Safety of G2-S16 Polyanionic Carbosilane Dendrimer as Possible HIV-1 Vaginal Microbicide.

The UNAIDS objective for 2020 was 500,000 new HIV-1 infections per year; however, the latest annual reported data confirmed 1.7 million new HIV-1 infections in that year. Those data evidences the need for new prevention strategies and prophylactic treatments. This prevention crisis occurred in spite of the knowledge and availability of efficient prevention strategies. The G2-S16 is a microbicidal polyanionic carbosilane dendrimer currently being tested for topical vaginal application, which has been shown to be efficient in the prevention of HIV-1 infection. However, safety tests were lacked. For this purpose, we injected intravenously G2-S16 dendrimer to CD1 mice, thereby analyzing the hemogram, blood biochemical markers of systemic damage, accumulation in the organs and organ-tissue damage in heart, spleen, kidney, liver and brain. This work shows that even if the G2-S16 dendrimer penetrates the epithelial tissue, it does not cause vaginal irritation or tissue damage. Moreover, the i.v. injection of the G2-S16 dendrimer did not cause a damaging effect on the studied organs and it did not modify the hemogram or the biochemical plasma markers. In conclusion, the G2-S16 dendrimer has a very good safety profile, indicating that this molecule can be a very safe and efficient vaginal microbicide.

Establishment of a miRNA profile in paediatric HIV-1 patients and its potential as a biomarker for effectiveness of the combined antiretroviral therapy.

miRNAs have been extensively studied in pathological conditions, including viral infections, such as those provoked by HIV-1. Several cellular and circulating miRNAs are altered during HIV-1 infection, with either beneficial effects on host defenses or enhanced virus infectivity. Blood samples were collected in sterile EDTA tubes and plasma was separated and stored, as were PBMCs. RNA was isolated and reverse-transcribed. Finally, the miRNA gene expression profile was assessed using TaqMan Array Human microRNA Card A v2.0. A comprehensive statistical analysis was performed on the results obtained. This is the first study on miRNAs in HIV-1 paediatric patients, and a miRNA profile differentiating patients starting combination antiretroviral therapy (cART) at different times after HIV-1 diagnosis was established. Thirty-four miRNAs were observed to have different expression levels between the control group and the cART group. The data indicates the need to start cART as soon as possible after the establishment of HIV-1 infection to assure the best outcome possible. Finally, the selected 34 miRNAs may be used as biomarkers for prognosis and assessing therapy effectiveness. However, more research must be conducted to establish adequate quantitative correlations.

Role of G2-S16 Polyanionic Carbosilane Dendrimer in the Prevention of Respiratory Syncytial Virus Infection In Vitro and In Vivo in Mice.

The respiratory syncytial virus (RSV) causes respiratory infection and bronchiolitis, requiring hospitalization mainly in infants. The interaction between RSV, envelope glycoproteins G and F, and cell surface heparan sulfate proteoglycans (HSPG) is required for binding and entry into the host cells. A G2-S16 polyanionic carbosilane dendrimer was identified as a possible RSV inhibitor. We speculated that the G2-S16 dendrimer adheres to the host cell-surface HSPG, acts through binding to HS receptors, and prevents further RSV infection. The G2-S16 dendrimer was non-toxic when applied intranasally to Balb/c mice, and interestingly enough, this G2-S16 dendrimer inhibits 85% RSV. Therefore, our G2-S16 dendrimer could be a candidate for developing a new possible therapy against RSV infection.

Cationic Dendrimer G2-S16 Inhibits Herpes Simplex Type 2 Infection and Protects Mice Vaginal Microbiome.

The G2-S16 polyanionic carbosilane dendrimer is a promising microbicide that inhibits HSV-2 infection in vitro and in vivo in mice models. This G2-S16 dendrimer inhibits HSV-2 infection even in the presence of semen. Murine models, such as BALB/c female mice, are generally used to characterize host-pathogen interactions within the vaginal tract. However, the composition of endogenous vaginal flora remains largely undefined with modern microbiome analyses. It is important to note that the G2-S16 dendrimer does not change healthy mouse vaginal microbiome where Pseudomonas (10.2-79.1%) and Janthinobacterium (0.7-13%) are the more abundant genera. The HSV-2 vaginally infected female mice showed a significant microbiome alteration because an increase of Staphylococcus (up to 98.8%) and Escherichia (30.76%) levels were observed becoming these bacteria the predominant genera. BALB/c female mice vaginally-treated with the G2-S16 dendrimer and infected with the HSV-2 maintained a healthy vaginal microbiome similar to uninfected female mice. Summarizing, the G2-S16 polyanionic carbosilane dendrimer inhibits the HSV-2 infection in the presence of semen and prevents the alteration of mice female vaginal microbiome.

