Sonia Zicari

A Multi-targeted Drug Candidate with Dual Anti-HIV and Anti-HSV Activity

Human immunodeficiency virus (HIV) infection is often accompanied by infection with other pathogens, in particular herpes simplex virus type 2 (HSV-2). The resulting coinfection is involved 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. Here, we report on the discovery of such a compound, designated PMEO-DAPym. We compared its properties with those of the structurally related and clinically used acyclic nucleoside phosphonates (ANPs) tenofovir and adefovir. We demonstrated the potent anti-HIV and -HSV activity of this drug in a diverse set of clinically relevant in vitro, ex vivo, and in vivo systems including (i) CD4+ T-lymphocyte (CEM) cell cultures, (ii) embryonic lung (HEL) cell cultures, (iii) organotypic epithelial raft cultures of primary human keratinocytes (PHKs), (iv) primary human monocyte/macrophage (M/M) cell cultures, (v) human ex vivo lymphoid tissue, and (vi) athymic nude mice. Upon conversion to its diphosphate metabolite, PMEO-DAPym markedly inhibits both HIV-1 reverse transcriptase (RT) and HSV DNA polymerase. However, in striking contrast to tenofovir and adefovir, it also acts as an efficient immunomodulator, inducing β-chemokines in PBMC cultures, in particular the CCR5 agonists MIP-1β, MIP-1α and RANTES but not the CXCR4 agonist SDF-1, without the need to be intracellularly metabolized. Such specific β-chemokine upregulation required new mRNA synthesis. The upregulation of β-chemokines was shown to be associated with a pronounced downmodulation of the HIV-1 coreceptor CCR5 which may result in prevention of HIV entry. PMEO-DAPym belongs conceptually to a new class of efficient multitargeted antivirals for concomitant dual-viral (HSV/HIV) infection therapy through inhibition of virus-specific pathways (i.e. the viral polymerases) and HIV transmission prevention through interference with host pathways (i.e. CCR5 receptor down regulation).

Extracellular Vesicles Carry HIV Env and Facilitate Hiv Infection of Human Lymphoid Tissue

Cells productively infected with HIV-1 release virions along with extracellular vesicles (EVs) whose biogenesis, size, and physical properties resemble those of retroviruses. Here, we found that a significant number of EVs (exosomes) released by HIV-1 infected cells carry gp120 (Env), a viral protein that mediates virus attachment and fusion to target cells, and also facilitates HIV infection in various indirect ways. Depletion of viral preparations of EVs, in particular of those that carry gp120, decreases viral infection of human lymphoid tissue ex vivo. Thus, EVs that carry Env identified in our work seem to facilitate HIV infection and therefore may constitute a new therapeutic target for antiviral strategy.

Vaginal Lactobacillus Inhibits HIV-1 Replication in Human Tissues Ex Vivo

Lactobacillus species, which dominate vaginal microbiota of healthy reproductive-age women, lower the risks of sexually transmitted infections, including the risk of human immunodeficiency virus (HIV) acquisition. The exact mechanisms of this protection remain to be understood. Here, we investigated these mechanisms in the context of human cervico-vaginal and lymphoid tissues ex vivo. We found that all six Lactobacillus strains tested in these systems significantly suppressed HIV type-1 (HIV-1) infection. We identified at least three factors that mediated this suppression: (i) Acidification of the medium. The pH of the undiluted medium conditioned by lactobacilli was between 3.8 and 4.6. Acidification of the culture medium with hydrochloric acid (HCl) to this pH in control experiments was sufficient to abrogate HIV-1 replication. However, the pH of the Lactobacillus-conditioned medium (CM) diluted fivefold, which reached ∼6.9, was also suppressive for HIV-1 infection, while in control experiments HIV-1 infection was not abrogated when the pH of the medium was brought to 6.9 through the use of HCl. This suggested the existence of other factors responsible for HIV-1 inhibition by lactobacilli. (ii) Lactic acid. There was a correlation between the concentration of lactic acid in the Lactobacillus-CM and its ability to suppress HIV-1 infection in human tissues ex vivo. Addition of lactic acid isomers D and L to tissue culture medium at the concentration that corresponded to their amount released by lactobacilli resulted in HIV-1 inhibition. Isomer L was produced in higher quantities than isomer D and was mostly responsible for HIV-1 inhibition. These results indicate that lactic acid, in particular its L-isomer, inhibits HIV-1 independently of lowering of the pH. (iii) Virucidal effect. Incubation of HIV-1 in Lactobacillus-CM significantly suppressed viral infectivity for human tissues ex vivo. Finally, lactobacilli adsorb HIV-1, serving as a sink decreasing the number of free virions. In summary, we found that lactobacilli inhibit HIV-1 replication in human tissue ex vivo by multiple mechanisms. Further studies are needed to evaluate the potential of altering the spectra of vaginal microbiota as an effective strategy to enhance vaginal health. Human tissues ex vivo may serve as a test system for these strategies.

