Longitudinal Viral Dynamics in Semen During Early HIV Infection.

Background: Multiple viruses coinfect the male genital tract, influencing each other's replication and perhaps affecting human immunodeficiency virus (HIV) pathogenesis and disease progression. Methods: This study included 453 longitudinal seminal samples from 195 HIV-infected men from the San Diego Primary Infection Resource Consortium and 67 seminal samples from HIV-negative healthy controls. Seminal HIV RNA and DNA from 7 human herpesviruses (HHVs) were measured by real-time polymerase chain reaction. Longitudinal shedding rates were determined by Kaplan-Meier survival analysis. Predictors of viral shedding were determined using backwards selection in a multivariable generalized estimating equation model. Results: HIV-infected participants presented significantly increased rates of seminal HHV shedding compared with HIV-uninfected controls. Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) were the most commonly detected HHV in semen of HIV-infected participants. Persistent shedding was more common for CMV and EBV when compared to other HHVs. With exception of HHV-7, HHV shedding was not significantly influenced by HIV RNA levels, CD4+ cell counts, or antiretroviral therapy. Presence of CMV, EBV, and herpes simplex virus (HSV) were independent predictors of genital HIV RNA shedding after adjusting for plasma HIV RNA and longitudinal measurements. Conclusions: Seminal replication of multiple HHVs is common in our HIV primary infection cohort. Genital replication of CMV and EBV was the most common and was significantly associated with seminal HIV RNA shedding. Prevalence of HSV shedding was lower and mostly intermittent, but its association with seminal HIV RNA was the strongest. Understanding the complex viral milieu in semen is important for HIV transmission but might also play a role in HIV pathogenesis and disease progression.

Replication of Human Herpesviruses Is Associated with Higher HIV DNA Levels during Antiretroviral Therapy Started at Early Phases of HIV Infection.

UNLABELLED: Asymptomatic replication of human herpesviruses (HHV) is frequent in HIV-infected men and is associated with increased T-cell activation and HIV disease progression. We hypothesized that the presence of replication of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) (the most frequently detected HHV) might influence HIV DNA decay during antiretroviral therapy (ART). We investigated 607 peripheral blood mononuclear cell (PBMC) samples from 107 CMV-seropositive, HIV-infected men who have sex with men, who started ART within a median of 3 months from their estimated date of infection (EDI) and were monitored for a median of 19 months thereafter. Levels of HIV, CMV, and EBV DNA and cellular HIV RNA were measured by droplet digital PCR (ddPCR) for each time point. Using a general linear mixed-effect regression model, we evaluated associations between the presence of detectable CMV DNA and EBV DNA levels and HIV DNA decay and cellular HIV RNA levels, while adjusting for peak HIV RNA, nadir CD4(+)count, CD4/CD8 ratio, CMV IgG levels, time from EDI to ART initiation, time from ART initiation to virologic suppression, detectable CMV DNA pre-ART, and age. The presence of intermittent CMV DNA in PBMC during ART was significantly associated with slower decay of HIV DNA (P= 0.011) but not with increased cellular HIV RNA transcription or more detectable 2-long terminal repeat circles. Higher levels of EBV DNA were also associated with higher levels of HIV DNA (P< 0.001) and increased unspliced cellular HIV RNA transcription (P= 0.010). These observations suggest that replication of HHV may help maintain a larger HIV DNA reservoir, but the underlying mechanisms remain unclear. IMPORTANCE: Over three-fourths of HIV-infected men have at least one actively replicating human herpesvirus (HHV) in their mucosal secretions at any one time. Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are the most common, and although it is often asymptomatic, such CMV and EBV replication is associated with higher levels of immune activation and HIV disease progression. We hypothesized that HHV-associated activation of HIV-infected CD4(+)T cells might lead to increased HIV DNA. This study found that detectable CMV in blood cells of HIV-infected men was associated with slower decay of HIV DNA even during antiretroviral therapy (ART) that was started during early HIV infection. Similarly, levels of EBV DNA were associated with higher levels of HIV DNA during ART. If this observation points to a causal pathway, interventions that control CMV and EBV replication may be able to reduce the HIV reservoir, which might be relevant to current HIV cure efforts.

