A new duplex qPCR-based method to quantify Mycoplasma mycoides in complex cell culture systems and host tissues.

Bacterial pathogen-host interactions are a complex process starting with adherence and colonization followed by a variety of interactions such as invasion or cytotoxicity on one hand and pathogen recognition, secretion of proinflammatory/antibacterial substances and enhancing the barrier function of epithelial layers on the other hand. Therefore, a variety of in vitro, ex vivo and in vivo models have been established to investigate these interactions. Some in vitro models are composed of different cell types and extracellular matrices such as tissue explants or precision cut lung slices. These complex in vitro models mimic the in vivo situation more realistically, however, they often require new and more sophisticated methods for quantification of experimental results. Here we describe a multiplex qPCR-based method to quantify the number of bacteria of Mycoplasma (M.) mycoides interacting with their hosts in an absolute manner as well as normalized to the number of host cells. We choose the adenylate kinase (adk) gene from the pathogen and the Carcinoembryonic antigen-related cell adhesion molecule 18 (CEACAM18) gene from the host to determine cell numbers by a TaqMan-based assay system. Absolute copy numbers of the genes are calculated according to a standard containing a defined number of plasmids containing the sequence which is amplified by the qPCR. The new multiplex qPCR therefore allows the quantification of M. mycoides interacting with host cells in suspension, monolayer, 3D cell culture systems as well as in host tissues.

Comparative analysis of ChAdOx1 nCoV-19 and Ad26.COV2.S SARS-CoV-2 vector vaccines.

Vector-based SARS-CoV-2 vaccines have been associated with vaccine- induced thrombosis with thrombocytopenia syndrome (VITT/TTS), but the causative factors are still unresolved. We comprehensively analyzed the ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Johnson and Johnson) vaccines. ChAdOx1 nCoV-19 contains significant amounts of host cell protein impurities, including functionally active proteasomes, and adenoviral proteins. A much smaller amount of impurities was found in Ad26.COV2.S. Platelet factor 4 formed complexes with ChAdOx1 nCoV-19 constituents, but not with purified virions from ChAdOx1 nCoV-19 or with Ad26.COV2.S. Vascular hyperpermeability was induced by ChAdOx nCoV-19 but not by Ad26.COV2.S. These differences in impurities together with EDTAinduced capillary leakage might contribute to the higher incidence rate of VITT associated with ChAdOx1 nCoV-19 compared to Ad26.COV2.S.

Point Mutations in the Glycoprotein Ectodomain of Field Rabies Viruses Mediate Cell Culture Adaptation through Improved Virus Release in a Host Cell Dependent and Independent Manner.

Molecular details of field rabies virus (RABV) adaptation to cell culture replication are insufficiently understood. A better understanding of adaptation may not only reveal requirements for efficient RABV replication in cell lines, but may also provide novel insights into RABV biology and adaptation-related loss of virulence and pathogenicity. Using two recombinant field rabies virus clones (rRABV Dog and rRABV Fox), we performed virus passages in three different cell lines to identify cell culture adaptive mutations. Ten passages were sufficient for the acquisition of adaptive mutations in the glycoprotein G and in the C-terminus of phosphoprotein P. Apart from the insertion of a glycosylation sequon via the mutation D247N in either virus, both acquired additional and cell line-specific mutations after passages on BHK (K425N) and MDCK-II (R346S or R350G) cells. As determined by virus replication kinetics, complementation, and immunofluorescence analysis, the major bottleneck in cell culture replication was the intracellular accumulation of field virus G protein, which was overcome after the acquisition of the adaptive mutations. Our data indicate that limited release of extracellular infectious virus at the plasma membrane is a defined characteristic of highly virulent field rabies viruses and we hypothesize that the observed suboptimal release of infectious virions is due to the inverse correlation of virus release and virulence in vivo.

Insights in ChAdOx1 nCoV-19 vaccine-induced immune thrombotic thrombocytopenia.

SARS-CoV-2 vaccine ChAdOx1 nCoV-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models, and analysis of VITT patient samples, we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the ethylenediaminetetraacetic acid (EDTA)-containing vaccine. Injected vaccine increased vascular leakage in mice, leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B-cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release neutrophil extracellular traps (NETs) in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drives thrombosis in VITT. The data support a 2-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.

The Erns Carboxyterminus: Much More Than a Membrane Anchor.

Pestiviruses express the unique essential envelope protein Erns, which exhibits RNase activity, is attached to membranes by a long amphipathic helix, and is partially secreted from infected cells. The RNase activity of Erns is directly connected with pestivirus virulence. Formation of homodimers and secretion of the protein are hypothesized to be important for its role as a virulence factor, which impairs the host's innate immune response to pestivirus infection. The unusual membrane anchor of Erns raises questions with regard to proteolytic processing of the viral polyprotein at the Erns carboxy-terminus. Moreover, the membrane anchor is crucial for establishing the critical equilibrium between retention and secretion and ensures intracellular accumulation of the protein at the site of virus budding so that it is available to serve both as structural component of the virion and factor controlling host immune reactions. In the present manuscript, we summarize published as well as new data on the molecular features of Erns including aspects of its interplay with the other two envelope proteins with a special focus on the biochemistry of the Erns membrane anchor.

