Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication. In this study, we aimed to further characterize the role of macrophage-derived EVs in immune responses against hepatitis C virus (HCV) and the potential of polyunsaturated fatty acids (PUFAs) to modulate this modality of innate immunity. To this end, EVs were isolated from interferon-stimulated macrophage cultures or from serum of patients with acute or chronic hepatitis C. EVs were characterized by electron microscopy, flow cytometry, RNA-sequencing, and Western blot analysis. The effect of EVs on replication of HCV was assessed in coculture models. Functional analyses were performed to assess the impact of PUFAs on EV-mediated antiviral immunity. We found that macrophages secreted various cytokines shortly after stimulation with type I and II IFN, which orchestrated a fast but short-lasting antiviral state. This rapid innate immune answer was followed by the production of macrophage-derived EVs, which induced a late, but long-lasting inhibitory effect on HCV replication. Of note, exposure of macrophages to PUFAs, which are important regulators of immune responses, dampened EV-mediated antiviral immune responses. Finally, EVs from patients with hepatitis C exhibited long-lasting antiviral activities during IFN therapy as well. The antiviral effect of EVs from Caucasian and Japanese patients differed, which may be explained by different nutritional uptake of PUFAs. In conclusion, our data indicate that macrophage-derived EVs mediate long-lasting inhibitory effects on HCV replication, which may bridge the time until efficient adaptive immune responses are established, and which can be blunted by PUFAs.

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For human hepatitis B virus eight distinct and two candidate genotypes are described. These genotypes differ with respect to geographic distribution, molecular virology and virus-associated pathogenesis. Comparative analysis of HBV genotypes revealed, with exception of HBV/G that shows impaired HBsAg release, that no fundamental disparities between genotypes exist regarding glycosylation, subcellular distribution, release of HBsAg and formation of subviral particles. However, there are distinctions regarding the proportion of L to M to S HBs proteins detected intra- and extracellularly for different genotypes. 2D electrophoresis revealed different posttranslational modification patterns for LHBs. In light of the relevance of HBsAg as diagnostic marker, detectability of purified recombinant HBsAg of various genotypes by HBsAg-specific detection systems licensed in Europe was investigated, showing similar sensitivities for genotypes included in this analysis. These data indicate that recombinant HBsAg reproducibly purified following a defined protocol might be used as an alternative to reference materials currently established.

Summary Background Naturally occurring variants with deletions or mutations in the C‐terminal PreS1 domain from hepatitis B virus (HBV) chronically infected patients have been shown to promote HBsAg retention, inhibit HBsAg secretion and change the extracellular appearance of PreS1‐containing HBV particles (filaments and virions). Aims To study the impact of N‐terminal deletion in preS1 domain on viral secretion and morphogenesis. Methods An HBV mutant with 15 amino acids (aa 25‐39) deletion in N‐terminal preS1 was isolated. Intracellular and extracellular HBsAg were quantified by Western blot. Subcellular HBsAg distribution was analysed by confocal laser scanning microscopy. The viral morphology was characterised by sucrose density gradient ultracentrifugation, Western blot, electron microscopy, HBV mixed ELISA and HBV particle gel essay. Results Expression of this mutant genome released higher amounts of HBsAg in the form of shorter filaments. A significant fraction of semi‐enveloped virions was observed in the supernatant that has been unprecedented so far. Stepwise insertion of aa 25‐31, aa 32‐39 and aa 25‐39 increased the length of filaments. The rescue of aa 25‐31 and aa 25‐39 drastically reduced the amounts of extracellular HBsAg and semi‐enveloped virions, while such effects could not be observed after insertion of aa 32‐39, arguing against a simple spacer function of this region. The deletion and rescued mutants do not differ in subcellular HBsAg distribution and colocalisation with ER, Golgi and multivesicular bodies markers arguing against differences in release pathways. Conclusion N‐terminal PreS1‐domain (aa 25‐31) determines HBsAg secretion and triggers proper assembly of PreS1‐containing particles.

Vaccine platforms that can be flexibly loaded with antigens can contribute to decrease response time to emerging infections. For many pathogens and chronic infections, induction of a robust cytotoxic T lymphocytes-mediated response is desirable to control infection. Antigen delivery into the cytoplasm of antigen presenting cells favors induction of cytotoxic T cells. By fusion of the cell-permeable translocation motif (TLM)-peptide to the capsid-forming core protein of hepatitis B virus, and by insertion of the strep-tag in the spike tip (a domain that protrudes from the surface of the capsid), cell-permeable carrier capsids were generated that can be flexibly loaded with various antigens. Loading with antigens was demonstrated by electron microscopy, density gradient centrifugation and surface plasmon resonance spectroscopy. Confocal immunofluorescence microscopy showed that cell-permeable carrier capsids mediate transfer of cargo antigen into the cytoplasm. Using cell-permeable carrier capsids loaded with ovalbumin as model antigen, activation of antigen presenting cells and ovalbumin-specific CD8+ T-cells, which correlates with enhanced specific killing activity, was found. This demonstrates the capacity of TLM-carrier-capsids to serve as universal antigen carrier to deliver antigens into the cytoplasm of antigen presenting cells, which leads to enhanced MHC class I-mediated presentation and induction of antigen-specific cytotoxic T lymphocytes response.