A. Anti-herpetic drug acyclovir as an anti-HIV drug Acyclovir (ACV) is a highly specific antiherpetic drug, currently widely used in particular against HSV-2 infection. HSV-2 is among the most common co-pathogens in HIV-1 infected persons and ACV is commonly used in HIV-1/HSV-2 coinfected patients to treat symptomatic HSV-2 infection. HSV-2 suppressive therapy using ACV (or its prodrug valacyclovir, valACV) reduced plasma HIV-1 RNA concentration by 0.25 to 1.23 logs in HIV-1/HSV-2 coinfected persons and delayed HIV-1 disease progressions. Since ACV has always been believed to be inactive against HIV-1, these results were explained by a decrease in generalized inflammation due to the suppression of HSV-2. Surprisingly, we found that in ex vivo human tissues, ACV suppresses HIV-1 by directly inhibiting HIV-1 reverse transcriptase provided that ACV is phosphorylated by thymidine kinase of HHVs present in these tissues. We hypothesized that if a similar mechanism occurs in vivo, the suppressive effect of ACV on HIV-1 replication should not be limited to HSV-2 coinfected individuals. Here, we tested this hypothesis in a randomized, placebo-controlled, cross-over trial. This trial aimed to evaluate the impact of valACV on HIV-1 viremia in HSV-2 seronegative persons not on antitretroviral therapy. Twenty-one HIV-infected male and female subjects were divided in two groups: Group A received 12 weeks of valACV 500 mg given twice daily by mouth followed by two weeks of no treatment then 12 weeks of placebo;Group B received 12 weeks of placebo followed by 12 weeks of valACV. We found that valACV reduced HIV-1 levels in plasma by 0.40 log10 copies/ml, demonstrating that ACV can suppress HIV-1 replication in the absence of HSV-2 coinfection. Plasma HIV-1 concentrations rebounded to pre-enrollment levels within 2 weeks of termination of valACV treatment, consistent with the short drug half-life and its lack of intracellular accumulation. Although the effect of valACV on HIV-1 RNA is modest, it is comparable to monotherapy with other antiretrovirals (e.g., zidovudine or stavudine monotherapy) and the reduction of HIV-1 plasma viral load mediated by ACV could be clinically beneficial. The reduction of HIV-1 load observed in our study provides evidence that the effect of ACV on HIV-1 load is not restricted to the presence of HSV-2 and suggests that ACV has a direct anti HIV-1 activity in vivo. In light of the ACV clinical data, an ACV chronic suppressive regimen may be beneficial for HIV-1 infected patients co-infected or not co-infected with HSV-2. B. Development of new anti-HIV compounds Despite intensive research to find new drugs, nucleoside reverse transcriptase inhibitors (NRTIs) remain at the central core of HIV-1 treatment. Among eight NRTIs that have been used, the most extensively studied is 3- azido-3-deoxythymidine (AZT, zidovudine, retrovir). However, this drug has significant side effects. Phosphonate derivatives of AZT showed significant decrease in toxicity and improvement of AZT therapeutic properties. Similar to AZT phosphonate derivatives, 3TC 5-H-phosphonate and 3TC 5-aminocarbonylphosphonate were found to be much less toxic than parent 3TC in cell cultures. Here, we describe a new phosphonate derivative, namely, 3TC-AZT heterodimer: This compound is a chimera of AZT-5- and 3TC-5-aminocarbonylphosphonate. At this initial stage of development of the new antiviral we characterized its structure and tested potential antiviral activity of the newly synthesized phosphonate chimera of AZT and 3TC. Since in vivo the critical pathogenic events of HIV infection occur in tissues that are not faithfully reflected by single cell cultures, we utilized a system of human lymphoid tissue ex vivo that retains tissue cytoarchitecture that was earlier developed in our laboratory. This system supports HIV-1 replication and has been shown to complement pre-clinical drug testing against different paathogens. Also, such a system reflects the in vivo donor-to-donor variability and allows various drug testing as a preliminary step before engaging in costly clinical trials. Blocks of human tonsillar tissue were treated with phosphonate derivatives overnight and then infected with a prototypical HIV-1 X4LAI.04. Compounds were present during the entire culture period. We found that the 3TC-AZT heterodimer significantly suppressed HIV-1 replication at the level that surpasses some of the clinically used antivirals. Moreover, it exhibited low toxicity towards various tissue lymphocytes. In conclusion, we showed that the development of bis phosphonate derivatives is feasible and that such a bis phosphonate of 3TC and AZT inhibited HIV-1 in human lymphoid tissue. In regards to its low toxicity and slow release of active compounds, the phosphonate strategy may be useful for the development of heterodimers of anti HIV-1 compounds. C. Antigenic composition of individual extracellular vesicles (EVs) Microvesicles, exosomes, and apoptotic bodies play an important role in cell-to-cell communication because different proteins, lipids and RNAs are specifically incorporated into these vesicles, which can be targeted to remote cells through receptor-ligand interactions. Release of EVs is part of normal physiological processes and is reported to change in pathologies. Since various cells supplying EVs to blood express different antigens, EVs produced by these cells are antigenically different. Analyses of blood EV composition, which have been performed predominantly in bulk, have revealed the presence of various cellular antigens in EVs but could not reflect the distribution of these antigens on individual EVs although such distribution may report on physiological conditions of the donor. Conventional flow cytometry cannot be applied to analysis of small particles like EVs. We now developed a new nanoparticle-based technique for analysis of surface proteins on single blood nano-sized (<300 nm) EVs. We used a regular commercial flow cytometer and magnetic nano particles (MNPs) to isolate fluorescence-labeled EVs and to separate them from non-bound fluorescent antibodies. Moreover, this analysis can be performed not only on EVs released by cells in culture, but also directly on blood plasma EVs. With MNPs coupled to antibodies against EV antigens, it is possible to focus on minor fractions of EVs that constitute few vesicles per microliter out of the large numbers EVs that are reported for normal blood plasma. Moreover, we were able to evaluate the distribution of antigens in these minor EV fractions. In particular, when we captured CD31-carrying EVs, we found that about half of these EVs co-expressed CD41 and CD63. Although CD63 is a highly prevalent antigen, our fine analysis of antigen distribution demonstrated that it is not carried by approximately 20% of the CD31+CD41+ EVs;thus these EVs form a separate fraction. In conclusion, here we performed a fine analysis of single blood EVs according to the distribution of antigens they carry. We demonstrated that the blood EV population is a mosaic, with various EVs carrying different combinations of antigens. None of these antigens can be claimed to be present on all EVs. These results would be impossible to obtain in a bulk analysis, which reports only on the general presence or absence of particular antigens in EV preparations. Because of the reproducibility of our analysis of distributions of individual blood EVs according to the combinations of antigens they carry, it is now possible to relate these distributions to the medical condition of an individual donor, as well as to search for EV antigenic patterns common to particular diseases. We now use this technique to analyze antigenic composition of EVs associated with pathologic pregnancies and cardio-vascular diseases as well as with HIV infection.
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