A. Seminal cytokines in male-to-female HIV-1 transmission The majority of HIV-1 infections in women occur through vaginal intercourse, in which virus-containing semen is deposited on the cervico-vaginal mucosa. Semen is more than a mere carrier of HIV-1, since it contains many biological factors, in particular cytokines that may affect HIV-1 transmission. The cytokine interleukin 7 (IL-7) is present in normal semen, and is further upregulated in the semen of HIV-infected men. To investigate the potential role of IL-7 in HIV-1 vaginal transmission we used a system of cervico-vaginal tissue ex vivo to simulate male-to-female transmission of HIV under controlled conditions. In particular, we deposited HIV-1 on cervico-vaginal tissue in combination with IL-7 at concentrations comparable with those measured in semen of HIV-1-infected individuals. We found that IL-7 significantly enhanced virus replication in ex vivo infected cervico-vaginal tissue from all the donors tested in this study, including one tissue donor in which there was no replication in the HIV-1 untreated tissue condition. This enhancement became detectable on day 6 postinfection and seems to be independent of the absolute level of viral replication, as it was also observed in tissues infected with a viral inoculum diluted 100 fold. The enhancement of HIV-1 replication by IL-7 was not restricted to laboratory strains but was also observed for primary isolates of all three tested clades. Analysis of T cells isolated from infected tissues showed that IL-7 reduced CD4+ T cell depletion preventing apoptosis, as shown by the decrease in the number of cells expressing the apoptotic marker APO2.7 and by the increase in the expression of the anti-apoptotic protein B-cell lymphoma (Bcl)-2. Also, IL-7 increased the fraction of cycling CD4+ T cells, as evidenced by staining for the nuclear factor Ki-67. High levels of seminal IL-7 in vivo may be relevant to the survival of the founder pool of HIV-1-infected cells in the cervico-vaginal mucosa at the initial stage of infection, promoting local expansion and dissemination of HIV infection. These findings raise the possibility that IL-7, alone or in combination with other molecules, can foster HIV male-to-female transmission and that the level of IL-7 in semen may determine how infectious a particular HIV-positive male is for a female sexual partner. Also, it is possible to use IL-7 as a target to prevent HIV transmission. B. Benchmarking of the system of cervico-vaginal tissue ex vivo and testing of new antivirals. In order to reach its primary cell targets in the female lower genital tract in the course of HIV-1 transmission the virus must circumvent mucosal barriers. These mucosal barrier components as well as the state of activation of HIV-1 target cells are strongly influenced by the menstrual cycle that is under the control of sexual hormones. In order to study this influence and to further benchmark the system of cervico-vaginal tissue ex vivo we studied in this system HIV-1 transmission as a function of the menstrual cycle. Cervical tissue explants from 22 HIV-1 seronegative women were exposed to R5 HIV-1 ex vivo. Eight tissue were productively infected in terms of HIV-1 p24Gag release in culture supernatants, whereas 14 were not. Nonetheless, both accumulation of HIV-1gag DNA and of p24Gag+ CD4+ T cells and macrophages occurred in both productive and, at lower levels, in nonproductive cervico-vaginal tissue. Nonproductive tissues differed from productive ones by a higher secretion of C-C motif chemokine ligand 3 (CCL3) and CCL5. A post-hoc analysis revealed that all productive cervico-vaginal tissues were established from women in their secretory phase of the menstrual cycle, whereas nonproductive tisues were derived from women either in their secretory (28%) or proliferative (36%) menstrual cycle phases or with an atrophic endometrium (36%). This demonstrates an association between the capacity of the ex vivo cervical tissue to support productive HIV-1 infection and the menstrual cycle phase of the donor. Our study also demonstrates that the system of cervico-vaginal tissue ex vivo faithfully reflects in vivo observation on the dependency of HIV transmission on the menstrual cycle and thus represents an adequate experimental system to study the mechanisms underlying the different susceptibility to HIV-1 infection in different stages of the menstrual cycle as well as other aspects of HIV infection. In particular, the system is now used as a platform to test newly-developed dual-targeted antivirals against HIV and herpesviruses. Indeed, HIV infection is often accompanied by infection with other pathogens, in particular herpes simplex virus type 2 (HSV-2) resulting in a vicious circle of mutual facilitations. Therefore, an important task is to develop a compound that is highly potent against both viruses to suppress their transmission and replication. We developed such a compound, an acyclic nucleoside phosphonate designated PMEO-DAPym. We compared its properties with those of the structurally related and clinically used acyclic nucleoside phosphonates tenofovir and adefovir. We demonstrated the potent anti-HIV and -HSV activity of this drug. PMEO-DAPym markedly inhibits both HIV-1 reverse transcriptase (RT) and HSV DNA polymerase. We did not limit testing this compound in cervico-vaginal tissue ex vivo, but also confirmed its anti HIV/HHV activity in several other experimental systems. In conclusion, we defined a distinct new subclass of acyclic nucleoside phosphonate, structurally and functionally different from the earlier developed and widely used tenofovir and adefovir, that has significant advantage over the commonly used drugs. C. Flow Virometry About 50 years ago, flow cytometry revolutionized immunology in particular and medicine in general, by making possible the distinction of individual cells by their antigenic spectra and therefore gaining unprecedented new insights into the mechanisms of immunity, including responses to infection by viruses.Unlike cells, viruses themselves are still characterized predominantly in bulk, in spite of the strong indications that viral particles may be as individualized as lymphocytes for example. We developed a new technology, flow virometry, which by using magnetic nanoparticles allows antigen detection on individual virions. The technology consists of binding magnetic nanoparticles to virions, staining virions with monoclonal antibodies, separating the formed complexes with magnetic columns and characterizing them with flow cytometers. With this technology we studied the distribution of two antigens (HLA-DR and LFA-1 that HIV-1 acquires from infected cells) on individual HIV-1virion. Unlike techniques reported earlier, flow virometry allows not only the demonstration of the presence of these antigens on viruses but, importantly, the analysis of the distribution of individual viral particles according to their antigen spectra. Our results revealed in the antigenic makeup of virions from a single preparation, a high antigenic heterogeneity, which cannot be detected with bulk analysis of viruses, the only method currently available. Moreover, in two preparations of the same HIV-1, but produced by different cells, the distribution of antigens among virions was different. In contrast, HIV-1 of two different HIV-1 genotypes replicating in the same cells became somewhat antigenically similar. The new nanotechnology is not restricted to the analysis of HIV but can be applied to the analysis of the individual antigenic makeup of any virus and may give new insights into basic mechanisms of viral infection leading to the development of new more precisely targeted antivirals.

Project Start
Project End
Budget Start
Budget End
Support Year
21
Fiscal Year
2013
Total Cost
$1,911,354
Indirect Cost
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State
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