570 Biowissenschaften; Biologie
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Mycobacterium tuberculosis (Mtb) poses a significant global health threat, necessitating the identification of protective immune responses across various host species to facilitate eradication efforts through novel vaccine strategies. While it is known that secretion of the cytokine interferon-gamma (IFNγ) by T cells is a primary anti-mycobacterial response across mammals with varying susceptibility to Mtb, knowledge about the downstream effects of IFNγ on macrophages from natural hosts and species-specific differences is incomplete. In this study, we systematically compared the IFNγ-mediated protection by primary macrophages from humans, cattle, and mice against Mtb. Utilizing Mtb growth assays, high-content microscopy, functional assays, RNAseq, and qPCR, we elucidated the similarities and differences in protective immune responses.
Our findings reveal that under comparable culture conditions, IFNγ induced a pro-inflammatory CXCL10-secreting macrophage phenotype in all three species, with numerous genes enriched responsible for pathogen detection, downstream signal transduction, and production of pro-inflammatory cytokines and chemokines. However, while IFNγ restricted intracellular Mtb growth in murine and bovine macrophages, it showed limited efficacy in human macrophages. Transcriptome analysis uncovered significant differences in IFNγ-mediated gene regulation among the species. Notably, ~77.7% of differentially expressed orthologous genes were species-specific upon IFNγ-stimulation and Mtb infection. Among these, numerous genes associated with the JAK-STAT signaling pathway. In particular, the transcription of NOS2 showed the most notable difference, with murine and bovine macrophages upregulating its transcription, while human macrophages did not. Different transcription of NOS2 was confirmed at the protein level and in functional assays, indicating conserved differential activation of NOS2, which was also confirmed additionally in freshly isolated monocytes and ex vivo activated alveolar macrophages across studied species.
In conclusion, our data suggest that IFNγ responses induce unique molecular processes despite a universal transcriptional immune response in macrophages and monocytes from the murine, human, or bovine host. These findings not only enhance our understanding of protective immunity against Mtb but also have implications for vaccine design. The necessity to investigate protective immune responses against Mtb in diverse natural hosts and experimental models is underscored, emphasizing the importance of considering species-specific responses in developing effective TB interventions.
The studies presented here in this thesis investigated the complex interplay of the human respiratory tract pathogens S. pneumoniae, S. aureus and IAV with human APCs from two different perspectives. On the one hand, our findings demonstrated that pneumococci were able to completely suppress the function of moDCs and alter the subsequent activation of T cells. This immune evasion strategy is mainly based on the release of the pneumococcal virulence factor Ply. Furthermore, pneumococcal lipoproteins are becoming increasingly prominent as potential new serotype independent vaccine candidates. We show the favorable basic potential of the pneumococcal lipoprotein DacB and the significant impact of the attachment of a lipid moiety in a human context. Both, the lipidated version of DacB and MetQ, demonstrated robust activation of human APCs and further induced the proliferation of human CD4+ T cells underscoring the potential adjuvant properties of the lipid moiety.
Coastal wetlands, including lagoons, marshes, and peatlands, are exceptionally important ecosystems that provide many ecosystem services and functions, and exert a substantial influence on global climate. As global climate change progresses, permafrost and ice sheets will continue to thaw and sea levels will rise, in turn causing inundation of coastal wetlands, such as thermokarst lakes, peatlands, and salt marshes. Inundation of sea water will also bring sulfate to freshwater systems, potentially reducing methane emissions, but also producing other negative ecosystems responses, such increased nutrient release, including dissolved organic carbon, ammonium, phosphate, iron, and many others. This thesis discusses the results of three studies from two coastal wetlands investigating the impact of sulfate on the biogeochemistry and microbial ecology of these sites, in conjunction with results from other studies.
In study 1, two thermokarst lakes and a thermokarst lagoon took the form of a natural laboratory to investigate how sulfate intrusion impacted the methane-cycling microbial community by altering the geochemistry of the lagoon. In addition to changes in the geochemistry, marine water intrusion also introduced and provided habitat for classical marine consortia of sulfate reducers and anaerobic methane oxidizers called ANME, which formed an effective filter on methane produced in the sediments. Sulfate intrusion also reformed the microbial community, and did not follow classical marine biogeochemical process zonation.
In study 2, the effects of brackish water rewetting on greenhouse gas dynamics in a previously drained and freshened peatland were investigated. Monitoring activities were conducted before and after rewetting to determine if brackish water rewetting was an effective way to reduce methane emissions in rewetted peatlands. Initial results showed that methane emissions were reduced compared to similar freshwater peatland rewetting projects, however carbon dioxide release remained higher than expected.
