Highly virulent Helicobacter pylori cause proinflammatory signaling inducing the transcriptional activation and secretion of cytokines such as IL-8 in epithelial cells. Responsible in part for this signaling is the cag pathogenicity island (cagPAI) that codetermines the risk for pathological sequelae of an H. pylori infection such as gastric cancer. The Cag type IV secretion system (CagT4SS), encoded on the cagPAI, can translocate various molecules into cells, the effector protein CagA, peptidoglycan metabolites and DNA. Although these transported molecules are known to contribute to cellular responses to some extent, a major part of the cagPAI-induced signaling leading to IL-8 secretion remains unexplained. We report here that biosynthesis of heptose-1,7-bisphosphate (HBP), an important intermediate metabolite of LPS inner heptose core, contributes in a major way to the H. pylori cagPAI-dependent induction of proinflammatory signaling and IL-8 secretion in human epithelial cells. Mutants defective in the genes required for synthesis of HBP exhibited a more than 95% reduction of IL-8 induction and impaired CagT4SS-dependent cellular signaling. The loss of HBP biosynthesis did not abolish the ability to translocate CagA. The human cellular adaptor TIFA, which was described before to mediate HBP-dependent activity in other Gram-negative bacteria, was crucial in the cagPAI- and HBP pathway-induced responses by H. pylori in different cell types. The active metabolite was present in H. pylori lysates but not enriched in bacterial supernatants. These novel results advance our mechanistic understanding of H. pylori cagPAI-dependent signaling mediated by intracellular pattern recognition receptors. They will also allow to better dissect immunomodulatory activities by H. pylori and to improve the possibilities of intervention in cagPAI- and inflammation-driven cancerogenesis.

T he human serotype 5 adenovirus (Ad5) is a nonenveloped linear double-stranded DNA virus associated with upper respiratory tract disease in humans. It has been extensively studied as a model for virus and host cell interactions. Replication-defective recombinant Ad5 vectors (rAdV) deleted in E1 and E3 coding domains have been characterized in gene therapy, vaccine, and oncolytic vector strategies in the murine model. Although nonpermissive for Ad5 replication, the murine model of rAdV infection provides a valuable resource for characterizing how the innate and adaptive immune response orchestrates an antiviral response to nonenveloped DNA viruses.Virus uptake by immune sentinel cells such as macrophage and dendritic cells is vital to initiating the antiviral immune response. In addition to antigen-presenting cells (APCs), other cell types, including endothelial cells or tissue-specific cells such as hepatocytes, when exposed to virus, also contribute to the host antiviral response. In vitro studies of isolated bone marrow-derived APCs or representative cell lines have revealed a cell-specific antiviral innate response, where activation of the type I interferon (IFN) cascade is a dominant feature (1-4). A valuable marker for early events in the antiviral recognition response is activation of the transcription factor interferon response factor 3 (IRF3). Following infection, cytosolic IRF3 undergoes phosphorylation as a primary response to adenovirus uptake. Activation occurs in a MyD88/TRIF-independent manner; it requires integrin-dependent endosomal entry, escape, and presentation of viral DNA to the cytosolic compartment (3).In rAdV-responsive murine cell lines, the STING/TBK1 cascade is required for IRF3 phosphorylation (5, 6). STING (7,8) functions as an adaptor linking DNA recognition signaling to activation of the TBK1 kinase. TBK1 activation (9) leads to C-terminal IRF3 phosphorylation, dimerization, and translocation to the nucleus (10, 11). In the nucleus, IRF3, in collaboration with additional transcription factors (NF-B and AP1), results in transcriptional activation of IRF3-responsive genes (including IFN-␤) (12). This sequence of events contributes to the primary antiviral response to adenovirus infection. The translation of primary response transcripts such as IFN-␤ leads to autocrine/paracrine secondary signaling. The combination of primary and secondary response functions leads to expression of a complete antiviral response, which is distinct for different cell types.Using various screening protocols, cell lines, and output assays, an extensive list of cytosolic DNA sensors, including DAI, RNA polymerase (Pol) III, IFI16, DDX41, and Aim 2, has been established (reviewed in reference 13). However, the DNA sensor involved in recognizing infection by adenovirus leading to early IRF3 activation has not been convincingly established. The recent identification of cyclic dinucleotide activation of STING (14-18) and the elegant discovery of cyclic-GMP-AMP synthase (cGAS) as a DNA sensor (19,20) provide an...