Modulation of Immune Responses by Extracellular Vesicles From Retinal Pigment Epithelium.

Purpose Extracellular vesicles (EV), such as exosomes, are important mediators of intercellular communication and have been implicated in modulation of the immune system. We investigated if EV released from retinal pigment epithelium (RPE) modulate immune responses in vitro. Methods Extracellular vesicles were isolated from ARPE-19 cultures stimulated or not with the inflammatory cytokines IL-1β, IFN-γ, and TNF-α. Isolated EV were characterized by nanoparticle flow and Western blot analyses. Retinal pigment epithelium–derived EV were cultured with human peripheral blood mononuclear cells, which were assayed for T-cell proliferation by 3H-thymidine incorporation. Retinal pigment epithelium–derived EV were also independently cultured with enriched lymphocytes or monocytes. Cell phenotype and cell death were evaluated by flow cytometric analysis. Cytokine levels were assayed in culture supernatants by multiplex bead analysis. Results The concentration of ARPE-derived EV from cytokine-stimulated cultures was slightly higher than from nonstimulated cultures. The size of EV was approximately 100 nm in both groups. Extracellular vesicles from both nonstimulated and cytokine-stimulated ARPE-19 significantly inhibited T-cell proliferation without affecting T-cell viability. Culture of EV from nonstimulated ARPE-19 with undifferentiated human monocytes induced an immunoregulatory phenotype with a significantly higher percentage of CD14++CD16+ monocytes and upregulation of TGF-β1. Culture of EV from cytokine-stimulated ARPE-19 cells with human monocytes induced upregulation of several proinflammatory cytokines and monocyte death. Conclusions Retinal pigment epithelium cells constitutively secrete EV in the size range of exosomes, with increased release from RPE cells stimulated with inflammatory cytokines. Extracellular vesicles from both nonstimulated and cytokine-stimulated RPE inhibited T-cell stimulation. Extracellular vesicles from nonstimulated ARPE-19 cells promoted an immunoregulatory CD14++CD16+ phenotype in human monocytes, while EV from cytokine-stimulated ARPE-19 cells caused human monocyte death. These findings suggest that RPE cells use EV to induce a downregulatory immune environment under homeostatic conditions. In an inflammatory milieu, RPE-derived EV may mitigate a potentially harmful inflammatory response through killing of monocytes.

A new antiviral: Chimeric 3TC–AZT phosphonate efficiently inhibits HIV-1 in human tissues ex vivo

Although more-recently developed antivirals target different molecules in the HIV-1 replication cycle, nucleoside reverse transcriptase inhibitors (NRTIs) remain central for HIV-1 therapy. Here, we test the anti-HIV activity of a phosphonate chimera of two well-known NRTIs, namely AZT and 3TC. We show that this newly synthesized compound suppressed HIV-1 infection in lymphoid tissue ex vivo more efficiently than did other phosphonates of NRTIs. Moreover, the new compound was not toxic for tissue cells, thus making the chimeric phosphonate strategy a valid approach for the development of anti HIV-1 compound heterodimers.

