Treating diabetes using novel biocompatible biomaterials for islet cell transplantation creating an immune privileged site

Diabetes mellitus ist eine metabolische Krankheit, die 422 Millionen Menschen weltweit betrifft. Dies bedeutet eine große finanzielle Belastung für weltweite Finanz- und Gesundheitssysteme. Typ 2 Diabetes PatientInnen leiden an einer Insulin Resistenz, welche oft auf ungesunde Ernährung, fehlende Bewegung und andere Umwelteinflüsse zurückzuführen ist. Typ 1 Diabetes PatientInnen sind von Insulin Injektionen abhängig, um fehlende Insulinproduktion zu kompensieren, welche durch die Zerstörung von pankreatischen Beta Zellen durch Autoantikörper verursacht wird. ELASTISLET ist ein innovativer Versuch pankreatische Beta Zellen zu transplantieren, um eine permanente und autonome Insulin Therapie, für Typ I Diabetiker, zu schaffen. Diese Inselzellen sind durch eine semi-artifizielle Hülle immunisoliert, welche aus Elastin-ähnlichen Rekombinanten (ELRs) besteht, die inert und permeabel für den Austausch von Nährstoffen, Sauerstoff, Glukose und Insulin ist. Dennoch soll diese Hülle Immunzellen und Antikörper von dieser immun privilegierten Nische ausschließen. Weiterst soll die Bildung einer fibrotischen Kapsel um das Implantat verhindert werden. Unser Ziel ist es zu testen, ob dieses Umhüllungsmaterial Immunreaktionen, Allergien oder Fremdkörperreaktionen, nach der in vivo Implantation in BALB/c Mäusen, hervorruft. Die differentiale Zellzählung, Zytokin Messung und Histologie zeigen, dass die ELRs keine Entzündungen oder Zytokin Produktion verursachen. Dennoch war der IgG1 Titer des Blutserums erhöht. Eine zweite Implantation des Materials hat keine verstärkte Antikörperproduktion verursacht. Diese Ergebnisse unterstützen unsere These, dass die ELRs für die Transplantation von Inselzellen geeignet sind. Es bleibt noch herauszufinden, ob dieses Material eine permanente und autonome Insulin Therapie durch ummantelte Inselzellen ermöglichen kann.Diabetes mellitus is a metabolic disease affecting 422 million people worldwide, which puts a massive financial burden on health systems and patients. Type 2 diabetes is characterized by progressing insulin resistance, due to unhealthy nutrition, lacking exercise and other environmental factors. Type 1 diabetes patients are dependent on insulin shots to compensate their lack of insulin, caused by the destruction of insulin producing beta cells by autoantibodies. ELASTISLET is an innovative attempt of transplanting functional pancreatic islets for establishing a long lasting and autonomous insulin therapy in type I diabetes patients. These islet cells are immunoisolated by a semi-artificial capsule composed of Elastin-like recombinamers (ELRs), which should be inert and semipermeable to exchange nutrients, oxygen, glucose and insulin. Nevertheless, immune cells and antibodies have to be isolated from the immune privileged site. Furthermore, the formation of a fibrotic capsule, around the implant, should be avoided. Our aim is to test whether this coating material is triggering an immune, allergic or foreign body response upon intraperitoneal and subcutaneous implantation in BALB/c mice in vivo. Differential cell count, cytokine measurement and skin histology suggest that the ELRs do not trigger inflammation or cytokine production. However, the serum IgG1 titer was elevated compared to the negative control. Moreover, a second implantation of the material did not cause an elicitation of IgG1 antibodies. These results support that the ELRs might be sufficient for islet cell transplantation. However, it remains to be determined, whether a long-term and self-contained insulin therapy can be established by transplanting these polymers as a coat for islet cells

Porcine Reproductive and Respiratory Syndrome Virus CD8⁺ T cell epitopes and recombination mechanisms

