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This protocol offers an ex vivo method for screening host-targeting antivirals (HTAs) using human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs). Unlike virus-targeting antivirals (VTAs), HTAs provide advantages in overcoming drug resistance and offering broad-spectrum protection, especially against rapidly mutating or newly emerging viruses. By focusing on PBMCs or pDCs, known for their high production of humoral factors such as Type I interferons (IFNs), the protocol enables the screening of antivirals that modulate immune responses against viruses. Targeting host pathways, especially innate immunity, allows for species-independent antiviral activity, reducing the likelihood of viral escape mutations. Additionally, the protocol's versatility makes it a powerful tool for testing potential antivirals against various viral pathogens, including emerging viruses, positioning it as an essential resource in both pandemic preparedness and broad-spectrum antiviral research. This approach differentiates itself from existing protocols by focusing on host immune modulation through pDCs, offering a novel avenue for HTA discovery. Key features ? Optimized protocol for screening HTAs against dengue virus (DENV), chikungunya virus (CHIKV), and Zika virus (ZIKV).? This protocol is ideal for screening soluble or intravenous-formulated compounds for evaluating their efficacy in experimental settings.? This protocol builds upon the method developed by Tsuji et al. [1] and extends its application to PBMCs and testing against DENV, CHIKV, and ZIKV.

Previously, we reported that Lactobacillus paragasseri SBT2055 (LG2055) activates plasmacytoid dendritic cells (pDCs) and induces interferon alpha (IFN-¦Á) in vitro. Our clinical trial suggested that LG2055 intake may enhance pDC activity, supporting immune maintenance and reducing subjective common cold symptoms. However, the precise mechanisms remain unclear. In this study, we investigated how LG2055 engages with pDCs to stimulate IFN-¦Á production. We evaluated LG2055-induced pDC activation using flow cytometry, ELISA, and phagocytosis assays. Human peripheral blood mononuclear cells (PBMCs) were stimulated with LG2055 and its components to evaluate immune responses. An in vitro M cell model was used to examine LG2055 translocation. We found that DNA extracted from LG2055 activated pDCs and enhanced IFN-¦Á production via Toll-like receptor 9 (TLR9). Phagocytosis assays demonstrated that LG2055 DNA was internalized by PBMC-derived pDCs, enabling TLR9-mediated signaling. Additionally, LG2055 translocated across M cells in vitro, suggesting potential transport into Peyer¡¯s patches, where it may interact with pDCs. These findings demonstrate that intestinal LG2055 can translocate across M cells, interact with pDCs, and exert immune-stimulatory effects to enhance host antiviral immunity. This study provides mechanistic insight into how dietary components support immune health and could inform the development of novel functional foods or therapeutic strategies.

Plasmacytoid dendritic cells (pDCs) are innate sensors of viral infections and important mediators of antiviral innate immunity through their ability to produce large amounts of IFN-α. Moreover, Toll-like receptor 7 (TLR7) and 9 (TLR9) ligands, such as HIV and CpG respectively, turn pDCs into TRAIL-expressing killer pDCs able to lyse HIV-infected CD4+ T cells. NK cells can regulate antiviral immunity by modulating pDC functions, and pDC production of IFN-α as well as cell-cell contact is required to promote NK cell functions. Impaired pDC-NK cell crosstalk was reported in the setting of HIV-1 infection, but the impact of HIV-1 on TRAIL expression and innate antiviral immunity during this crosstalk is unknown. Here, we report that low concentrations of CCR5-tropic HIV-1Ba-L promote the release of pro-inflammatory cytokines such as IFN-α, TNF-α, IFN-γ and IL-12, and CCR5-interacting chemokines (MIP-1α and MIP-1β) in NK-pDCs co-cultures. At high HIV-1BaL concentrations, the addition of NK cells did not promote the release of these mediators, suggesting that once efficiently triggered by the virus, pDCs could not integrate new activating signals delivered by NK cells. However, high HIV-1BaL concentrations were required to trigger IFN-α-mediated TRAIL expression at the surface of both pDCs and NK cells during their crosstalk. Interestingly, we identified the alarmin HMGB1, released at pDC-NK cell synapse, as an essential trigger for the secretion of IFN-α and IFN-related soluble mediators during the interplay of HIV-1 exposed pDCs with NK cells. Moreover, HMGB1 was found crucial for mTRAIL translocation to the plasma membrane of both pDCs and NK cells during their crosstalk following pDC exposure to HIV-1. Data from serum analyses of circulating HMGB1, HMGB1-specific antibodies, sTRAIL and IP-10 in a cohort of 67 HIV-1+ patients argue for the in vivo relevance of these observations. Altogether, these findings identify HMGB1 as a trigger for IFN-α-mediated TRAIL expression at the surface of pDCs and NK cells, and they suggest a novel mechanism of innate control of HIV-1 infection.