Cytomegalovirus replication in semen is associated with higher levels of proviral HIV DNA and CD4+ T cell activation during antiretroviral treatment.

Asymptomatic cytomegalovirus (CMV) replication occurs frequently in the genital tract in untreated HIV-infected men and is associated with increased immune activation and HIV disease progression. To determine the connections between CMV-associated immune activation and the size of the viral reservoir, we evaluated the interactions between (i) asymptomatic seminal CMV replication, (ii) levels of T cell activation and proliferation in blood, and (iii) the size and transcriptional activity of the HIV DNA reservoir in blood from 53 HIV-infected men on long-term antiretroviral therapy (ART) with suppressed HIV RNA in blood plasma. We found that asymptomatic CMV shedding in semen was associated with significantly higher levels of proliferating and activated CD4(+) T cells in blood (P < 0.01). Subjects with detectable CMV in semen had approximately five times higher average levels of HIV DNA in blood CD4(+) T cells than subjects with no CMV. There was also a trend for CMV shedders to have increased cellular (multiply spliced) HIV RNA transcription (P = 0.068) compared to participants without CMV, but it is unclear if this transcription pattern is associated with residual HIV replication. In multivariate analysis, the presence of seminal plasma CMV (P = 0.04), detectable 2-long terminal repeat (2-LTR), and lower nadir CD4(+) (P < 0.01) were independent predictors of higher levels of proviral HIV DNA in blood. Interventions aimed at reducing seminal CMV and associated immune activation may be important for HIV curative strategies. Future studies of anti-CMV therapeutics will help to establish causality and determine the mechanisms underlying these described associations. Importance: Almost all individuals infected with HIV are also infected with cytomegalovirus (CMV), and the replication dynamics of the two viruses likely influence each other. This study investigated interactions between asymptomatic CMV replication within the male genital tract, levels of inflammation in blood, and the size of the HIV DNA reservoir in 53 HIV-infected men on long-term antiretroviral therapy (ART) with suppressed HIV RNA in blood plasma. In support of our primary hypothesis, shedding of CMV DNA in semen was associated with increased activation and proliferation of T cells in blood and also significantly higher levels of HIV DNA in blood cells. These results suggest that CMV reactivation might play a role in the maintenance of the HIV DNA reservoir during suppressive ART and that it could be a target of pharmacologic intervention in future studies.

Virologic correlates of anti-CMV IgG levels in HIV-1-infected men.

In human immunodeficiency virus (HIV)-infected individuals, higher levels of anti-cytomegalovirus (CMV) immunoglobulin G (IgG) antibody have been associated with increased immune activation, increased HIV transmission, cardiovascular complications, and neurocognitive impairment. However, the mechanism of these observations is unknown. This analysis of 228 HIV-infected men found that higher CMV IgG levels were positively associated with older age and antiretroviral treatment. Higher frequency of detectable CMV in peripheral blood mononuclear cells and recurrent seminal CMV reactivations were associated with lower plasma CMV IgG levels, suggesting that immune response to CMV rather than direct effect of viral replication is likely responsible for adverse clinical outcome observed in other studies.

Role of seminal shedding of herpesviruses in HIV Type 1 Transmission.

To investigate the role of genital shedding of herpesviruses in human immunodeficiency virus type 1 (HIV) transmission, we compared 20 HIV-infected men who did and 26 who did not transmit HIV to their sex partners. As described previously, HIV transmission was associated with the potential source partner having higher levels of HIV RNA in blood and semen, having lower CD4(+) T cell counts, having bacterial coinfections in the genital tract, and not using antiretroviral therapy. This study extended these findings by observing significant associations between HIV transmission and the following characteristics, especially among therapy-naive potential source partners: seminal cytomegalovirus (CMV) shedding, seminal Epstein-Barr virus shedding, and levels of anti CMV immunoglobulin in blood plasma.

Cytomegalovirus DNA in semen and blood is associated with higher levels of proviral HIV DNA.