Neutralizing antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry into cells via surface-expressed angiotensin-converting enzyme 2 (ACE2). We used a surrogate virus neutralization test (sVNT) and SARS-CoV-2 S protein-pseudotyped vesicular stomatitis virus (VSV) vector-based neutralization assay (pVNT) to assess the degree to which serum antibodies from coronavirus disease 2019 (COVID-19) convalescent patients interfere with the binding of SARS-CoV-2 S to ACE2. Both tests revealed neutralizing anti-SARS-CoV-2 S antibodies in the sera of ~90% of mildly and 100% of severely affected COVID-19 convalescent patients. Importantly, sVNT and pVNT results correlated strongly with each other and to the levels of anti-SARS-CoV-2 S1 IgG and IgA antibodies. Moreover, levels of neutralizing antibodies correlated with the duration and severity of clinical symptoms but not with patient age. Compared to pVNT, sVNT is less sophisticated and does not require any biosafety labs. Since this assay is also much faster and cheaper, sVNT will not only be important for evaluating the prevalence of neutralizing antibodies in a population but also for identifying promising plasma donors for successful passive antibody therapy.

A fraction of COVID-19 convalescent individuals mount a potent antibody response to SARS-CoV-2 with cross-reactivity to SARS-CoV-1. To uncover their humoral response in detail, we performed single B-cell analysis from 10 SARS-CoV-2 elite neutralizers. We isolated and analyzed 126 monoclonal antibodies, many of which were sarbecovirus cross-reactive, with some displaying merbecovirus- and embecovirus-reactivity. Several isolated broadly neutralizing antibodies were effective against B.1.1.7, B1.351, B.1.429, B.1.617, B.1.617.2 variants and 19 prominent potential escape sites. Furthermore, assembly of 716,806 SARS-CoV-2 sequences predicted emerging escape variants, which were also effectively neutralized. One of these broadly neutralizing potent antibodies, R40-1G8, is a IGHV3-53 RBD-Class-1 antibody. Remarkably, Cryo-EM analysis revealed that R40-1G8 has a flexible binding mode, targeting both ‘up’ and ‘down’ conformations of the RBD. Given the threat of emerging SARS-CoV-2 variants, we demonstrate that elite neutralizers are a valuable source for isolating ultrapotent antibody candidates to prevent and treat SARS-CoV-2 infection.

Early recognition of viral infection by sentinel immune cells is key to induction of the innate and adaptive arms of antiviral immunity. In the case of adenovirus (Ad) and recombinant adenoviral vectors (rAdV), early recognition by antigen-presenting cells (APCs) (macrophages and dendritic cells) generates an antiviral response that is biased toward a type I interferon (IFN) pathway (8,32,49). Multiple viral components contribute to anti-Ad recognition by APCs, including viral capsid proteins (6, 34, 38), virus-dependent transcription (46), and the viral genome (8,29,31,32,49). In the murine model, recognition of the doublestranded DNA (dsDNA) viral genome occurs in a cell type-specific manner, where the Toll-like receptor 9 (TLR9) endosomal receptor responds to rAd DNA in plasmacytoid dendritic cells (DCs) (49), while a cytosolic DNA sensor is implicated in primary murine macrophages and classical dendritic cells (29,32,49).Depending on the cellular environment, the murine APC response to rAdV can trigger distinct antiviral cascades. The classic antiviral interferon response is initiated by activation of interferon response factor 3 (IRF3) and contributes to type I IFN gene expression. Activated IRF3 in combination with NF-B and ATF-2/ cJUN binds to the beta interferon (IFN-␤) promoter, leading to early expression of IFN-␤ mRNA as well as a number of other IRF3-dependent transcription units (31). Secretion of type I IFNs (and other chemokines and cytokines) by the activated cell leads to paracrine-autocrine signaling, which amplifies the antiviral response. Type I interferon binding to the IFN-␣/␤ receptor triggers Janus kinase phosphorylation of Stat1 and STAT2, which combine with IRF9 to form the heterotrimeric ISGF3 transcription factor. In the case of macrophage and conventional dendritic cells, the culmination of the primary and secondary antiviral cascades is APC maturation from a naïve to a mature phenotype (8,31,32).A second antiviral DNA response leads to inflammasome formation (29) and is characterized by caspase-1 cleavage of prointerleukin-1 beta (pro-IL-1␤) to IL-1␤ (as well as processing of pro-IL-18 to IL-18) (reviewed in references 22 and 33). In the case of rAdV, initial studies characterizing inflammasome activation were carried out using primary murine macrophage primed with lipopolysaccharide (LPS) (29). rAdV stimulation of the Caspase-1/IL-1␤ inflammasome pathway was dependent on both NODlike receptor 3 (NLRP3) and apoptosis-associated speck-like protein (ASC). Macrophages derived from either ASC or NLRP3 knockout (KO) mice were compromised in inflammasome activation by rAdV, but IRF3 activation remained intact. In contrast to viral infection, when viral DNA (vDNA) was introduced through chemical transduction, ASC but not NLRP3 was required for Caspase-1 cleavage and secretion of IL-1␤. Neither ASC nor NLRP3 contains known DNA binding domains. For both inflammasome and interferon antiviral response cascades, a cytosolic DNA sensor has been proposed, but the nature of the Ad DNA sensor has n...

