gradient centrifugation over Ficoll-Hypaque solution (Ficoll-Paque Plus; Amersham Biosciences, Piscataway, NJ) for 10 minutes at 850g, and blood plasma and peripheral blood mononuclear cells (PBMCs) were separated by layering onto Ficoll-Hypaque solution and centrifuged at 500g for 30 minutes. Seminal and blood plasma samples were flash frozen at −80°C, and seminal mononuclear cells and PMBCs were cryopreserved in 10% dimethyl sulfoxide/fetal bovine serum and stored at −150°C.
Blood and semen plasma HIV-1 VLs were measured using the Versant HIV-1 RNA 3.0 assay (Bayer Diagnostics; lower limit of detection, 50 RNA copies/mL), as previously described.38,39 Serologic testing was performed for HSV-2 immunoglobulin G (IgG) (Kalon Biological, Kalon, Aldershot, United Kingdom) and CMV IgG (AxSYM CMV IgG assay; Abbott Laboratories, Abbott Park, IL). CMV DNA levels were assayed by real-time polymerase chain reaction (lower limit of quantification, 6 DNA copies/mL in a diluted semen sample) on a Roche Lightcycler (Roche Diagnostics). Because RPMI was often spilled during semen collection, virus levels, cytokine levels, and all calculations were corrected for dilution under the assumption of a semen volume of 2 mL, as previously described.5
Cellular Phenotyping and Soluble Immune Proteins
Previously cryopreserved blood and semen mononuclear cells were thawed, washed, and stained with CD3, CD4, CD25, and FoxP3 (panel 1) and CD3, CD8, CD38, and CD69 (panel 2) as per manufacturer's instructions (BD Biosciences, Mississauga, Ontario, Canada). CD4+CD25+ T cells expressing FoxP3 were defined as regulatory T cells (Tregs).40,41 Events were acquired and analyzed on the FACSCalibur system with CellQuest software (BD Biosciences) using FlowJo version 8.6 (TreeStar Inc, Ashland, OR). Seminal cytokine and chemokine levels were measured using SearchLight Multiplex Immunoassay Kits (Aushon, Billerica, MA), including interferon-γ (IFN-γ), interleukin (IL)-1β, IL-6, IL-8, IL-12p70, monokine induced by interferon-gamma (MIG), monocyte chemotactic protein-1 (MCP-1), interferon gamma-induced protein 10 (IP-10), regulated upon activation normal T cell expressed and presumably secreted (RANTES), and tumor necrosis factor alpha (TNF-α). Aliquots of seminal plasma were thawed and diluted 1:2 and 1:50 and run as per manufacturer's specifications in duplicate.
Ex Vivo Stimulation and Proliferative Immune Responses
Ex vivo HIV- and CMV-specific proliferations were assessed as previously described.42 PBMCs (8 × 106) were resuspended in 8 mL of media and incubated with carboxyfluorescein diacetate succinimidyl ester (Molecular Probes, Eugene, OR) for 5 minutes at a final concentration of 1.5 μM at room temperature. Carboxyfluorescein diacetate succinimidyl ester–labeled cells were washed, resuspended in a sterile 24-well tissue culture plate at 106 PBMCs per well, and incubated at 37°C, 5% CO2 in media, 2 μg/mL staphylococcal enterotoxin-B (Sigma Aldrich Canada, Ontario, Canada), and 0.1 μg/mL per peptide with Gag, Pol, Env, Mix (pool of accessory proteins Rev, Nef, Tat, Vif, Vpr, and Vpu), pp65, and IE-1. On day 5, cells were harvested, washed, and stained with CD3-phycoerythrin, CD4-peridinin-chlorophyll-protein complex, and CD8-allophycocyanin (BD Pharmingen, San Jose, CA). A positive response was defined as proliferation exceeding 0.05% of gated cells and background levels of proliferation by at least 2-fold. Background proliferation was subtracted from antigen-specific proliferation in all reported results.
The primary clinical end point was the change in semen HIV RNA levels from baseline to the completion of valganciclovir treatment at 2 weeks. Secondary clinical end points included (1) change in semen HIV VL from baseline to the week 8 visit (ie, 6 weeks after valganciclovir cessation) and (2) change in semen CMV levels from baseline to week 2 and week 8 visits. SPSS for Windows (Version 18.0) was used for all statistical analyses. Wilcoxon signed rank nonparametric tests were performed for all comparisons within individuals over time, and correlations were determined by Spearman rank test. Virus-specific immune responses were analyzed both as discrete variables, defined as present/absent (see definition, above), and as continuous variables with background responses subtracted. Statistical significance was defined as a P value ≤0.05.