Over three-fourths of human immunodeficiency virus (HIV)-infected men who have sex with men (MSM) have at least one herpesvirus detected in their semen, and cytomegalovirus (CMV) is the most prevalent. The presence of CMV is associated with higher T-cell immune activation and with HIV disease progression in treated and untreated individuals. In this study of 113 antiretroviral (ART)-naive HIV-infected MSM, we found that CMV replication in blood and semen was associated with higher levels of HIV DNA in peripheral blood mononuclear cells. These observations suggest that interventions aimed to reduce CMV replication and, thus, systemic immune activation could decrease the size of the latent HIV reservoir.

Shedding of HIV and human herpesviruses in the semen of effectively treated HIV-1-infected men who have sex with men.

BACKGROUND: Current antiretroviral therapy (ART) suppresses human immunodeficiency virus (HIV) in blood to undetectable levels in most infected individuals; however, some men shed HIV in semen despite suppressed levels in blood. METHODS: This study included 114 chronically HIV type 1-infected men who have sex with men, who were receiving ART with blood plasma HIV <500 copies/mL. Asymptomatic participants were screened for bacterial sexually transmitted infections (STIs) and nonspecific genital inflammation. Levels of HIV and 7 human herpesviruses were measured by real-time polymerase chain reaction in seminal plasma. Predictors of HIV seminal shedding were determined for the entire cohort, and on the subset of 100 subjects with blood plasma HIV <50 copies/mL. RESULTS: Eleven subjects (9.6%) had detectable levels of seminal HIV (median, 2.1 log10 copies/mL), and 72 (63.2%) had at least 1 herpesvirus detected in their seminal plasma. Detectable levels of seminal HIV were present more often in persons with plasma HIV between 50 and 500 copies/mL compared to those <50 copies/mL (P values adjusted for false discovery rate [FDR] = 0.08). There was a trend for high-level cytomegalovirus (CMV; >4 log10 DNA copies/mL; FDR-adjusted P = .08), and presence of Epstein-Barr virus (FDR-adjusted P = .06) in semen to be associated with detectable seminal HIV levels. In a subanalysis of 100 subjects with blood plasma HIV <50 copies/mL, high levels of CMV in semen was the only significant predictor for seminal HIV shedding. CONCLUSIONS: Low-level HIV replication in blood and high-level seminal CMV shedding, but not presence of asymptomatic STIs, is associated with seminal shedding of HIV in men receiving ART, conferring a potential risk for HIV transmission.

Associations between virologic and immunologic dynamics in blood and in the male genital tract.

To determine the influence of asymptomatic genital viral infections on the cellular components of semen and blood, we evaluated the associations between the numbers and activation statuses of CD4+ and CD8+ T lymphocytes in both compartments and the seminal levels of cytomegalovirus (CMV), herpes simplex virus (HSV), and human immunodeficiency virus 1 (HIV). Paired blood and semen samples were collected from 36 HIV-infected antiretroviral-naïve individuals and from 40 HIV-uninfected participants. We performed multiparameter flow cytometry analysis (CD45, CD45RA, CD3, CD4, CD8, and CD38) of seminal and blood cellular components and measured HIV RNA and CMV and HSV DNA levels in seminal and blood plasma by real-time PCR. Compared to HIV-uninfected participants, in the seminal compartment HIV-infected participants had higher levels of CMV (P < 0.05), higher numbers of total CD3+ (P < 0.01) and CD8+ subset (P < 0.01) T lymphocytes, and higher CD4+ and CD8+ T lymphocyte activation (RA-CD38+) (P < 0.01). Seminal CMV levels positively correlated with absolute numbers of CD4+ and CD8+ T cells in semen (P < 0.05) and with the activation status of CD4+ T cells in semen and in blood (P < 0.01). HIV levels in semen (P < 0.05) and blood (P < 0.01) were positively associated with T-cell activation in blood. Activation of CD8+ T cells in blood remained an independent predictor of HIV levels in semen in multivariate analysis. The virologic milieu in the male genital tract strongly influences the recruitment and activation of immune cells in semen and may also modulate T-cell immune activation in blood. These factors likely influence replication dynamics, sexual transmission risk, and disease outcomes for all three viruses.