PhD thesis - University of Veterinary Medicine Vienna - 2023PRRSV is an enveloped single-stranded RNA virus of positive polarity, and one of the most devastating porcine pathogens worldwide. Clinical signs of infected animals include respiratory disease and reproductive disorders, causing huge production and financial losses of affected farms. MLV vaccines are available and widely used, but they are often not cross-protective against heterologous virus strains and able to recombine with wild-type or other vaccine strains. These two issues are the focus of this thesis: The search for cross-protective PRRSV-1 epitopes with the potential to elicit a CD8+ T cell response, and the investigation of recombination mechanism of PRRSV-1 wild type and MLVs strains. For the identification of PRRSV-1 epitopes with the potential to restimulate CD8+ T cells, we isolated MHC-I/peptide complexes of PRRSV-1 infected PAMs by immunoprecipitation. Furthermore, we analyzed the bound peptides with LC-MS/MS and compared them to the pig proteome. For the confirmation of the immunogenicity of these epitopes, we conducted in vitro restimulation assays of PBMCs followed by an ICS and flow cytometry. We were able to successfully establish a convenient MHC-I/peptide complex isolation protocol for the LC-MS/MS identification of PRRSV-1 epitopes. Furthermore, we conducted a workflow for the restimulation of PBMCs with these identified peptides to measure CD8+ T cell cytokine responses. Our analyses reveal the most confident MS matches of MHC-I-bound peptides derive from the PRRSV-1 nsps of ORF1. Additionally, we confirmed the elicitation of an IFNγ response by CD8+ T cells after restimulation with several of these peptides. At last, we compared these peptide sequences with proteomes of other PRRSV strains and detected the conservation of several epitopes in other PRRSV-1, PRRSV-2, and PRRSV vaccine strains. We enforce a more detailed research of the PRRSV immunopeptidome by investigating other strains and other SLA-I haplotypes to gain a deeper understanding of MHC-I presentation toward CD8+ T cells after infection. The second part of the thesis focuses on PRRSV recombination mechanisms. After the isolation and sequencing of three recombinant PRRSV-1 strains from farms with mild to severe PRRS cases, our attention was drawn towards a specific MLV vaccine strain. Recombination analyses of these isolates confirmed the recombination of three different wild-type strain with the same MLV strain at the beginning of ORF5. RNA structure predictions revealed a conserved stem-loop within ORF5, which might be a plausible cause of the RdRp to switch temples during replication upon the co-infection of the same animal with different strains. We suggest a more detailed exploration of this stem-loops structure and function and underline the surveillance of PRRSV recombination in the swine population. Taken together, we suggest a more thorough investigation of the PRRSV immunopeptidome for the development of PRRSV vaccines with the ability to elicit a cross-protective CD8+ T cell response, in order to eliminate the risk of introducing more recombinant strains in the field.PhD thesis - University of Veterinary Medicine Vienna - 2023PRRSV is an enveloped single-stranded RNA virus of positive polarity, and one of the most devastating porcine pathogens worldwide. Clinical signs of infected animals include respiratory disease and reproductive disorders, causing huge production and financial losses of affected farms. MLV vaccines are available and widely used, but they are often not cross-protective against heterologous virus strains and able to recombine with wild-type or other vaccine strains. These two issues are the focus of this thesis: The search for cross-protective PRRSV-1 epitopes with the potential to elicit a CD8+ T cell response, and the investigation of recombination mechanism of PRRSV-1 wild type and MLVs strains. For the identification of PRRSV-1 epitopes with the potential to restimulate CD8+ T cells, we isolated MHC-I/peptide complexes of PRRSV-1 infected PAMs by immunoprecipitation. Furthermore, we analyzed the bound peptides with LC-MS/MS and compared them to the pig proteome. For the confirmation of the immunogenicity of these epitopes, we conducted in vitro restimulation assays of PBMCs followed by an ICS and flow cytometry. We were able to successfully establish a convenient MHC-I/peptide complex isolation protocol for the LC-MS/MS identification of PRRSV-1 epitopes. Furthermore, we conducted a workflow for the restimulation of PBMCs with these identified peptides to measure CD8+ T cell cytokine responses. Our analyses reveal the most confident MS matches of MHC-I-bound peptides derive from the PRRSV-1 nsps of ORF1. Additionally, we confirmed the elicitation of an IFNγ response by CD8+ T cells after restimulation with several of these peptides. At last, we compared these peptide sequences with proteomes of other