Chlamydia psittaci-Infected Dendritic Cells Communicate with NK Cells via Exosomes To Activate Antibacterial Immunity.

Dendritic cells (DCs) and natural killer (NK) cells are critically involved in the early response against various bacterial microbes. Functional activation of infected DCs and NK cell-mediated gamma interferon (IFN-γ) secretion essentially contribute to the protective immunity against Chlamydia How DCs and NK cells cooperate during the antichlamydial response is not fully understood. Therefore, in the present study, we investigated the functional interplay between Chlamydia-infected DCs and NK cells. Our biochemical and cell biological experiments show that Chlamydia psittaci-infected DCs display enhanced exosome release. We find that such extracellular vesicles (referred to as dexosomes) do not contain infectious bacterial material but strongly induce IFN-γ production by NK cells. This directly affects C. psittaci growth in infected target cells. Furthermore, NK cell-released IFN-γ in cooperation with tumor necrosis factor alpha (TNF-α) and/or dexosomes augments apoptosis of both noninfected and infected epithelial cells. Thus, the combined effect of dexosomes and proinflammatory cytokines restricts C. psittaci growth and attenuates bacterial subversion of apoptotic host cell death. In conclusion, this provides new insights into the functional cooperation between DCs, dexosomes, and NK cells in the early steps of antichlamydial defense.

NK Cell-Mediated Processing Of Chlamydia psittaci Drives Potent Anti-Bacterial Th1 Immunity.

Natural killer (NK) cells are innate immune cells critically involved in the early immune response against various pathogens including chlamydia. Here, we demonstrate that chlamydia-infected NK cells prevent the intracellular establishment and growth of the bacteria. Upon infection, they display functional maturation characterized by enhanced IFN-γ secretion, CD146 induction, PKCϴ activation, and granule secretion. Eventually, chlamydia are released in a non-infectious, highly immunogenic form driving a potent Th1 immune response. Further, anti-chlamydial antibodies generated during immunization neutralize the infection of epithelial cells. The release of chlamydia from NK cells requires PKCϴ function and active degranulation, while granule-associated granzyme B drives the loss of chlamydial infectivity. Cellular infection and bacterial release can be undergone repeatedly and do not affect NK cell function. Strikingly, NK cells passing through such an infection cycle significantly improve their cytotoxicity. Thus, NK cells not only protect themselves against productive chlamydial infections but also actively trigger potent anti-bacterial responses.

Characterization of gene deletion mutants of Cyprinid herpesvirus 3 (koi herpesvirus) lacking the immunogenic envelope glycoproteins pORF25, pORF65, pORF148 and pORF149.

Cyprinid herpesvirus 3 (CyHV-3) or koi herpesvirus is a global pathogen causing mass mortality in koi and common carp, against which improved vaccines are urgently needed. In this study we investigated the role of four nonessential, but immunogenic envelope glycoproteins encoded by members of the ORF25 gene family (ORF25, ORF65, ORF148 and ORF149) during CyHV-3 replication. Single deletion of ORF65 did not affect in vitro replication, and deletion of ORF148 even slightly enhanced virus growth on common carp brain (CCB) cells. Deletions of ORF25 or ORF149 led to reduced plaque sizes and virus titers, which was due to delayed entry into host cells. An ORF148/ORF149 double deletion mutant exhibited wild-type like growth indicating opposing functions of the two proteins. Electron microscopy of CCB cells infected with either mutant did not indicate any effects on virion formation and maturation in nucleus or cytoplasm, nor on release of enveloped particles. The ORF148, ORF149 and double deletion mutants were also tested in animal experiments using juvenile carp, and proved to be insufficiently attenuated for use as live virus vaccines. However, surviving fish were protected against challenge with wild-type CyHV-3, demonstrating that these antibody inducing proteins are dispensable for an efficient immune response in vivo.

High level expression of the Drosophila Toll receptor ectodomain and crystallization of its complex with the morphogen Spätzle.

Drosophila Toll receptors are involved in embryonic development and in the immune response of adult flies. In both processes, the Toll receptor ligand is the NGF-like cystine knot protein Spätzle. Here we present the expression of Toll receptor ectodomain in Schneider cells at high yields and demonstrate a high affinity interaction with the refolded and trypsin-processed Spätzle cystine knot domain dimer. Poorly and anisotropically diffracting crystals of the complex could be improved by deglycosylation and dehydration, paving the way for structural analyses of the Toll-Spätzle interaction.