Impact of seminal cytomegalovirus replication on HIV-1 dynamics between blood and semen.

The genital tract of individuals infected with human immunodeficiency virus type 1 (HIV-1) is an anatomic compartment that supports local HIV-1 and cytomegalovirus (CMV) replication. This study investigated the association of seminal CMV replication with changes in HIV-1 clonal expansion, evolution and phylogenetic compartmentalization between blood and semen. Fourteen paired blood and semen samples were analyzed from four untreated subjects. Clonal sequences (n = 607) were generated from extracted HIV-1 RNA (env C2-V3 region), and HIV-1 and CMV levels were measured in the seminal plasma by real-time PCR. Sequence alignments were evaluated for: (i) viral compartmentalization between semen and blood samples using Slatkin-Maddison and F(ST) methods, (ii) different nucleotide substitution rates in semen and blood, and (iii) association between proportions of clonal HIV-1 sequences in each compartment and seminal CMV levels. Half of the semen samples had detectable CMV DNA, with at least one CMV positive sample for each patient. Seminal CMV DNA levels correlated positively with seminal HIV-1 RNA levels (Spearman P = 0.05). A trend towards an association between compartmentalization of HIV-1 sequences sampled from blood and semen and presence of seminal CMV was observed (Cochran Q test P = 0.12). Evolutionary rates between semen and blood HIV-1 populations did not differ significantly, and there was no significant association between seminal CMV DNA levels and the frequency of non-unique clonal HIV-1 sequences in the semen. In conclusion, the effects of CMV replication on HIV-1 viral and immunologic dynamics within the male genital tract are not significant enough to perturb evolution or disrupt compartmentalization in the genital tract.

Herpes viruses and HIV-1 drug resistance mutations influence the virologic and immunologic milieu of the male genital tract.

OBJECTIVES: To further understand the role that chronic viral infections of the male genital tract play on HIV-1 dynamics and replication. DESIGN: Retrospective, observational study including 236 paired semen and blood samples collected from 115 recently HIV-1 infected antiretroviral naive men who have sex with men. METHODS: In this study, we evaluated the association of seminal HIV-1 shedding to coinfections with seven herpes viruses, blood plasma HIV-1 RNA levels, CD4 T-cell counts, presence of transmitted drug resistance mutations (DRMs) in HIV-1 pol, participants' age and stage of HIV-infection using multivariate generalized estimating equation methods. Associations between herpes virus shedding, seminal HIV-1 levels, number and immune activation of seminal T-cells was also investigated (Mann-Whitney). RESULTS: Seminal herpes virus shedding was observed in 75.7% of individuals. Blood HIV-1 RNA levels (P < 0.01) and seminal cytomegalovirus (CMV) and human herpes virus (HHV)-8 levels (P < 0.05) were independent predictors of detectable seminal HIV-1 RNA; higher seminal HIV-1 levels were associated with CMV and Epstein-Barr virus (EBV) seminal shedding, and absence of DRM (P < 0.05). CMV and EBV seminal shedding was associated with higher number of seminal T-lymphocytes, but only presence of seminal CMV DNA was associated with increased immune activation of T-lymphocytes in semen and blood. CONCLUSION: Despite high median CD4 T-cells numbers, we found a high frequency of herpes viruses seminal shedding in our cohort. Shedding of CMV, EBV and HHV-8 and absence of DRM were associated with increased frequency of HIV-1 shedding and/or higher levels of HIV-1 RNA in semen, which are likely important cofactors for HIV-1 transmission.

Sexual transmission of predicted CXCR4-tropic HIV-1 likely originating from the source partner's seminal cells.

We present a case of sexual transmission of HIV-1 predicted to have CXCR4-tropism during male-to-male sexual exposure. Phylogenetic analyses exclude cell-free virus in the seminal plasma of the source partner and possibly point to the seminal cells as the origin of the transmission event.