Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein

RNA-dependent RNA polymerases (RdRps) play a key role in the life cycle of RNA viruses and impact their immunobiology. The arenavirus lymphocytic choriomeningitis virus (LCMV) strain Clone 13 provides a benchmark model for studying chronic infection. A major genetic determinant for its ability to persist maps to a single amino acid exchange in the viral L protein, which exhibits RdRp activity, yet its functional consequences remain elusive. To unravel the L protein interactions with the host proteome, we engineered infectious L protein-tagged LCMV virions by reverse genetics. A subsequent mass-spectrometric analysis of L protein pulldowns from infected human cells revealed a comprehensive network of interacting host proteins. The obtained LCMV L protein interactome was bioinformatically integrated with known host protein interactors of RdRps from other RNA viruses, emphasizing interconnected modules of human proteins. Functional characterization of selected interactors highlighted proviral (DDX3X) as well as antiviral (NKRF, TRIM21) host factors. To corroborate these findings, we infected Trim21-/-mice with LCMV and found impaired virus control in chronic infection. These results provide insights into the complex interactions of the arenavirus LCMV and other viral RdRps with the host proteome and contribute to a better molecular understanding of how chronic viruses interact with their host

The role of antigen cross-presentation in the vaccine-induced activation of cytotoxic T-lymphocytes

Antigen delivery systems that are efficient in inducing CD8+ T-cell mediated immune responses are required for the development of novel vaccines against intracellular pathogens and cancer. Although many new approaches were already investigated in mice and also humans, low immunogenicity of tested vaccines is still a major challenge in the field. Therefore it is important to study the molecular and cellular function of particular vaccines, in order to develop strategies that overcome this limitation. Beside “directpresentation” of endogenous proteins, antigen “cross-presentation” by professional antigen presenting cells (APCs) is an essential pathway for the major histocompatibility complex (MHC) class I presentation of exogenous antigens. It becomes more and more evident that cross-presentation is not only involved in physiological responses to various pathogens and malignancies, but also mediates specific cytotoxicity in response to vaccines. This thesis aimed on analyzing the molecular and cellular requirements for antigen cross-presentation, especially in response to selected vaccines. In chapter I we characterized the cell types involved in the cross-presentation of antigens that are encapsulated into a particulate biodegradable vaccine. These poly(lactic-co-glycolic) acid (PLGA) microspheres are approved as drug delivery system in humans and display promising properties to serve as therapeutic vaccine against cancer. In the murine system, we could show that cross-presentation of PLGA MS-encapsulated antigen can be performed by both dendritic cells (DCs) and macrophages. In contrast to the current idea that CD8+ DCs are the important cell type in cross-presentation we provide evidence for a dominant role of CD8- DCs and macrophages. In chapter II we investigated the intracellular fate of PLGA MS after phagocytosis by APCs. Based on the encapsulation of inorganic nanocrystals, we introduce a novel method to label PLGA MS for electron and fluorescent microscopy, as well as magnetic cell sorting. Using these tools, we were able to show that PLGA MS do not enter the cytosol after uptake, but are stored in lysosomal vesicles for at least 72h. Since cross-presentation of encapsulated antigen already occurred at much earlier time points, our data have implications on the intracellular cross-presentation pathway. In chapter III we studied the molecular requirements concerning antigen-stability on the efficiency of DNA vaccines and immune responses induced by recombinant vaccinia virus (VV). We could show that targeting antigens for rapid degradation increases direct-presentation in vitro, but in contrast inhibits in vivo responses to DNA vaccination and recombinant VV. The results shown here argue for a dominant role of cross-presentation in the systems analyzed. In chapter IV we investigated the role of heat-shock proteins (HSPs) and other cellular factors on the cross-presentation of cell-associated antigen. We provide evidence that the stable full-length protein, but not antigenic peptides, is the source of antigen transfer to APCs. At the same time we exclude several HSPs as mediators for cross-presentation in this system. Finally we show biochemical strategies to find interaction partners of a viral antigen that can mediate the transfer to APCs. Taken together the data accumulated in this thesis contribute to the development of novel strategies to enhance immune responses following vaccination

Abolishing carceral geographies?

No abstract available

Challenges in developing personalized neoantigen cancer vaccines

The recent success of cancer immunotherapies has highlighted the benefit of harnessing the immune system for cancer treatment. Vaccines have a long history of promoting immunity to pathogens and, consequently, vaccines targeting cancer neoantigens have been championed as a tool to direct and amplify immune responses against tumours while sparing healthy tissue. In recent years, extensive preclinical research and more than one hundred clinical trials have tested different strategies of neoantigen discovery and vaccine formulations. However, despite the enthusiasm for neoantigen vaccines, proof of unequivocal efficacy has remained beyond reach for the majority of clinical trials. In this Review, we focus on the key obstacles pertaining to vaccine design and tumour environment that remain to be overcome in order to unleash the true potential of neoantigen vaccines in cancer therapy.</p

Near-Infrared Bioluminescence Imaging of Macrophage Sensors for Cancer Detection In Vivo

