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.