Hepatitis C virus-specific cellular immune responses in individuals with no evidence of infection

The detection of hepatitis C virus (HCV)-specific T cell responses in HCV-uninfected, presumably unexposed, subjects could be due to an underestimation of the frequency of spontaneously resolving infections, as most acute HCV infections are clinically silent. To address this hypothesis, HCV-specific cellular immune responses were characterized, in individuals negative for an HCV PCR assay and humoral response, with (n = 32) or without (n = 33) risk of exposure to HCV. Uninfected volunteers (n = 20) with a chronically HCV-infected partner were included as positive controls for potential exposure to HCV and HCV infection, respectively. HCV-specific T cell responses in freshly isolated peripheral blood mononuclear cells were studied ex vivo by ELISPOT and CFSE-based proliferation assays using panels of HCV Core and NS3-derived peptides. A pool of unrelated peptides was used as a negative control, and a peptide mix of human cytomegalovirus, Epstein-Bar virus and Influenza virus as a positive control. Overall, 20% of presumably HCV-uninfected subject tested had detectable T-cell responses to the virus, a rate much higher than previous estimates of HCV prevalence in developed countries. This result would be consistent with unapparent primary HCV infections that either cleared spontaneously or remained undetected by conventional serological assays

In vivo cellular tropism of gorilla simian foamy virus in blood of infected humans.

International audienceSimian foamy viruses (SFV) are retroviruses that are widespread among nonhuman primates. SFV can be transmitted to humans, giving rise to a persistent infection. Only a few data are available concerning the distribution of SFV in human blood cells. Here we purified blood mononuclear cell subsets from 11 individuals infected with a Gorilla gorilla SFV strain and quantified SFV DNA levels by quantitative PCR. SFV DNA was detected in the majority of the CD8(+), CD4(+), and CD19(+) lymphocyte samples and rarely in CD14(+) monocyte and CD56(+) NK lymphocyte samples. The median (interquartile range [IQR]) SFV DNA counts were 16.0 (11.0 to 49.8), 11.3 (5.9 to 28.3), and 17.2 (2.0 to 25.2) copies/10(5) cells in CD8(+) T lymphocytes, CD4(+) T lymphocytes, and CD19(+) B lymphocytes, respectively. In the CD4 compartment, SFV DNA was detected in both memory and naive CD4(+) T lymphocytes. SFV DNA levels in CD4(+) T cells were positively correlated with the duration of the infection. Our study shows with a quantitative method that CD8(+), CD4(+), and B lymphocytes are major cellular targets of SFV in the blood of infected humans.Investigation of SFV infections in humans is important due to the origin of human immunodeficiency viruses (HIV) and human T cell lymphotropic viruses (HTLV) from cross-species transmission of their simian counterparts to humans. Surprisingly little is known about many aspects of the biology of SFV in infected humans, including quantitative data concerning the cellular targets of SFV in vivo. Here we show that the distribution of SFV DNA among the different leukocyte populations is not homogeneous and that viral load in CD4(+) T lymphocytes is correlated with the duration of infection. These new data will help in understanding the biology of retroviral infections in humans and can be useful in the growing field of SFV-based gene therapy

Latency, tropism and genetic variation of Simian Foamy Virus in blood and saliva from infected Humans

International audienceSimian foamy viruses (SFV) are widespread retroviruses among non-human primates (NHP). SFV actively replicate in the oral cavity of NHP and can be transmitted to humans through NHP bites, in whom they establish a persistent infection. We aimed to study three major properties of these zoonotic retroviruses: replicative status, tropism and variability. In 14 hunters from Cameroon previously shown to be infected with a gorilla SFV strain, viral DNA could be detected by quantitative polymerase chain reaction in most samples of peripheral blood mononuclear cells (PBMCs) and saliva. The SFV DNA levels were 7.1±6.0 SFV DNA copies/105 cells in PBMCs and 2.4±4.3 SFV DNA copies/105 cells in saliva. In contrast, no SFV RNA was detected by qRT-PCR in either PBMCs or saliva. PBMCs populations (T4, T8, B, NK lymphocytes and monocytes) were sorted with magnetic beads before quantification of SFV DNA. Our preliminary results showed the presence of SFV DNA in all PBMCs populations at different levels. We finally assessed the viral diversity in vivo. Although intra-individual SFV genetic variation was low (<0,5%) we detected some viral diversity in 3 out of 9 individuals. In one subject, genetic variation might be associated with coinfection with 2 SFV strains, while in the two other subjects, variations seemed to derive from APOBEC3 editing with a high rate of G-to-A substitutions. Our study demonstrates that SFV infection is mostly latent in PBMCs and in saliva. Such a scenario may explain the putative lack of secondary human-to-human transmissions of SFV