PRRSV strains and detected the conservation of several epitopes in other PRRSV-1, PRRSV-2, and PRRSV vaccine strains. We enforce a more detailed research of the PRRSV immunopeptidome by investigating other strains and other SLA-I haplotypes to gain a deeper understanding of MHC-I presentation toward CD8+ T cells after infection. The second part of the thesis focuses on PRRSV recombination mechanisms. After the isolation and sequencing of three recombinant PRRSV-1 strains from farms with mild to severe PRRS cases, our attention was drawn towards a specific MLV vaccine strain. Recombination analyses of these isolates confirmed the recombination of three different wild-type strain with the same MLV strain at the beginning of ORF5. RNA structure predictions revealed a conserved stem-loop within ORF5, which might be a plausible cause of the RdRp to switch temples during replication upon the co-infection of the same animal with different strains. We suggest a more detailed exploration of this stem-loops structure and function and underline the surveillance of PRRSV recombination in the swine population. Taken together, we suggest a more thorough investigation of the PRRSV immunopeptidome for the development of PRRSV vaccines with the ability to elicit a cross-protective CD8+ T cell response, in order to eliminate the risk of introducing more recombinant strains in the field.PhD Thesis - Veterinärmedizinische Universität Wien - 2023PRRSV ist ein umhülltes, einzelsträngiges RNA-Virus mit positiver Polarität und einer der verheerendsten Krankheitserreger bei Schweinen weltweit. Zu den klinischen Symptomen infizierter Tiere gehören Atemwegserkrankungen und Fortpflanzungsstörungen, die in den betroffenen Betrieben enorme finanzielle Verluste verursachen. Modifizierte Lebendimpfstoffe sind verfügbar und weit verbreitet, aber sie sind oft nicht kreuzprotektiv gegen heterologe Virusstämme und können mit Wildtyp- oder anderen Impfstämmen rekombinieren. Diese beiden Probleme stehen im Mittelpunkt dieser Arbeit: Die Suche nach PRRSV-spezifischen MHC-I Epitopen, die das Potenzial haben, eine CD8+ T-Zell-Antwort hervorzurufen, und die Suche nach dem Rekombinationsmechanismus von PRRSV-1 Impf- und Wildtyp-Stämmen. Zur Identifizierung von PRRSV-1 Epitopen die CD8+ T-Zellen stimulieren, isolierten wir MHC-I/Peptid-Komplexe von PRRSV-1-infizierten Alveolarmakrophagen durch Immunpräzipitation. Dann analysierten wir die isolierten Peptide mit LC-MS/MS und verglichen sie mit dem Proteom des Schweins. Zur Bestätigung der Immunogenität dieser Epitope führten wir in vitro Restimulationsversuche mit PBMCs durch, gefolgt von einer intrazellulärem Zytokinfärbung und Durchflusszytometrie. Es gelang uns, ein erfolgreiches MHC-I/Peptidkomplex-Isolierungsprotokoll für die LC-MS/MS-Identifizierung von PRRSV-1 Epitopen zu entwickeln. Außerdem führten wir eine PBMC-Restimulation mit diesen identifizierten Peptiden durch, um die Zytokinreaktionen von CD8+ T-Zellen zu messen. Unsere Analysen zeigen, dass die zuverlässigsten MS-Übereinstimmungen der MHC-I-gebundenen Peptide von den PRRSV-1 Nichtstrukturproteinen vom Leseraster 1 stammen. Darüber hinaus bestätigten wir die Generierung einer IFNγ-Antwort durch CD8+ T-Zellen nach Restimulation mit mehreren dieser Peptide. Schließlich verglichen wir diese Peptidsequenzen mit den Proteom anderer PRRSV-Stämme und stellten fest, dass mehrere unserer Epitope in anderen PRRSV-1-, PRRSV-2- und PRRSV-Impfstoff-Stämmen konserviert sind. In der Zukunft beabsichtigen wir eine detailliertere Analyse des PRRSV-Immunopeptidoms, indem wir andere Virusstämme und andere SLA-I-Haplotypen untersuchen, um ein tieferes Verständnis der MHC-I-Präsentation gegenüber CD8+ T-Zellen nach der Infektion zu gewinnen. Der zweite Teil der Arbeit befasst sich mit Rekombinationsmechanismen von PRRSV. Nach der Isolierung und Sequenzierung von drei rekombinanten PRRSV-1-Stämmen aus Betrieben mit leichten bis schweren PRRS-Fällen wurde unsere Aufmerksamkeit auf einen spezifischen modifizierten Lebendimpfstoffstamm gelenkt. Rekombinationsanalysen dieser Isolate bestätigten die Rekombination von drei verschiedenen Wildtyp-Stämmen mit demselben Impfstamm am Anfang vom Leseraster 5. RNA-Strukturvorhersagen ergaben eine konservierte Haarnadelstruktur innerhalb des Leserasters 5, die ein plausibler Grund dafür sein könnte, dass das RdRp während der Replikation, bei der Koinfektion desselben Tieres mit verschiedenen Stämmen, die RNA-Vorlage wechselt. Wir schlagen eine genauere Untersuchung der Struktur und Funktion dieser Stammschleife vor und unterstreichen die genauere Überwachung von rekombinanten PRRSV Stämmen in der Schweinepopulation. Zusammenfassend schlagen wir eine gründlichere Untersuchung des PRRSV Immunopeptidoms vor, um Impfstoffe zu entwickeln, die in der Lage sind, eine kreuzprotektive CD8+ T-Zellen-Antwort auszulösen, um das Risiko der Einführung weiterer rekombinanter Stämme in der Praxis zu vermeiden