Study Demographics and Clinical Characteristics
Twenty-one chronically HIV-infected men (>6 months) were enrolled into this observational, open-label clinical study (Table 1). Participants remained ART naive for the duration of the study. All participants were men who have sex with men; one individual had also received a blood transfusion in the past. The median age was 42 years (range 22–63 years), and the median duration of HIV infection was 3 years (range 1–18 years). All participants were hepatitis B and C uninfected, and none had a clinical history of anal or genital herpes; 2 subjects (9.5%) were seropositive for HSV-2. Median CD4+ and CD8+ T-cell counts at enrollment were 505 and 1000/mm3, respectively.
All study participants completed the 14-day course of valganciclovir with 100% self-reported compliance. One subject (4.7%) reported diarrhea and headache at baseline. At week 2, self-reported adverse events included diarrhea (9.5%), vomiting (4.7%), difficulty sleeping (4.7%), headache (9.5%), fatigue (4.7%), rash (9.5%), and constipation (4.7%). All symptoms resolved by week 8. Median hemoglobin levels fell from 151 to 145 g/L (P = 0.01) after 2 weeks on treatment and then increased to 148 g/L by week 8.
Impact of Valganciclovir on the HIV and CMV VLs in Blood and Semen
At baseline, 11 of 21 (52%) men had detectable CMV DNA in semen, with a median seminal CMV VL of 4.3 log10 copies per milliliter. All men had detectable HIV RNA in both semen and blood at baseline: median baseline HIV levels were 3.3 log10 copies per milliliter in semen and 3.7 log10 copies per milliliter in blood. The blood HIV VL was inversely related to the peripheral blood CD4 T-cell count (r2 = −0.50, P = 0.021) and was positively correlated with the HIV VL in semen (r2 = 0.71, P < 0.001). After 2 weeks of valganciclovir therapy, both the HIV and CMV VLs decreased significantly in semen (median 3.3–2.65 log10 copies/mL, P = 0.005; median 4.3–0 log10 copies/mL, P = 0.003, respectively; Fig. 1). In addition, the blood HIV VL was significantly lower (median 3.7–3.2 log10 copies/mL, P = 0.001; Fig. 1C). Valganciclovir therapy was then stopped, and 6 weeks later (at week 8), the semen HIV VL increased significantly and did not differ from baseline levels (median 2.92 log10 copies/mL, P = 0.106 vs. baseline); the same pattern was observed for the blood HIV RNA VL (median 3.83 log10 copies/mL, P = 0.106 vs. baseline). However, the reduced semen CMV levels were sustained after therapy cessation (median 0 log10 copies/mL, P = 0.004 vs. baseline), and the number of participants with undetectable semen CMV VL tended to increase over time (from 10 at baseline to 11 at week 2, P = 1.0, and to 14 at week 8, P = 0.12 vs. baseline).
Valganciclovir Therapy and T-Cell Characteristics
We assessed the impact of valganciclovir therapy on T cells in the semen and blood. There was a progressive reduction in the immune activation of semen CD8+ T cells that continued after therapy completion: coexpression of CD38/CD69 was seen on 46.5% of semen CD8+ T cells at baseline, 38.8% at week 2 (P = 0.341 vs. baseline), and 14.3% at week 8 (P = 0.033 vs. baseline; Fig. 2A). No alteration in T-cell immune activation was apparent in the peripheral blood (medians 3.0%, 3.3%, and 2.7%, respectively; all P > 0.3). The frequency of Tregs was unchanged in semen over the course of the study but progressively increased in the peripheral blood from baseline to week 2 (median 0.37% vs. 0.47%, P = 0.042) and to week 8 (median 0.61%, P = 0.007 vs. baseline; Fig. 2B). T-cell immune activation in blood correlated with the median HIV VL in both blood and semen (r2 = 0.57, P = 0.008 and r2 = 0.49, P = 0.029, respectively), but Treg frequency was not associated with HIV or CMV levels in semen or blood (data not shown). Valganciclovir therapy was not associated with changes in the blood CD4+ T-cell count (medians 515, 525, and 521/mm3 at weeks 0, 2, and 6, respectively; all P > 0.5).