Inhibitors of the Interferon Response Increase the Replication of Gorilla Simian Foamy Viruses

International audienceSimian foamy viruses (SFVs) are complex retroviruses widespread throughout nonhuman primates. SFVs can also be transmitted to humans, mostly through bites. We previously observed that the primary zoonotic gorilla SFV strains much more slowly than laboratory-adapted chimpanzee strains. Here, we tested the hypothesis that SFV growth is limited by interferon (IFN)-induced restriction factors using inhibitors of cellular signaling pathways involved in type I IFN induction or action. Inhibitors of JAK1/2 (Ruxolitinib) and TBK-1 (BMX795) led to a 2 to > 20-fold higher percentage of infected BHK-1 and HT1080 cells. However, replication of the laboratory-adapted prototype foamy virus was not sensitive to these molecules, and IKK2 inhibitors had no effect on any of the SFV strains. In conclusion, the addition of small molecules that inhibit type I IFN response to the culture medium can be used as a simple and efficient method to enhance the replication of zoonotic gorilla SFV

Absence of oxysterol-like side effects in human monocytic cells treated with phytosterols and oxyphytosterols

Oxysterols, found in some commonly consumed foods, can induce a wide range of cytotoxic effects, which have been extensively studied. On the other hand, the side effects of phytosterols and oxyphytosterols are less well-known. Over the past few years, different types of foods have been enriched with phytosterols on the basis of the properties of these compounds that reduce circulating cholesterol levels in certain experimental conditions. It is therefore important to gain better knowledge of the risks and benefits of this type of diet. In this study, conducted in human monocytic U937 cells, the ability of phytosterols (sitosterol, campesterol) and oxyphytosterols (7 beta-hydroxysitosterol, 7-ketositosterol) to induce cell death, polar lipid accumulation, and pro-inflammatory cytokine (MCP-1; IL-8) secretion was determined and compared to that of oxysterols (7-ketocholesterol, 7 beta-hydroxycholesterol). Phytosterols and oxyphytosterols had no significant effects on the parameters studied; only 7 beta-hydroxysitosterol slightly increased cell death, whereas at the concentration used (20 mu g/mL), strong cytotoxic effects were observed with the oxysterols. With sitosterol, campesterol, and 7-ketositosterol, IL-8 secretion was decreased, and with campesterol the intracellular polar lipid level was reduced. The data show that phytosterols and oxyphytosterols have no oxysterol-like side effects, and they rather argue in favor of phytosterols' beneficial effects

Plasma antibodies from humans infected with zoonotic simian foamy virus do not inhibit cell-to-cell transmission of the virus despite binding to the surface of infected cells

International audienceZoonotic simian foamy viruses (SFV) establish lifelong infection in their human hosts. Despite repeated transmission of SFV from nonhuman primates to humans, neither transmission between human hosts nor severe clinical manifestations have been reported. We aim to study the immune responses elicited by chronic infection with this retrovirus and previously reported that SFV-infected individuals generate potent neutralizing antibodies that block cell infection by viral particles. Here, we assessed whether human plasma antibodies block SFV cell-to-cell transmission and present the first description of cell-to-cell spreading of zoonotic gorilla SFV. We set-up a microtitration assay to quantify the ability of plasma samples from 20 Central African individuals infected with gorilla SFV and 9 uninfected controls to block cell-associated transmission of zoonotic gorilla SFV strains. We used flow-based cell cytometry and fluorescence microscopy to study envelope protein (Env) localization and the capacity of plasma antibodies to bind to infected cells. We visualized the cell-to-cell spread of SFV by real-time live imaging of a GFP-expressing prototype foamy virus (CI-PFV) strain. None of the samples neutralized cell-associated SFV infection, despite the inhibition of cell-free virus. We detected gorilla SFV Env in the perinuclear region, cytoplasmic vesicles and at the cell surface. We found that plasma antibodies bind to Env located at the surface of cells infected with primary gorilla SFV strains. Extracellular labeling of SFV proteins by human plasma samples showed patchy staining at the base of the cell and dense continuous staining at the cell apex, as well as staining in the intercellular connections that formed when previously connected cells separated from each other. In conclusion, SFV-specific antibodies from infected humans do not block cell-to-cell transmission, at least in vitro , despite their capacity to bind to the surface of infected cells

Case-control study of the immune status of humans infected with zoonotic gorilla simian foamy viruses

