Phenotypical Screening of an MMV Open Box Library and Identification of Compounds with Antiviral Activity against St. Louis Encephalitis Virus.

St. Louis encephalitis virus (SLEV) is a neglected mosquito-borne Flavivirus that may cause severe neurological disease in humans and other animals. There are no specific treatments against SLEV infection or disease approved for human use, and drug repurposing may represent an opportunity to accelerate the development of treatments against SLEV. Here we present a scalable, medium-throughput phenotypic cell culture-based screening assay on Vero CCL81 cells to identify bioactive compounds that could be repurposed against SLEV infection. We screened eighty compounds from the Medicines for Malaria Venture (MMV) COVID Box library to identify nine (11%) compounds that protected cell cultures from SLEV-induced cytopathic effects, with low- to mid-micromolar potencies. We validated six hit compounds using viral plaque-forming assays to find that the compounds ABT-239, Amiodarone, Fluphenazine, Posaconazole, Triparanol, and Vidofludimus presented varied levels of antiviral activity and selectivity depending on the mammalian cell type used for testing. Importantly, we identified and validated the antiviral activity of the anti-flavivirus nucleoside analog 7DMA against SLEV. Triparanol and Fluphenazine reduced infectious viral loads in both Vero CCL81 and HBEC-5i cell cultures and, similar to the other validated compounds, are likely to exert antiviral activity through a molecular target in the host.

COVID-19 inactivated and non-replicating viral vector vaccines induce regulatory training phenotype in human monocytes under epigenetic control.

Background: Trained immunity is the enhanced innate immune response resulting from exposure to pathogens or vaccines against an unrelated pathogen stimulus. Certain vaccines induce a memory like response in monocytes and NK cells, leading to modulation in cytokine production, metabolic changes, and modifications in histone patterns. Here, we hypothesized that vaccination against SARS-CoV-2 could induce the training of monocytes in addition to stimulating the adaptive immune response. Methods: Therefore, we aimed to investigate the immunophenotyping, cytokine and metabolic profile of monocytes from individuals who were completely immunized with two doses of inactivated COVID-19 vaccine or non-replicating viral vector vaccine. Subsequently, we investigated the epigenetic mechanisms underlying monocyte immune training. As a model of inflammatorychallenge, to understand if the monocytes were trained by vaccination and how they were trained, cells were stimulated in vitro with the endotoxin LPS, an unrelated stimulus that would provoke the effects of training. Results: When challenged in vitro, monocytes from vaccinated individuals produced less TNF-α and those who received inactivated vaccine produced less IL-6, whereas vaccination with non-replicating viral vector vaccine induced more IL-10. Inactivated vaccine increased classical monocyte frequency, and both groups showed higher CD163 expression, a hallmark of trained immunity. We observed increased expression of genes involved in glycolysis and reduced IRG1 expression in vaccinated subjects, a gene associated with the tolerance phenotype in monocytes. We observed that both vaccines reduced the chromatin accessibility of genes associated with the inflammatory response, the inactivated COVID-19 vaccine trained monocytes to a regulatory phenotype mediated by histone modifications in the IL6 and IL10 genes, while the non-replicating viral vector COVID-19 vaccine trained monocytes to a regulatory phenotype, mediated by histone modifications in the IL6, IL10, TNF, and CCL2 genes. Conclusions: Our findings support the recognized importance of adopting vaccination against SARS CoV-2, which has been shown to be effective in enhancing the adaptive immune response against the virus and reducing mortality and morbidity rates. Here, we provide evidence that vaccination also modulates the innate immune response by controlling the detrimental inflammatory response to unrelated pathogen stimulation.

Identification of insect-specific flaviviruses in areas of Brazil and Paraguay experiencing endemic arbovirus transmission and the description of a novel flavivirus infecting Sabethes belisarioi.

Viral infection was examined with pan-flavivirus and pan-alphavirus sets of primers in mosquitoes collected in four South American regions with confirmed pathogenic arbovirus circulation. Positive pools for flavivirus infection were sequenced and screened for specific arboviruses, which were not detected. However, NS5 gene sequencing showed that most sequences corresponded to the insect-specific Culex flavivirus. One sequence retrieved from an Aedes albopictus pool grouped with the insect-specific Aedes flavivirus and two Sabethes belisarioi pools were infected by a previously unknown flavivirus, tentatively named Sabethes flavivirus (SbFV). Phylogenetic inference placed SbFV as ancestral to a clade formed by Culiseta flavivirus, Mercadeo, and Calbertado. SbFV polyprotein showed an average aminoacidic identity of 51% in comparison to these flaviviruses. In vitro studies suggest that SbFV infects insect cells, but not vertebrate cells, therefore, we propose it as a new insect-specific flavivirus. These results highlight the wide distribution of insect-specific flaviviruses concomitant with the circulation of emergent arboviruses.

MyD88 Adapter-like (Mal)/TIRAP Is Required for Cytokine Production by Splenic Ly6CloTLR2hi but Not by Ly6ChiTLR2hi Monocytes during Trypanosoma cruzi Infection.

This study continues to explore the plasticity of Toll-like receptor 2 (TLR2) previously described in immune response during Trypanosoma cruzi infection. Here, we have shown that Ly6ChiTLR2hi monocytes were involved in TNF-α and IL-12 production, whereas Ly6CloTLR2hi monocytes were mainly committed to IL-10 and TNF-α production during T. cruzi infection independently of TLR agonist used (i.e. TLR2 or TLR9 agonists). Another difference between the monocyte populations is that the adapter Mal (encoded by TIRAP) has appeared crucial for the cytokine production by Ly6Clo but not by Ly6Chi monocytes. The protein Mal was necessary to induce cytokine synthesis by Ly6Clo monocytes after triggering TLR2 or TLR9. Finally, our data have suggested that TLR2, TLR9, and Mal/TIRAP controlled differentially the emergence of the different TLR2hi monocyte populations in the spleen. In summary, this study highlights the central role of the TLR2/Mal tandem in the distinct activity among the monocyte subsets during T. cruzi infection. Such findings provide a basis for understanding the challenge posed by the use of TLR2 agonist in immunotherapy.

Neutrophil paralysis in Plasmodium vivax malaria.

BACKGROUND: The activation of innate immune responses by Plasmodium vivax results in activation of effector cells and an excessive production of pro-inflammatory cytokines that may culminate in deleterious effects. Here, we examined the activation and function of neutrophils during acute episodes of malaria. MATERIALS AND METHODS: Blood samples were collected from P. vivax-infected patients at admission (day 0) and 30-45 days after treatment with chloroquine and primaquine. Expression of activation markers and cytokine levels produced by highly purified monocytes and neutrophils were measured by the Cytometric Bead Assay. Phagocytic activity, superoxide production, chemotaxis and the presence of G protein-coupled receptor (GRK2) were also evaluated in neutrophils from malaria patients. PRINCIPAL FINDINGS: Both monocytes and neutrophils from P. vivax-infected patients were highly activated. While monocytes were found to be the main source of cytokines in response to TLR ligands, neutrophils showed enhanced phagocytic activity and superoxide production. Interestingly, neutrophils from the malaria patients expressed high levels of GRK2, low levels of CXCR2, and displayed impaired chemotaxis towards IL-8 (CXCL8). CONCLUSION: Activated neutrophils from malaria patients are a poor source of pro-inflammatory cytokines and display reduced chemotactic activity, suggesting a possible mechanism for an enhanced susceptibility to secondary bacterial infection during malaria.