Study 3 examined whether the microbial community from the same field site as study 2 was adapted to the new conditions two years post-rewetting. In an incubation experiment, soil samples from three depths were subjected to two different salinity regimes, representing the range of expected salinities experienced by the microbial community since rewetting over the course of 3 months, with special attention paid to differences in biogeochemistry and the microbial community. Contrary to expectations, the largest influence on biogeochemistry and the microbial community came from legacy sulfate deposits from when the site was previously connected to the Baltic Sea as a brackish marsh in the early 1900s.
The overall picture of sulfate intrusion and methane cycling is somewhat clouded. In general, increased sulfate concentrations do lower methane emissions, but also induces increased releases of other nutrients. One part of the larger solution to curtailing methane emissions in natural systems, especially those impacted by anthropogenic activities, could be increased methanotrophy which is not necessarily reliant on sulfate. A plan forward is proposed in the synthesis, which encourages more research into the topic of sulfate introduction to freshwater systems and a different way to tackle methane emissions through encouraging natural mitigation through microbial methanotrophy.
Throughout evolutionary history, species have always been faced with changing environments, but the unprecedented rate of change induced by current human-induced climate change poses significant challenges to many recent organisms. Besides altering large-scale weather patterns and increasing the frequency and intensity of extreme weather events, climate change is characterised by a significant rise in global temperatures. Projections indicate an increase of 1.5°C to 5°C by the end of the century, largely depending on the extent of greenhouse gas emissions. As temperature is intimately linked to nearly all biological processes, global warming may impact many aspects of animal physiology, including thermoregulation, reproduction, and morphology.
In this doctoral thesis, I investigate how global warming affects the physiology of Bechstein’s bats (Myotis bechsteinii), a typical European forest-dwelling species of high conservation concern. I focus particularly on the mechanisms underlying a previously observed increase in body size among individuals born during warmer summers. Using individualised long-term data on morphological and reproductive traits from four wild bat colonies, combined with two field experiments, together with my co-authors, I examined the effects of temperature on body size and metabolism across multiple reproductive seasons.
In a first experiment, we artificially raised and maintained roost temperatures at temperatures within the thermoneutral zone of the bats, simulating optimal thermal conditions for growth. Directly heating the bat boxes that were used as day roosts during the maternity season, allowed us to isolate the effects of temperature on body size from potential confounding factors such as insect availability. Our findings reveal that body size in Bechstein’s bats is directly and positively related to warmer roost temperature, likely due to reduced thermoregulatory costs, and thus, increased energy availability for growth. In other words, bats in heated roosts should have exhibited greater thermoregulatory efficiency, requiring less energy to maintain normothermia, which allowed more energy to be allocated toward juvenile growth.
In a second experiment, we aimed to unveil the underlying mechanisms that drive the established direct effect of temperature on body size in M. bechsteinii. In order to do so, we used field respirometry to assess the metabolic response of communally roosting Bechstein’s bats to roost temperatures during two lactation periods. Notably, average daily metabolic rates were not different on heated than on unheated days, suggesting that the saved energy from thermoregulating more efficiently is directly reinvested in other aspects, such as growth and/or maternal care. Furthermore, the results highlight the capacity of Bechstein’s bats to cope with cooler temperatures through metabolic and behavioural mechanisms such as torpor, digestion-induced thermogenesis and social thermoregulation. However, as temperature exceeds the thermoneutral zone, the bats’ metabolic rate increased sharply, suggesting that individuals may struggle to cope with extended periods of extreme heat. Prolonged exposure to high temperatures could significantly elevate energy expenditure and lead to dehydration, jeopardising bat populations in an ever warming climate. Thermal imaging data also indicated minimal torpor use during lactation in heated colonies, with only two instances of torpor observed during the whole lactation period in one colony. A reduction in torpor use may alleviate the negative effects of torpor on milk production and growth processes and thus additionally promote offspring development.
In a third analysis, we leveraged 14 years of individualised data to test whether heating had an effect on body condition in reproductive females, additionally comparing them to non-reproductive females. Interestingly, despite the thermoregulatory benefits, mothers from heated colonies did not show improved body condition compared to those from unheated colonies, suggesting that the energy saved in warmer conditions is instead allocated to maternal care, further enhancing juvenile growth. We additionally show that body condition in spring is higher in those females that had offspring the subsequent summer than non-reproductive females, suggesting that resources left after hibernation may act as capital for the decision whether or not to reproduce in a given year.
In conclusion, the studies I present in this thesis provide key insights into the mechanisms behind the temperature-dependent increase in body size in Bechstein’s bats and, more broadly, how temperature influences their physiology, growth, and reproduction. These findings highlight the complex trade-offs between the apparent immediate benefits of warmer conditions for growth and the potential associated long-term negative effects posed by global warming. Given the ongoing climate crisis, understanding these physiological responses is crucial for the conservation of temperate-zone bat species such as Bechstein’s bats.