This work was presented at the 26th conference on retroviruses and opportunistic infections (CROI) held in Seattle in 2019.International audienceBackground. Zoonotic simian foamy viruses (SFVs) establish persistent infections in humans, for whom the long-term consequences for health are poorly described. Here, we aimed to characterize blood-cell phenotypes and plasma biomarkers associated with gorilla SFV infection in humans. Methods. We used a case-control design to compare 15 Cameroonian hunters infected with gorilla SFV to 15 controls matched for age and ethnicity. A flow cytometry-based phenotypic study and quantification of soluble immune biomarkers were carried out on blood samples from all participants. Wilcoxon signed rank tests were used to compare cases and controls. Results. Cases had a significantly higher percentage of CD8 T lymphocytes than controls (median: 17.6% vs. 13.7%, P = 0.03), but similar levels of B, NK, and CD4 T lymphocytes. Cases also had a lower proportion of recent CD4 thymic emigrants (10.9% vs. 18.6%, P = 0.05), a higher proportion of programmed death receptor 1 (PD-1) expressing memory CD4 T lymphocytes (31.7% vs. 24.7%, P = 0.001), and higher plasma levels of the soluble CD163 scavenger receptor (0.84 vs 0.59 µg/mL, P = 0.003) than controls. Conclusions. We show, for the first time, that chronic infection with SFV is associated with T lymphocyte differentiation and monocyte activation

Phospholipidosis and down-regulation of the PI3-K/PDK-1/Akt signalling pathway are vitamin E inhibitable events associated with 7-ketocholesterol-induced apoptosis

International audienceAmong the oxysterols accumulating in atherosclerotic plaque, 7-ketocholesterol (7KC) is a potent apoptotic inducer, which favours myelin figure formation and polar lipid accumulation. This investigation performed on U937 cells consisted in characterizing the myelin figure formation process; determining the effects of 7KC on the PI3-K/PDK-1/Akt signalling pathway; evaluating the activities of vitamin E (Vit-E) (α-tocopherol) on the formation of myelin figures and the PI3-K/PDK-1/Akt signalling pathway and assessing the effects of PI3-K inhibitors (LY-294002, 3-methyladenine) on the activity of Vit-E on cell death and polar lipid accumulation. The ultrastructural and biochemical characteristics of myelin figures (multilamellar cytoplasmic inclusions rich in phospholipids and 7KC present in acidic vesicles and the reversibility of these alterations) support the hypothesis that 7KC is an inducer of phospholipidosis. This oxysterol also induces important changes in lipid content and/or organization of the cytoplasmic membrane demonstrated with merocyanine 540 and fluorescence anisotropy, a loss of PI3-K activity and dephosphorylation of PDK-1 and Akt. It is noteworthy that Vit-E was able to counteract phospholipidosis and certain apoptotic associated events (caspase activation, lysosomal degradation) to restore PI3-K activity and to prevent PDK-1 and Akt dephosphorylation. When Vit-E was associated with LY-294002 or 3-methyladenine, impairment of 7KC-induced apoptosis was inhibited, and accumulation of polar lipids was less counteracted. Thus, 7KC-induced apoptosis is a PI3-K-dependent event, and Vit-E up- and down-regulates PI3-K activity and phospholipidosis, respectively

Measurement of inflammatory cytokines by multicytokine assay in tears of patients with glaucoma topically treated with chronic drugs

International audienceAim To investigate the ocular surface inflammatory response to chronic topical treatments in patients with glaucoma by measuring the cytokine level in tears using multiplex bead analysis. Methods Tear samples were collected from 21 patients with glaucoma and 12 healthy volunteers. Tears were analysed for the presence of 17 cytokines: interleukin (IL)1β, IL2, IL4, IL5, IL6, IL7, IL8, IL10, IL12, IL13, IL17, granulocyte‐colony stimulating factor, granulocyte‐macrophage stimulating factor, interferon (INF)γ, monocyte chemotactic protein (MCP)1, macrophage inflammatory protein 1β and tumour necrosis factor (TNF)α. The cytokines in each sample of tears were measured using multiplex bead analysis with microspheres as solid support for immunoassays. Results In the tears of treated patients, proinflammatory cytokines (IL1β, IL6, IL12, TNFα) were significantly increased compared with controls. T helper (Th)1 (INFγ, IL2) and Th2 (IL5, IL10, IL4) type cytokines were also significantly higher (p<0.05); however, the most marked increase was observed with Th1 cytokines. The expression of chemokine IL8 and MCP1 was also increased in the treated group. Conclusion This study shows that pro‐inflammatory cytokine secretion by conjunctival cells is increased in response to topical treatments for glaucoma. The characterisation of cytokines in tears was previously limited by the small volume attainable, a limitation that has been overcome by multiplex analysis