Impact of Valganciclovir Therapy on Virus-Specific T-Cell Proliferation
From baseline to week 8, there was a progressive increase in T-cell responses to CMV IE-1 (median CD4+ T-cell proliferation: 0.15% vs. 0.7%, P = 0.002; median CD8+ T-cell proliferation: 0.2% vs. 1.14%, P = 0.006; Fig. 3A) and a trend to increased pp65-specific responses (median CD4+ T-cell proliferation: 0.26% vs. 0.96%, P = 0.064; median CD8+ T-cell proliferation: 0.36% vs. 0.76%, P = 0.087; Fig. 3B). No association was seen between semen CMV VL and CMV-specific T-cell responses.
The impact of valganciclovir therapy on HIV-specific responses was less consistent. HIV-specific CD4+ T-cell proliferation to the pol peptide pool was 2.11% at baseline, 3.63% at week 2 (P = 0.6), and 18.1% at week 8 (P = 0.064 vs. baseline; Fig. 3C), and CD8+ T-cell responses to pol also tended to increase over the course of the study (median 1.91% at baseline, 5.19% at week 2, P = 0.778; 13.9% at week 8, P = 0.055 vs. baseline). No association of pol responses with peripheral blood or semen HIV VL was observed. Env-specific T-cell responses behaved in a similar way: median CD4+ and CD8+ T-cell responses were 1.8% and 2.33% at baseline, 4.22% and 3.07% at week 2 (P = 0.233 and P = 0.256, respectively), and 9.49% and 7.98% at week 8 (P = 0.028 for both vs. baseline; Fig. 3D). Blood HIV VL was inversely correlated with env-specific CD4+ T-cell responses at baseline (r2 = −0.609, P = 0.016) and at week 8 (r2 = −0.620, P = 0.005). However, we observed no effect of valganciclovir therapy on immune responses to HIV gag or accessory proteins and no association of those responses with the HIV VL in blood or semen. Valganciclovir therapy had no effect on the levels of background T-cell proliferation.
Semen Cytokine Levels and HIV VL
Semen concentrations of the cytokines/chemokines IFN-γ, IL-1β, IL-6, IL-8, IL-12p70, MIG, MCP-1, IP-10, RANTES, and TNF-α were evaluated at each study visit. At baseline, levels of IL-8 (r2 = 0.504, P = 0.02), IFN-γ (r2 = 0.515, P = 0.017), and MCP-1 (r2 = 0.603, P = 0.004) were associated with the semen HIV VL. No consistent associations were seen between semen cytokines/chemokines and either semen CMV DNA levels or blood HIV RNA levels (data not shown). The relationship between semen cytokines/chemokines and the semen HIV VL was consistent throughout the study. At week 2, IL-1β (P < 0.001), IL-6 (P = 0.002), IL-8 (P = 0.023), IFN-γ (P < 0.001), and MCP-1 (P = 0.017) were associated with seminal HIV VL; at week 8, a positive association was seen with IL-1β (P < 0.001), IL-6 (P = 0.042), IL-8 (P < 0.001), IFN-γ (P < 0.001), MCP-1 (P < 0.001), and MIG (P = 0.001). However, valganciclovir therapy was not associated with any significant change in semen cytokine/chemokine levels (data not shown).
Compartmentalized genital reactivation of CMV has been associated with increased HIV levels in both the semen and the female genital tract, independent of the blood VL,5,43 and with sexual HIV transmission.17 However, cross-sectional studies cannot determine the direction of causation in the relationship between semen CMV and HIV levels. To address this issue, we performed a prospective, unblinded clinical study on the impact of oral valganciclovir on CMV levels in semen and HIV levels in semen and blood.31,33 Valganciclovir was associated with reduced HIV levels in both the semen and blood, reduced CMV levels in semen, enhanced blood CMV-specific T-cell responses, and reduced semen T-cell immune activation.
Interestingly, the reductions in HIV and CMV VLs seemed to be independent of each other. Specifically, semen and blood levels of HIV only fell while participants were taking valganciclovir and rapidly returned to baseline when treatment was stopped. However, there were much more durable reductions in the semen CMV VL and prolonged reductions in semen T-cell immune activation and enhancement of CMV-specific T-cell proliferation. The rapid return of the semen and blood HIV VLs to baseline upon valganciclovir cessation, despite persistent reductions in the semen CMV VL and in immune parameters, suggests that the impact of valganciclovir on HIV levels was not mediated through reduced seminal CMV levels or secondary immune alterations.