Hypoxic environmental conditions such as flooding and the resulting oxygen limitation for plant roots interrupt oxidative phosphorylation and lead to an energy crisis in plants. The energy shortage eventually results in the breakdown of cellular transmembrane gradients and cell death. The degradation of sucrose and starch and an increase in anaerobic glycolysis are well-described mechanisms to provide energy and survive this crisis. Lactic acid and ethanolic fermentation recycle reducing equivalents to maintain ATP synthesis. However, alternative reducing equivalent oxidation mechanisms (Chapter I), alternative energy and carbon sources (Chapter II) and regulation of energy consumption (Chapter III) can be crucial for survival under low oxygen conditions. To answer this hypothesis, emissions of nitric oxide (NO) under low-oxygen conditions (mechanism – Chapter I), changes in lipid composition under hypoxic conditions (energy source – Chapter II) and a redox-reactive and highly conserved cysteine residue of a plasma membrane (PM) P-type H+-ATPase (energy consumption – Chapter III) were investigated.
The thesis confirmed the importance of nitrite (NO2-) as an alternative electron acceptor resulting in NO emissions of plant roots during low-oxygen stress. Firstly, the chemiluminescence-based detection of oxygen-dependent NO emissions in in vivo studies for roots of tomato, barley and tobacco is reported using a root reactor (Chapter I). The large amounts of low-oxygen NO emission in the root reactor suggest a role in generation of ATP. NO2- reduction appears to be involved in the mitochondrial electron transfer chain (mETC) and therefore may contribute to ATP generation. In response to oxygen depletion, an escape strategy of the studied species seems likely, including oxygen supply systems. In this strategy, additional ATP is invested to integrate structural changes. However, hypoxic tomato plants seem to rely on glycolysis and fermentation. They do not utilize lipid degradation and β-oxidation as energy source which was confirmed in HPLC-MS-MS analysis studies of hypoxic tomato roots (Chapter II). In terms of energy source, neither a depletion of lipids nor an increase in lipid degradation was observed. Triacylglycerol (TG) was more abundant and the degree of unsaturation of fatty acids increased due to hypoxic conditions (Chapter II). TGs play a role as intermediates in lipid metabolism. Subsequent utilization of carbon and energy sources during prolonged oxygen scarcity is possible. However, an altered activity of PM P-type H+-ATPase due to hypoxic-induced, posttranslational NO modification was not confirmed. A possible S-nitrosylation site at the conserved cysteine residue was not verified in the biochemical mutagenesis study described in Chapter III. Regulation of PM P-type H+-ATPase activity via cytoplasmatic ATP-level may be possible in plants.
The maintenance of proteins and cell functions appears to be essential under conditions of low-oxygen and high levels of reactive nitrogen species (RNS) such as NO (Chapter I). The induction of antioxidant systems under low-oxygen stress is well known. In this thesis, the highly conserved cysteine near the P-site of the PM P-type H+-ATPase was shown to act as an endogenous antioxidant (Chapter III). This underlines the importance of this enzyme for the cell. While the changes in lipid composition observed under hypoxia highlight the importance of maintaining membrane integrity and fluidity as in the case of plastids (Chapter II). Membrane adaptation results in increased TG abundance which led to the formation of lipid droplets. TG may scavenge toxic intermediates to prevent cell death. Lipid droplets provide a binding site for enzymes involved in adaptation and may even protect unsaturated fatty acids under high reactive oxygen species (ROS) and RNS environments by being incorporated into their core.
Under low-oxygen conditions, various strategies have evolved to provide sufficient energy for adaptation and survival while protecting vital cellular functions in plants. The increase in flooding and heavy rainfall due to climate change poses a challenge to crops and their harvest. Plant-based stress-responses may even be a factor impacting the climate (Chapter I). In order to stabilize food production, plant breeding may need to rethink breading strategies in certain areas. With more heavy rains and same annual rain fall a quiescence strategy via growth retention may sometimes be better than an escape strategy to save crucial energy under time-limited stress conditions. A better understanding of survival mechanisms, as provided in this thesis, may help to develop strategies that strengthen plants under mild low-oxygen conditions.
This thesis aimed at the identification, engineering, and application of NAD(P)H dependent oxidoreductases such as Baeyer-Villiger monooxygenases (BVMOs) and enoate reductases (EREDs) due to their importance as biocatalysts in the chemical and pharmaceutical industry. To facilitate the implementation of biocatalysis in industrial processes, improved co-factor regeneration strategies as well as tools for the high-throughput characterization of enzymes are greatly demanded. Therefore, the combination of target biocatalytic reactions with the direct electrochemical regeneration of NAD(P)H co-factors have been investigated herein. Furthermore, the bioinformatic-assisted identification of novel oxidoreductases and their tailoring by
protein engineering not only expands the number of available enzymes, the
presented biocatalysis-based strategies offer promising alternatives to established chemical reaction schemes that are compliant with ‘The 12 Principles of Green Chemistry’.