Melanoma is an aggressive type of skin cancer with a poor prognosis after it gets metastasized. The early detection of malignant melanoma is critical for effective therapy. Because melanoma often resembles moles, routine skin check-up may help for timely identification of suspicious areas. Recently, it has been shown that the interplay of melanoma cells with the immune system can help develop efficient therapeutic strategies. Here, we leveraged engineered macrophages (BMC2) as cell-based sensors for metastatic melanoma. To perform dual-color bioluminescence imaging (BLI) in vivo, macrophages were engineered to express a green click beetle luciferase (CBG2) and a near-infrared fluorescent dye (DiR), and B16F10 melanoma cells were instead engineered to express a near-infrared click beetle luciferase (CBR2). Using real-time in vivo dual-color BLI and near-infrared fluorescence (FL) imaging, we could demonstrate that macrophages were able to sense and substantially accumulate in subcutaneous and metastatic melanoma tissues at 72 h after systemic injections. Together, we showed the potentiality to use optical imaging technologies to track circulating macrophages for the non-invasive detection of metastatic melanoma

Microencapsulation of inorganic nanocrystals into PLGA microsphere vaccines enables their intracellular localization in dendritic cells by electron and fluorescence microscopy

Biodegradable poly-(D,L-lactide-co-glycolide) microspheres (PLGA-MS) are approved as a drug delivery system in humans and represent a promising antigen delivery device for immunotherapy against cancer. Immune responses following PLGA-MS vaccination require cross-presentation of encapsulated antigen by professional antigen presenting cells (APCs). While the potential of PLGA-MS as vaccine formulations is well established, the intracellular pathway of cross-presentation following phagocytosis of PLGA-MS is still under debate. A part of the controversy stems from the difficulty in unambiguously identifying PLGA-MS within cells. Here we show a novel strategy for the efficient encapsulation of inorganic nanocrystals (NCs) into PLGA-MS as a tool to study their intracellular localization. We microencapsulated NCs as an electron dense marker to study the intracellular localization of PLGA-MS by transmission electron microscopy (TEM) and as fluorescent labels for confocal laser scanning microscopy. Using this method, we found PLGA-MS to be rapidly taken up by dendritic cells and macrophages. Co-localization with the lysosomal marker LAMP1 showed a lysosomal storage of PLGA-MS for over two days after uptake, long after the initiation of cross-presentation had occurred. Our data argue against an escape of PLGA-MS from the endosome as has previously been suggested as a mechanism to facilitate cross-presentation of PLGA-MS encapsulated antigen

Stable Antigen is most effective for eliciting CD8 T-Cell responses after DNA vaccination and infection with recombinant Vaccinia Virus in vivo

The induction of strong CD8 T-cell responses against infectious diseases and cancer has remained a major challenge. Depending on the source of antigen and the infectious agent, priming of CD8 T cells requires direct and/or cross-presentation of antigenic peptides on major histocompatibility complex (MHC) class I molecules by professional antigen-presenting cells (APCs).However, both pathways show distinct preferences concerning antigen stability. Whereas direct presentation was shown to efficiently present peptides derived from rapidly degraded proteins, cross-presentation is dependent on long-lived antigen species.In this report, we analyzed the role of antigen stability on DNA vaccination and recombinant vaccinia virus (VV) infection using altered versions of the same antigen. The long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) can be targeted for degradation by N-terminal fusion to ubiquitin or, as we show here, to the ubiquitin-like modifier FAT10. Direct presentation by cells either transfected with NP-encoding plasmids or infected with recombinant VV in vitro was enhanced in the presence of short-lived antigens. In vivo, however, the highest induction of NP-specific CD8 T-cell responses was achieved in the presence of long-lived NP. Our experiments provide evidence that targeting antigens for proteasomal degradation does not improve the immunogenicity of DNA vaccines and recombinant VVs. Rather, it is the long-lived antigen that is superior for the efficient activation of MHC class I-restricted immune responses in vivo. Hence, our results suggest a dominant role for antigen cross-priming in DNA vaccination and recombinant VV infection

Sec22b and Stx4 Depletion Has No Major Effect on Cross-Presentation of PLGA Microsphere-Encapsulated Antigen and a Synthetic Long Peptide In Vitro

The induction of CTL responses by vaccines is important to combat infectious diseases and cancer. Biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres and synthetic long peptides are efficiently internalized by professional APCs and prime CTL responses after cross-presentation of Ags on MHC class I molecules. Specifically, they mainly use the cytosolic pathway of cross-presentation that requires endosomal escape, proteasomal processing, and subsequent MHC class I loading of Ags in the endoplasmic reticulum (ER) and/or the endosome. The vesicle SNARE protein Sec22b has been described as important for this pathway by mediating vesical trafficking for the delivery of ER-derived proteins to the endosome. As this function has also been challenged, we investigated the role of Sec22b in cross-presentation of the PLGA microsphere-encapsulated model Ag OVA and a related synthetic long peptide. Using CRISPR/Cas9-mediated genome editing, we generated Sec22b knockouts in two murine C57BL/6-derived APC lines and found no evidence for an essential role of Sec22b. Although pending experimental evidence, the target SNARE protein syntaxin 4 (Stx4) has been suggested to promote cross-presentation by interacting with Sec22b for the fusion of ER-derived vesicles with the endosome. In the current study, we show that, similar to Sec22b, Stx4 knockout in murine APCs had very limited effects on cross-presentation under the conditions tested. This study contributes to characterizing cross-presentation of two promising Ag delivery systems and adds to the discussion about the role of Sec22b/Stx4 in related pathways. Our data point toward SNARE protein redundancy in the cytosolic pathway of cross-presentation.</p