A Conserved Stem-Loop Structure within ORF5 Is a Frequent Recombination Hotspot for Porcine Reproductive and Respiratory Syndrome Virus 1 (PRRSV-1) with a Particular Modified Live Virus (MLV) Strain

The emergence of recombinant PRRSV strains has been observed for more than a decade. These recombinant viruses are characterized by a genome that contains genetic material from at least two different parental strains. Due to the advanced sequencing techniques and a growing number of data bank entries, the role of PRRSV recombinants has become increasingly important since they are sometimes associated with clinical outbreaks. Chimeric viruses observed more recently are products of PRRSV wild-type and vaccine strains. Here, we report on three PRRSV-1 isolates from geographically distant farms with differing clinical manifestations. A sequencing and recombination analysis revealed that these strains are crossovers between different wild-type strains and the same modified live virus vaccine strain. Interestingly, the recombination breakpoint of all analyzed isolates appears at the beginning of open reading frame 5 (ORF5). RNA structure predictions indicate a conserved stem loop in close proximity to the recombination hotspot, which is a plausible cause of a polymerase template switch during RNA replication. Further research into the mechanisms of the stem loop is needed to help understand the PRRSV recombination process and the role of MLVs as parental strains

Identification of MHC-I-Presented Porcine Respiratory and Reproductive Syndrome Virus (PRRSV) Peptides Reveals Immunogenic Epitopes within Several Non-Structural Proteins Recognized by CD8+ T Cells

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most relevant porcine pathogens worldwide. Active control of the disease relies on modified live virus vaccines (MLVs), as most inactivated vaccines provide very limited protection. Neutralizing antibodies occur late in infection; therefore, CD8+ T cells are considered important correlates of protection and are a frequent focus of investigation. Our aim was to identify viral peptides naturally bound by the class I major histocompatibility complex (MHC-I) and to confirm their ability to stimulate CD8+ T cells. For this purpose, we immunoprecipitated MHC-I/peptide complexes of PRRSV (strain AUT15-33) -infected cells (SLA-I Lr-Hp 35.0/24 mod) to isolate the viral epitopes and analyzed them with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Furthermore, we employed these identified peptides to stimulate peripheral blood mononuclear cells (PBMCs) of previously PRRSV-infected pigs and measured the PRRSV-specific CD8+ T-cell response with an intracellular cytokine staining (ICS). Our data revealed that PRRSV non-structural proteins (NSPs), encoded in open reading frame 1a and 1b (ORF1), present the major source of MHC-I-presented peptides. Additionally, we show that our identified epitopes are able to trigger IFNγ responses in vitro. These findings are a basis for understanding the proteasomal degradation of PRRSV proteins, the cellular ability to display them via MHC-I, and their potential to restimulate CD8+ T cells

ADAM17 Is an Essential Factor for the Infection of Bovine Cells with Pestiviruses

The entry of BVDV into bovine cells was studied using CRIB cells (cells resistant to infection with bovine viral diarrhea virus [BVDV]) that have evolved from MDBK cells by a spontaneous loss of susceptibility to BVDV. Recently, larger genetic deletions were reported but no correlation of the affected genes and the resistance to BVDV infection could be established. The metalloprotease ADAM17 was reported as an essential attachment factor for the related classical swine fever virus (CSFV). To assess whether ADAM17 might be involved in the resistance of CRIB-1 cells to pestiviruses, we analyzed its expression in CRIB-1 and MDBK cells. While ADAM17 protein was detectable in MBDK cells, it was absent from CRIB-1 cells. No functional full-length ADAM17 mRNA could be detected in CRIB cells and genetic analysis revealed the presence of two defective alleles. Transcomplementation of functional ADAM17 derived from MDBK cells in CRIB-1 cells resulted in a nearly complete reversion of their resistance to pestiviral infection. Our results demonstrate that ADAM17 is a key cellular factor for the pestivirus resistance of CRIB-1 cells and establishes its essential role for a broader range of pestiviruses

New Emergence of the Novel Pestivirus Linda Virus in a Pig Farm in Carinthia, Austria