Monocytes but Not Lymphocytes Carrying HIV-1 on Their Surface Transmit Infection to Human Tissue Ex Vivo

ABSTRACT Unprotected sexual intercourse with HIV-infected men is the major cause of new infections. HIV virions are released into semen by various cells of the male genital tract, as well as by infected monocytes and lymphocytes present in semen. Some of these virions may attach to the surfaces of cells, infected or uninfected. We investigated whether cells carrying attached HIV on their surfaces can transmit infection. We addressed this question in a model system of human tissue exposed ex vivo to monocytes and lymphocytes carrying HIV on their surfaces. We gamma irradiated the cells to prevent their productive infection. In spite of comparable amounts of HIV attached to monocytes and lymphocytes, only monocytes were capable of transmitting infection and triggering productive infection in tissue. This HIV-1 transmission was mediated by cell-cell contacts. Our experiments suggest that in vivo, HIV attached to infected or uninfected monocytes, which far outnumber lymphocytes in HIV-infected semen, may contribute to sexual transmission of HIV from men to their partners. IMPORTANCE The vast majority of new HIV infections occur through sexual transmission, in which HIV is transferred from the semen of an infected male to an uninfected partner. In semen, HIV-1 particles may exist as free-floating virions; inside infected cells; or attached to the surfaces of cells, whether they are infected or not. Here, we investigated whether HIV attached to the surfaces of monocytes or lymphocytes could transmit infection to human tissue. Incubation of human tissue with monocyte-attached HIV resulted in productive tissue infection. In contrast, there was no infection of tissues when they were incubated with lymphocyte-attached HIV-1. Our results highlight the important role that seminal monocytes may play in HIV transmission in vivo, especially since monocytes far outnumber lymphocytes in the semen of HIV-infected individuals.

Virtual screening of acyclovir derivatives as potential antiviral agents: design, synthesis and biological evaluation of new acyclic nucleoside ProTides

Following our findings on the anti-human immunodeficiency virus (HIV) activity of acyclovir (ACV) phosphate prodrugs, we herein report the ProTide approach applied to a series of acyclic nucleosides aimed at the identification of novel and selective antiviral, in particular anti-HIV agents. Acyclic nucleoside analogues used in this study were identified through a virtual screening using HIV-reverse transcriptase (RT), adenylate/guanylate kinase, and human DNA polymerase γ. A total of 39 new phosphate prodrugs were synthesized and evaluated against HIV-1 (in vitro and ex vivo human tonsillar tissue system) and human herpes viruses. Several ProTide compounds showed substantial potency against HIV-1 at low micromolar range while the parent nucleosides were not effective. Also, pronounced inhibition of herpesvirus replication was observed. A carboxypeptidase-mediated hydrolysis study was performed for a selection of compounds to assess the formation of putative metabolites and support the biological activity observed.

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Human cytomegalovirus-infected cells release extracellular vesicles that carry viral surface proteins

Extracellular vesicles (EVs) released by virus-infected cells typically incorporate host and viral components inside the vesicles (cargo molecules). Here, we investigated if human cytomegalovirus (HCMV) proteins are incorporated in EV outer membrane released by HCMV-infected cells. We separated EVs from HCMV using an iodixanol step-gradient and found that the separated vesicles carried EV markers such as the tetraspanin CD63 and Rab27A. Flow analysis of individual EVs demonstrated that on average, 15 ± 3.7% of EVs were positive for gB, 5.3 ± 2.3% were positive for gH and 3.74 ± 1.5% were positive for both gB and gH. In light of previous findings demonstrating HIV envelope proteins in EV membranes, the presence of viral protein at the surface of EVs released by HCMV-infected cells indicated that viral membrane proteins incorporated in EVs released by virus-infected cells may be a general phenomenon.

Dual-targeted anti-TB/anti-HIV heterodimers

&NA; HIV and M. tuberculosis are two intersecting epidemics making the search for new dual action drugs against both pathogens extremely important. Here, we report on the synthesis and suppressive activities of five dual‐targeted HIV/TB compounds. These compounds are heterodimers of AZT, as anti‐HIV molecules, and 5‐substituted uracil derivatives, as anti‐TB molecules. We found that these compounds inhibit the growth of M. tuberculosis and suppress the replication of HIV in human cell cultures and human lymphoid tissues ex vivo. We identified one particular heterodimer that inhibited both HIV and the drug‐resistant TB strain MS‐115 most potently. This compound demonstrated low toxicity and had no cytostatic effect on cells in culture, constituting an ideal candidate for future development and further in vivo testing.