Synergistic Activation of Latent HIV-1 Expression by Novel Histone Deacetylase Inhibitors and Bryostatin-1.

Viral reactivation from latently infected cells has become a promising therapeutic approach to eradicate HIV. Due to the complexity of the viral latency, combinations of efficient and available drugs targeting different pathways of latency are needed. In this work, we evaluated the effect of various combinations of bryostatin-1 (BRY) and novel histone deacetylase inhibitors (HDACIs) on HIV-reactivation and on cellular phenotype. The lymphocyte (J89GFP) or monocyte/macrophage (THP89GFP) latently infected cell lines were treated with BRY, panobinostat (PNB) and romidepsin (RMD) either alone or in combination. Thus, the effect on the viral reactivation was evaluated. We calculated the combination index for each drug combination; the BRY/HDACIs showed a synergistic HIV-reactivation profile in the majority of the combinations tested, whereas non-synergistic effects were observed when PNB was mixed with RMD. Indeed, the 75% effective concentrations of BRY, PNB and RMD were reduced in these combinations. Moreover, primary CD4 T cells treated with such drug combinations presented similar activation and proliferation profiles in comparison with single drug treated cells. Summing up, combinations between BRY, PNB and/or RMD presented a synergistic profile by inducing virus expression in HIV-latently infected cells, rendering these combinations an attractive novel and safe option for future clinical trials.

Prevention vaginally of HIV-1 transmission in humanized BLT mice and mode of antiviral action of polyanionic carbosilane dendrimer G2-S16.

The development of a safe, effective, and low-priced topical microbicide to prevent HIV-1 sexual transmission is urgently needed. The emerging field of nanotechnology plays an important role in addressing this challenge. We demonstrate that topical vaginal administration of 3% G2-S16 prevents HIV-1JR-CSF transmission in humanized (h)-BLT mice in 84% with no presence of HIV-1 RNA and vaginal lesions. Second-generation polyanionic carbosilane dendrimer G2-S16 with silica core and 16 sulfonate end-groups exerts anti-HIV-1 activity at an early stage of viral replication, blocking the gp120/CD4 interaction, acting on the virus, and inhibiting the cell-to-cell HIV-1 transmission, confirming its multifactorial and non-specific ability. This study represents the first demonstration that transmission of HIV-1 can be efficiently blocked by vaginally applied G2-S16 in h-BLT mice. These findings provide a step forward in the development of G2-S16-based vaginal microbicides to prevent vaginal HIV-1 transmission in humans. FROM THE CLINICAL EDITOR: HIV infections remain a significant problem worldwide and the major route of transmission is through sexual activity. In this article, the authors developed an antiviral agent containing polyanionic carbosilane dendrimer with silica core and 16 sulfonate end-groups. When applied vaginally, this was shown to exert anti-HIV protection. These positive findings may offer hope in the fight against the spread of HIV epidemic.

Triple combination of carbosilane dendrimers, tenofovir and maraviroc as potential microbicide to prevent HIV-1 sexual transmission.

AIM: To research the synergistic activity by triple combinations of carbosilane dendrimers with tenofovir and maraviroc as topical microbicide. METHODS: Cytotoxicity, anti-HIV-1 activity, vaginal irritation and histological analysis of triple combinations were determined. Analysis of combined effects and the median effective concentration were performed using CalcuSyn software. RESULTS: Combinations showed a greater broad-spectrum anti-HIV-1 activity than the single-drug, and preserved this activity in acid environment or seminal fluid. The strongest combinations were G2-STE16/G2-S24P/tenofovir, G2-STE16/G2-S16/maraviroc and G2-STE16/tenofovir/maraviroc at 2:2:1, 10:10:1 10:5:1 ratios, respectively. They demonstrated strong synergistic activity profile due to the weighted average combination indices varied between 0.06 and 0.38. No irritation was detected in female BALB/c mice. CONCLUSION: The three-drug combination increases their antiviral potency and act synergistically as potential microbicide.

Prostaglandin E2 reduces the release and infectivity of new cell-free virions and cell-to-cell HIV-1 transfer.

