A. Human immunodeficiency virus and locally shed herpesviruses reprogram the seminal cytokine network. HIV sexual transmission from males to their partners occurs through various complex stages (gatekeepers) that control which HIV-1 variant is transmitted to start the infection. During the last year we focused on one of the earliest of these mechanisms that operate in semen of HIV-1 infected men. Semen is not only a mere passive transporter of virions but also plays an active role in HIV-1 transmission through cytokines and other biological factors. We addressed the relationship between the cytokine milieu and coinfecting viruses in the male genital tract of HIV-1-infected individuals by comparing the levels of 21 cytokines/chemokines as well as the loads of HIV-1 and six coinfecting herpesviruses in seminal and blood plasmas from HIV-1-infected and uninfected men. Semen plasma and blood were collected from 50 HIV-1-infected and thirty-three HIV-uninfected healthy volunteers enrolled at the All-India Institute of Medical Sciences in Delhi, India. We found that semen and blood are two separate immunological compartments, in which concentrations of cytokines and loads of coinfecting herpesviruses are profoundly different. In particular, we found that semen is enriched in IL-1 alpha, IL-7, IL-8, MIP-3 alpha, MCP-1, MIG, IP-10, SDF-1 alpha, and TGF-beta, while blood is enriched in IL-2, IL-16, MIP-1 beta, and eotaxin. Upon HIV infection, the levels of blood and semen cytokines were significantly altered, thus affecting the compartmentalization of the semen and blood cytokine networks. HIV-1 infection changes the seminal cytokine spectrum by upregulating 16 out of the 21 measured cytokines while in blood two cytokines were downregulated and three were upregulated. As a result, HIV-1 infection emphasizes the cytokine compartmentalization between blood and semen. Since the cytokine network and its modulation in semen is different from that in blood, the upregulated seminal cytokines seem to be produced locally in the male genital tract. Thus, the changes in cytokine levels upon HIV-1 infection may reflect a profound dysregulation of the functional state of immune cells resident in the male genital tract. The seminal cytokine network already altered by HIV-1 infection is further altered by herpesviruses reactivated in the male genital tract. Our data indicate that such reactivation may occur locally, since approximately 75% and 54% of the patients, respectively, had CMV and EBV seminal shedding in the absence of blood plasma viremia. Moreover, the median load of CMV in seminal plasma was more than 100 times higher than in blood plasma. Our work indicates that the source of the EBV and CMV seminal shedding is not a systemic reactivation of the infection accompanied by a spill-over of virus from the blood, but rather their specific and compartmentalized reactivation in the male genital tract, further indicating that these two compartments are immunologically different. The seminal cytokine spectra altered by local infection with HIV-1, CMV, EBV, and probably other viruses may favor selection of particular HIV-1 variants. Coinfecting viruses and cytokines present in semen may alter not only HIV-1 replication and evolution in the male genital tract but also the immunological landscape of the female genital tract when delivered with the ejaculate. These alterations modify the recruitment and activation status of immune cells therein and determine the efficiency of male-to-female HIV transmission and should, thus, be considered as another target in HIV-1 transmission prevention strategies. B. Dual-targeted antivirals: topical tenofovir as dual-targeted anti-human immunodeficiency virus and anti-herpesvirus microbicide. Various new compounds have been tested over the last few years as vaginal microbicides. Recently, instead of testing new compounds as potential microbicides, tenofovir, an efficient nucleotide HIV reverse transcriptase (RT) inhibitor widely used in HIV therapy, was formulated as a 1% gel and became the first example of a microbicide convincingly diminishing HIV-1 transmission. Surprisingly, a significant reduction of the risk of acquisition of herpes simplex virus type 2 (HSV-2), a common HIV-1 copathogen, which facilitates HIV transmission, was also observed. This is important since herpesviruses facilitate transmission of HIV-1 and adversely affect the clinical course of HIV disease. The effect of tenofovir gel on HSV in vivo was unanticipated, since tenofovir was not known to exhibit anti-HSV activity. Now, we report on the resolution of this apparent contradiction. We provide compelling evidence that at the concentrations achieved intravaginally by the topical administration of a 1% gel, tenofovir exhibits a direct anti-herpetic activity. The effect of tenofovir on the replication of both laboratory and clinical HSV-1 and HSV-2 isolates was investigated in infected human tissues ex vivo. Tenofovir suppressed replication of HSV-1F, HSV-2G and HSV-2MS in a dose-dependent manner. At a concentration of 66 mcg/ml tenofovir reduced HSV-1F, HSV-2G and HSV-2MS replication by 99+/-0.1%, 87+/-12% and 91.7+/-3.2%, respectively, compared to infected donor matched-untreated tissue. Tenofovir inhibited replication of HSV in HIV-1 coinfected tissues as well. Tenofovir was not found to be toxic based on the lack of depletion of tonsillar cell subsets and on the unaffected release of 15 cytokines. The antiherpetic effect of tenofovir is not a general property of nucleoside reverse transcriptase inhibitors since there was no effect of lamivudine (3TC) on HSV replication. Furthermore, we deciphered the molecular mechanism of the anti-herpetic activity of tenofovir: tenofovir diphosphate, to which tenofovir is converted in various human cell types, efficiently inhibits HSV DNA polymerase. Thus, the antiretroviral drug tenofovir is endowed with a direct antiherpetic activity at drug concentrations that are comparable with those that are achieved in cervicovaginal fluid following the administration of 1% tenofovir gel. We hypothesize that the discrepancy between the earlier reported lack of significant antiherpetic activity and the recent clinical data is explained by the striking differences in drug concentrations between systemic and topical applications of tenofovir. Our findings provide a direct explanation for the dual anti-HIV/HSV activity reported in vivo. In conclusion, tenofovir, which was designed to suppress HIV-1 rather than herpesviruses, at the high concentrations that are achieved with topical application suppress not only HIV-1 but HSV as well. Moreover, tenofovir affects HSV directly rather than through complex indirect mechanisms. Antiviral activities of a variety of existing drugs should be revisited as potential microbicides with dual or multiple antiviral properties. These data and the therapeutic principles emerging from our study are important for the development of new drug formulations and administration protocols to design and/or optimize future microbicide trials.
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