Linda virus (LindaV) was first identified in a pig farm in Styria, Austria in 2015 and associated with congenital tremor (CT) type A-II in newborn piglets. Since then, only one more LindaV affected farm was retrospectively discovered 10 km away from the initially affected farm. Here, we report the recent outbreak of a novel LindaV strain in a farrow-to-finish farm in the federal state Carinthia, Austria. No connection between this farm and the previously affected farms could be discovered. The outbreak was characterized by severe CT cases in several litters and high preweaning mortality. A herd visit two months after the onset of clinical symptoms followed by a diagnostic workup revealed the presence of several viremic six-week-old nursery pigs. These animals shed large amounts of virus via feces and saliva, implying an important epidemiological role for within- and between-herd virus transmission. The novel LindaV strain was isolated and genetically characterized. The findings underline a low prevalence of LindaV in the Austrian pig population and highlight the threat when introduced into a pig herd. Furthermore, the results urge the need to better understand the routes of persistence and transmission of this enigmatic pestivirus in the pig population

DataSheet_1_Influence of PRRSV-1 vaccination and infection on mononuclear immune cells at the maternal-fetal interface.docx

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating viruses for the global swine industry. Infection during late gestation causes reproductive failure but the local immune response in utero remains poorly understood. In this study, an experimental PRRSV-infection model with two different PRRSV-1 field isolates was used to investigate the immune cell phenotypes at the maternal-fetal interface during late gestation. In addition, phenotypic changes induced by a modified live virus (MLV, ReproCyc® PRRS EU) vaccine were studied. Vaccinated (n = 12) and non-vaccinated pregnant gilts (n = 12) were challenged with either one of the PRRSV-1 field isolates (low vs. high virulent, LV or HV) or sham-inoculated at day 84 of gestation. Twenty-one days post infection all gilts were euthanized and the fetal preservation status for all fetuses per litter was assessed. Leukocytes from the maternal-fetal interface were isolated and PRRSV-induced changes were investigated using ex vivo phenotyping by flow cytometry. PRRSV load in tissue from the maternal endometrium (ME) and fetal placenta (FP) was determined by RT-qPCR. In the ME, a vast increase in CD8β T cells with CD8αposCD27dim early effector phenotype was found for fetuses from the non-vaccinated LV and HV-challenged gilts, compared to non-treated and vaccinated-only controls. HV-challenged fetuses also showed significant increases of lymphocytes with effector phenotypes in the FP, including NKp46pos NK cells, CD8αhigh γδ T cells, as well as CD8αposCD27pos/dim CD4 and CD8 T cells. In vaccinated animals, this common activation of effector phenotypes was more confined and the fetal preservation status significantly improved. Furthermore, a negative correlation between the viral load and CD163highCD169pos mononuclear phagocytic cells was observed in the FP of HV-infected animals. These results suggest that the strong expansion of effector lymphocytes in gilts that were only infected causes immune-pathogenesis rather than protection. In contrast, the attenuated MLV seems to dampen this effect, yet presumably induces memory cells that limit reproductive failure. This work provides valuable insights into changes of local immune cell phenotypes following PRRSV vaccination and infection.</p

Identification of MHC-I-Presented Porcine Respiratory and Reproductive Syndrome Virus (PRRSV) Peptides Reveals Immunogenic Epitopes within Several Non-Structural Proteins Recognized by CD8+ T Cells

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most relevant porcine pathogens worldwide. Active control of the disease relies on modified live virus vaccines (MLVs), as most inactivated vaccines provide very limited protection. Neutralizing antibodies occur late in infection; therefore, CD8+ T cells are considered important correlates of protection and are a frequent focus of investigation. Our aim was to identify viral peptides naturally bound by the class I major histocompatibility complex (MHC-I) and to confirm their ability to stimulate CD8+ T cells. For this purpose, we immunoprecipitated MHC-I/peptide complexes of PRRSV (strain AUT15-33) -infected cells (SLA-I Lr-Hp 35.0/24 mod) to isolate the viral epitopes and analyzed them with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Furthermore, we employed these identified peptides to stimulate peripheral blood mononuclear cells (PBMCs) of previously PRRSV-infected pigs and measured the PRRSV-specific CD8+ T-cell response with an intracellular cytokine staining (ICS). Our data revealed that PRRSV non-structural proteins (NSPs), encoded in open reading frame 1a and 1b (ORF1), present the major source of MHC-I-presented peptides. Additionally, we show that our identified epitopes are able to trigger IFNγ responses in vitro. These findings are a basis for understanding the proteasomal degradation of PRRSV proteins, the cellular ability to display them via MHC-I, and their potential to restimulate CD8+ T cells