BACKGROUND: The course of human immunodeficiency virus type-1 (HIV-1) infection is influenced by a complex interplay between viral and host factors. HIV infection stimulates several proinflammatory genes, such as cyclooxigense-2 (COX-2), which leads to an increase in prostaglandin (PG) levels in the plasma of HIV-1-infected patients. These genes play an indeterminate role in HIV replication and pathogenesis. The effect of prostaglandin E2 (PGE2) on HIV infection is quite controversial and even contradictory, so we sought to determine the role of PGE2 and the signal transduction pathways involved in HIV infection to elucidate possible new targets for antiretrovirals. RESULTS: Our results suggest that PGE2 post-infection treatment acts in the late stages of the viral cycle to reduce HIV replication. Interestingly, viral protein synthesis was not affected, but a loss of progeny virus production was observed. No modulation of CD4 CXCR4 and CCR5 receptor expression, cell proliferation, or activation after PGE2 treatment was detected. Moreover, PGE2 induced an increase in intracellular cAMP (cyclic AMP) levels through the EP2/EP4 receptors. PGE2 effects were mimicked by dbcAMP and by a specific Epac (exchange protein directly activated by cyclic AMP) agonist, 8-Cpt-cAMP. Treatment with PGE2 increased Rap1 activity, decreased RhoA activity and subsequently reduced the polymerization of actin by approximately 30% compared with untreated cells. In connection with this finding, polarized viral assembly platforms enriched in Gag were disrupted, altering HIV cell-to-cell transfer and the infectivity of new virions. CONCLUSIONS: Our results demonstrate that PGE2, through Epac and Rap activation, alters the transport of newly synthesized HIV-1 components to the assembly site, reducing the release and infectivity of new cell-free virions and cell-to-cell HIV-1 transfer.

Gene therapy in HIV-infected cells to decrease viral impact by using an alternative delivery method.

The ability of dendrimer 2G-[Si{O(CH(2))(2)N(Me)(2) (+)(CH(2))(2)NMe(3) (+)(I(-))(2)}](8) (NN16) to transfect a wide range of cell types, as well as the possible biomedical application in direct or indirect inhibition of HIV replication, was investigated. Cells implicated in HIV infection such as primary peripheral blood mononuclear cells (PBMC) and immortalized suspension cells (lymphocytes), primary macrophages and dendritic cells, and immortalized adherent cells (astrocytes and trophoblasts) were analyzed. Dendrimer toxicity was evaluated by mitochondrial activity, cell membrane rupture, release of lactate dehydrogenase, erythrocyte hemolysis, and the effect on global gene expression profiles using whole-genome human microarrays. Cellular uptake of genetic material was determined using flow cytometry and confocal microscopy. Transfection efficiency and gene knockdown was investigated using dendrimer-delivered antisense oligonucleotides and small interfering RNA (siRNA). Very little cytotoxicity was detected in a variety of cells relevant to HIV infection and erythrocytes after NN16 dendrimer treatment. Imaging of cellular uptake showed high transfection efficiency of genetic material in all cells tested. Interestingly, NN16 further enhanced the reduction of HIV protein 24 antigen release by antisense oligonucleotides due to improved transfection efficiency. Finally, the dendrimer complexed with siRNA exhibited therapeutic potential by specifically inhibiting cyclooxygenase-2 gene expression in HIV-infected nervous system cells. NN16 dendrimers demonstrated the ability to transfect genetic material into a vast array of cells relevant to HIV pathology, combining high efficacy with low toxicity. These results suggest that NN16 dendrimers have the potential to be used as a versatile non-viral vector for gene therapy against HIV infection.

Progesterone inhibits HIV-1 replication in human trophoblast cells through inhibition of autocrine tumor necrosis factor secretion.

BACKGROUND: Progesterone levels are higher in placental barriers during pregnancy, but the effect of progesterone on human immunodeficiency virus type 1 (HIV-1) infection in placental cells has not been addressed. We hypothesize that progesterone may affect HIV infection. METHODS: Purified trophoblastic cells and trophoblastic cell lines were infected or transfected with HIV-1, and the effect of progesterone was analyzed. Viral replication was measured by viral p24 or viral load quantification. Nuclear factor kappa -B (NF- kappa B) or long terminal repeat (LTR)-dependent transcription was measured by luciferase assays. Expression of chemokine receptors was analyzed by flow cytometry. Tumor necrosis factor (TNF) messenger RNA was assessed by reverse-transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR. RESULTS: Progesterone inhibits HIV-1 replication in placental cells at the concentration found in the placental interface, at a postentry step, and does not affect cell surface expression of chemokine receptors. Progesterone did not inhibit basal or induced LTR transcription or NF- kappa B activation. TNF synthesis in placental cells is induced by HIV-1 infection that, in an autocrine manner, activates viral replication, because neutralizing anti-TNF antibodies block it. Progesterone inhibits the induction of TNF synthesis by viral infection and virus or gp-120-induced TNF transcription. CONCLUSION: Our results demonstrate that progesterone inhibits HIV-1 replication in placental cells by reducing TNF levels, which are required for optimal viral replication.