Replication of CMV, EBV and KSHV persists in many antiretroviral-suppressed, HIV-infected patients to a greater degree than in HIV-uninfected adults 
. T cell activation is also consistently higher in antiretroviral-suppressed patients compared to HIV negatives, and when present is often associated with suboptimal gains in absolute CD4+ T cells. To explore the possible causal role of persistent herpesvirus replication on immune activation and immune reconstitution during suppressive antiretroviral therapy, we selected two distinct groups of patients: those with relatively normal CD8+ T cell activation/high absolute CD4+ T cell counts and those with high CD8+ T cell activation/low absolute CD4+ T cell counts. Using very sensitive measures of herpesvirus persistence, we found no consistent association between the level of viral DNA and immune activation. We also found no evidence suggesting that residual viral replication during suppressive antiretroviral therapy was causally associated with either high T cell activation. The proportions of all CMV, EBV or KSHV DNA positive specimens between the cases and controls were essentially the same, as were the proportions of subjects in each group that intermittently or continuously shed CMV, EBV or KSHV DNA in saliva. In addition, the median number of copies of CMV, EBV and KSHV DNA longitudinally in cases was virtually identical to that in controls.
Miller, et al, previously reported results using a similar PCR assay to detect CMV, EBV and KSHV in similar unstimulated whole saliva specimens from 58 HIV-infected patients, most of whom had HIV replication suppressed by antiretroviral therapy 
. In that study, 31%, 57% and 90% of saliva samples were positive for CMV, KSHV and EBV DNA, respectively, compared to 37%, 26% and 71% in our study. Perhaps, the lower number of positive KSHV and EBV specimens we observed was a result of our including only patients whose HIV replication had been completely suppressed below the limit of plasma detectability by commercial assay for at least one year.
Similar to the results from Miller et al, we noticed that median CMV levels were lower than median KSHV levels, which in turn were lower than median EBV levels (). This suggests that even though herpesvirus shedding is often considered a categorical event (i.e., a person either has undetectable viral load or is actively shedding virus), the amount of virus that can be transmitted orally by a person experiencing viral reactivation differs considerably for the different herpesviruses.
This pilot study has several important limitations. First and foremost, the sample size was small, and thus the potential Type II error is likely substantial. However, we avoided confounding factors by requiring prolonged HIV RNA undetectability in plasma for study eligibility and excluding patients with chronic HCV infection. Second, we selected our cases and controls based on more than one factor (i.e., the groups differed based on both CD8+ T cell activation and absolute CD4+ T cell counts). This study design would have been particularly problematic had we observed differences between the two groups. Since it is unlikely that low absolute CD4+ T cell counts may have blunted the impact of herpes viruses on immune activation, it is also unlikely that the negative results observed in this study were due to our study design. Third, we only measured human herpesvirus replication in two reservoirs, saliva and PBMC. Assaying specimens from other reservoirs (e.g., genital secretions, urine, plasma) may have demonstrated a difference between the two groups. In addition, the saliva and PBMC specimens we assayed were obtained infrequently (only once every four months). Sampling more frequently, for example by daily oral swabs 
, might reveal differences between these two groups of patients in the frequency and duration of herpesvirus replication. Since it is impractical to assay all potential reservoirs of human herpesvirus daily, an alternative method to determine the role of human herpesvirus replication in driving abnormal T cell activation in HIV-infected patients would be to administer therapy with an agent with broad anti-herpesvirus activity, such as valganciclovir, and then measure the effect on T cell activation and absolute CD4+ T cell count in a randomized, controlled manner. We are currently performing such a trial.
Given the strong association between inflammation/immune activation and disease outcomes in treated HIV disease, defining the mechanisms associated with HIV-associated inflammation is of high importance. Our preliminary data do not provide strong evidence for a role of persistent high level herpesvirus replication.
Other possible mechanisms includes the following:
- Persistent microbial translocation. It is now well-appreciated that HIV causes irreversible damage to the gut mucosal integrity early in the course of disease, leading to translocation of gut microbial products into the system circulation . Circulating lipopolysaccharide and bacterial DNA have each been reported to correlate with measures of T cell activation in HIV-infected patients , .
- Residual HIV viremia. Another factor leading to persistent abnormal immune activation in antiretroviral-suppressed patients could be persistent low level replication of HIV. Several studies have reported that by using an ultra-ultrasensitive HIV RNA PCR assay, low levels of plasma HIV viremia (below the detectability of currently available commercial HIV RNA assays) can be detected in the majority of HIV-infected patients successfully treated with antiretroviral therapy –. Results of these studies indicate that the level of this residual viremia remains stable for at least up to seven years , . Of note, the duration of suppressive antiretroviral therapy in the controls we studied tended to be longer than in the cases, which could be consistent with the controls having a lower level of HIV replication than the cases.
- Persistant immune dysregulation. Finally, the damage to the immune system done by uncontrolled HIV replication before antiretroviral therapy is initiated, for example in altering T cell diversity in adults with limited thymic reserve for repopulating diverse naïve T cells  or reducing the number of regulatory T cells –, could result in persistent T cell dysregulation that is antigen-independent.
In summary, future studies to examine the effect of suppressing CMV, EBV and KSHV with drugs such as valganciclovir 
may better clarify the role of persistent human herpesvirus replication in driving abnormal T cell activation and resultant incomplete immune reconstitution in patients with HIV infection. Furthermore, observational studies that measure HIV RNA by ultra-ultrasensitive assays 
, circulating lipopolysaccharide and/or bacterial DNA, and measurements of regulatory T cells or T cell superfamily representation (a method to characterize T cell diversity) and correlate these parameters with measurements of T cell activation and absolute CD4+ T cell counts in cohorts of long-term, antiretroviral suppressed patients could help elucidate the etiologic contributions of microbial translocation, low level HIV replication, and persistent T cell dysregulation to this pathologic process.