Our findings are analogous to clinical trials demonstrating significant acyclovir/valacyclovir-associated reductions in the HIV VL of participants coinfected by HIV and HSV-2.2,29 Valacyclovir lowered HIV levels in both blood and genital secretions, and this effect was more rapid than would be expected if it were secondary to reduced HSV-2–associated inflammation.44 Although acyclovir directly inhibits HIV-1 reverse transcriptase by inhibiting chain elongation,26 there was no evidence for viral mutational escape despite prolonged, incomplete virus suppression with acyclovir, arguing against a direct drug effect.45 Therefore, the mechanism of the HIV VL reduction in HIV-HSV-2–coinfected individuals remains unclear.46 While the mechanism(s) of HIV interaction with HSV-2 and CMV may be quite distinct, there is a similar mechanism of action for acyclovir (against HSV-2) and ganciclovir (against both HSV-2 and CMV).47 Therefore, a direct antiviral effect of ganciclovir on HIV would be plausible, although to our knowledge such an effect has not previously been demonstrated in vitro.
Whether or not valganciclovir directly mediated the observed reductions in HIV VL, it was also associated with substantial immune alterations in both the blood and semen compartments. In contrast to a recent trial demonstrating valganciclovir-associated reductions in systemic T-cell activation among men on HIV therapy,14 the reductions in CD8+ T-cell activation that we observed were limited to the semen. An increase in blood Treg frequency was apparent, perhaps representing a host response to the enhanced CMV- and HIV-specific T-cell responses that were observed after valganciclovir therapy. This valganciclovir-associated enhancement of CMV-specific T-cell responses in blood might also explain the relatively prolonged posttreatment reduction in semen CMV VL. The effects of valganciclovir on HIV-specific responses were less consistent. We saw no impact of therapy on Gag-specific responses, which have been associated with enhanced host HIV immune control.48 Env-specific responses were enhanced at week 6, perhaps in response to the HIV VL increase, but these have been associated with poor host outcomes.48 Semen proinflammatory cytokines were consistently associated with the semen HIV VL, confirming previous findings,39 but were unaltered by valganciclovir therapy. This again suggests that the reduction in HIV semen levels was a direct effect of valganciclovir, although the strength of this conclusion is tempered by the inherent variability in semen cytokine measurements.
Our study does have limitations. It is possible that valganciclovir may have induced immune and HIV VL changes through an effect on other herpesviruses. Tissue reactivation of HHV-6 is associated with the increased HIV levels,49 and the semen HIV VL has been correlated with the reactivation of herpesviruses HHV-8, Epstein-Barr virus, and CMV.7 Valganciclovir has antiviral effects on HHV-6 in vitro50 and reduced oral shedding of HHV-851 and Epstein-Barr virus.14 Therefore, it remains possible that the effects we observed were related to suppression of an additional unmeasured herpesvirus, although this would not include HSV-2: exclusion of participants with previous genital/anal herpes meant that the seroprevalence of this infection was lower than expected19,52,53 and an impact of valganciclovir on HSV-2 does not explain our results. The sample size was relatively small, and the duration of therapy was shorter than a previous trial in ART-treated participants.14 Although the distinct treatment effects that we observed on immunology and HIV VL suggest that this was not a major barrier, it is possible that longer therapy and/or a larger sample size would provide additional important results. Finally, we did not assay CMV DNA in blood because the low expected frequency of CMV detection54 meant that we were not powered to assess of the impact of valganciclovir on blood CMV levels.
In summary, CMV infection is a nearly ubiquitous in HIV-infected men who have sex with men.55 Genital CMV reactivation is common and has been associated with increased HIV levels in the genital tract,5,43 suggesting that it may play a role in HIV sexual transmission. Despite a relatively small sample size, our observational clinical study demonstrated a clear, substantial effect of valganciclovir in reducing both HIV and CMV levels in semen and blood. Interestingly, although valganciclovir treatment lowered mucosal immune activation and enhanced CMV-specific T-cell responses, the timing of these changes suggests that the reduction in HIV blood and semen VLs may have been mediated by a direct effect of valganciclovir on HIV. This study in ART-naive participants provides interesting insights into the interaction between HIV and CMV. However, the current standard of care is to start ART as soon as is practically feasible, and our results do not justify the deferral of ART in favor of therapy against herpesviruses.
The authors thank all the study participants for their time and dedication to this study and the staff at Maple Leaf Medical Clinic in Toronto, Canada, for their research support. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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