The Latency-Associated Nuclear Antigen (LANA) is required for latent replication and persistence of Kaposi´s Sarcoma-associated Herpesvirus (KSHV)/human herpesvirus-8 (HHV-8). It acts via replicating and tethering the virus episome to the host chromatin and exert other functions. We conceived a new approach for the discovery of antiviral drugs to inhibit the interaction between LANA and the viral genome. We applied a biophysical screening cascade and identified the first LANA binders from small, structurally diverse compound libraries. Starting from a fragment-sized scaffold, we generated optimized hits via fragment growing using a dedicated fluorescence polarization-based assay as the structure-activity-relationship driver. We improved compound potency to the double-digit micromolar range. Importantly, we qualified the resulting hit through orthogonal methods employing EMSA, STD-NMR and MST methodologies. This optimized hit provides an ideal starting point for subsequent hit-to-lead campaigns providing evident target-binding, suitable ligand efficiencies and favorable physicochemical properties.

The human gastric pathogen Helicobacter pylori activates human epithelial cells by a particular combination of mechanisms, including NOD1 and ALPK1-TIFA activation. These mechanisms are characterized by a strong participation of the bacterial cag pathogenicity island, which forms a type IV secretion system (CagT4SS) that enables the bacteria to transport proteins and diverse bacterial metabolites, including DNA, glycans, and cell wall components, into human host cells. Building on previous findings, we sought to determine the contribution of lipopolysaccharide inner core heptose metabolites (ADP-heptose) in the activation of human phagocytic cells by H. pylori. Using human monocyte/macrophage-like Thp-1 cells and human primary monocytes and macrophages, we were able to determine that a substantial part of early phagocytic cell activation, including NF-κB activation and IL-8 production, by live H. pylori is triggered by bacterial heptose metabolites. This effect was very pronounced in Thp-1 cells exposed to bacterial purified lysates or pure ADP-heptose, in the absence of other bacterial MAMPs, and was significantly reduced upon TIFA knock-down. Pure ADP-heptose on its own was able to strongly activate Thp-1 cells and human primary monocytes/macrophages. Comprehensive transcriptome analysis of Thp-1 cells co-incubated with live H. pylori or pure ADP-heptose confirmed a signature of ADP-heptose-dependent transcript activation in monocyte/macrophages. Bacterial enzyme-treated lysates (ETL) and pure ADP-heptose–dependent activation differentiated monocytes into macrophages of predominantly M1 type. In Thp-1 cells, the active CagT4SS was less required for the heptose-induced proinflammatory response than in epithelial cells, while active heptose biosynthesis or pure ADP-heptose was required and sufficient for their early innate response and NF-κB activation. The present data suggest that early activation and maturation of incoming and resident phagocytic cells (monocytes, macrophages) in the H. pylori–colonized stomach strongly depend on bacterial LPS inner core heptose metabolites, also with a significant contribution of an active CagT4SS.

Neutralizing antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry into cells using surface-expressed angiotensin-converting enzyme 2 (ACE2). We developed a surrogate neutralization test (sVNT) to assess at what degree serum antibodies interfere with the binding of SARS-CoV-2-S-RBD to ACE2. The sVNT revealed neutralizing anti-SARS-CoV-2-S antibodies in the sera of 90% of mildly and 100% of severely affected coronavirus-disease-2019 (COVID-19) convalescent patients. Importantly, sVNT results correlated strongly to the results from pseudotyped-vesicular stomatitis virus-vector-based neutralization assay and to levels of anti-SARS-CoV-2-S1 IgG and IgA antibodies. Moreover, levels of neutralizing antibodies also correlated to duration and severity of clinical symptoms, but not patient age or gender. These findings together with the sVNT will not only be important for evaluating the prevalence of neutralizing antibodies in a population but also for identifying promising plasma donors for successful passive antibody therapy.