In Article I, the direct electrochemical NADH regeneration was explored on the
cathode of a carbon electrode, modified with Toray paper sprayed with carbon
nanoparticles. The nicotinamide product distribution was investigated and the
electrochemical flow cell reactor was combined with an enzymatic reduction reaction to validate the method.
Article II focuses on the discovery and characterization of BVMOs, through the
bioinformatic analysis of sequence similarity networks (SSNs). An uncharacterized BVMO from Halopolyspora algeriensis (BVMOHalo) was identified after in silico clustering of protein sequences and characterized biochemically (e.g., pH optimum, organic solvent tolerance). Substrate scope elucidation enabled the annotation of BVMOHalo as aliphatic ketone monooxygenase. Expanding from the findings in Article II, Article III features the first genetically encoded biosensor system to monitor BVMO activity. This biosensor implementation accelerated the screening of a focused library of active site mutants of BVMOHalo and the identification of ester forming variants, overcoming the low-throughput analysis of BVMO reaction products by chromatographic methods. Together, the presented articles address key
challenges in biocatalysis, ranging from the recycling of co-factors and the
identification and evolution of novel enzymes.
Article I: Product distribution of steady-state and pulsed electrochemical regeneration of 1,4-NADH and integration with enzymatic reaction
Mohammed Ali Saif Al-Shaibani , Thaleia Sakoleva , Luka A.
Živković, Harry P. Austin , Mark Dörr , Liane Hilfert , Edgar Haak, Uwe
T. Bornscheuer, Tanja Vidaković-Koch, ChemistryOpen, 2024, e202400064
This article describes the direct electrochemical regeneration of NADH on carbon electrodes, modified with carbon nanoparticles sprayed Toray paper. The product distribution under steady-state and dynamic conditions was investigated and proved that the dynamic operation offered a faster mode, yielding the desired 1,4-NADH isomer. In parallel, side products such as different NAD dimers, the enzymatically inactive isomer 1,6-NADH, and the formation of ADP-ribose represent disadvantages of the investigated electrochemical co-factor recycling method. In addition to this, the different nicotinamide side products are enzymatic inhibitors, negatively impacting product yields.
Article II: Discovery and characterization of a Baeyer-Villiger monooxygenase using sequence similarity network analysis
Thaleia Sakoleva, Harry P. Austin, Chrysoula Tzima, Mark Dörr, Uwe T. Bornscheuer, ChemBioChem, 2023, e202200746
This article describes the discovery of the novel BVMOHalo by SSN analysis. The
sequence was retrieved after protein clustering. The enzyme was successfully
expressed in Escherichia coli (E. coli) and purified. Substrate scope investigation suggested that BVMOHalo prefers linear aliphatic ketones. Optimal reaction conditions were determined in different buffer systems at varying pH, temperatures, and in the presence of commonly used organic co-solvents. Finally, the enzyme remained stable after five days of storage, which is remarkable since BVMOs regularly suffer from short half-lives.
Article III: Biosensor-guided engineering of a Baeyer-Villiger monooxygenase for aliphatic ester production
Thaleia Sakoleva, Florian Vesenmaier, Lena Koch, Jarne E. Schunke, Kay Novak, Sascha Grobe, Mark Dörr, Uwe T. Bornscheuer, Thomas Bayer, ChemBioChem, e202400712.
This article describes the engineering of the catalytic site of BVMOHalo and the detection of active variants using a bioluminescence-based assay. Aliphatic ketones were converted into the corresponding esters by this BVMO. Subsequently, esters are transformed through an artificial enzyme cascade, consisting of an esterase and an alcohol dehydrogenase (ADH), yielding aldehydes as cascade products. The aldehyde is then substrate for a bacterial luciferase (LuxAB), which emits light through the oxidation of aldehydes to the corresponding carboxylates. This biosensor setup was used to guide the selection of ester-forming variants of BVMOHalo – not only representing the first genetically encoded biosensor system to monitor the product formation in BVMO-catalyzed reactions but offering great potential to accelerate protein and metabolic engineering campaigns in the future.
Klebsiella pneumoniae, a Gram-negative, non-motile, encapsulated, rod-shaped enterobacteria is a formidable opportunistic pathogen causing most hospital-acquired nosocomial infections such as pneumonia, urinary tract infections, and bloodstream infections. In recent years, the emergence of multi-resistant hypervirulent strains has posed a severe challenge to healthcare systems worldwide. To address this crisis, further researches on K. pneumoniae and exploration of potential antimicrobial approaches are called.
In this study, biochemical, structural, and physiological characterizations of a putative classical deacetylase homolog, HdaH, annotated in K. pneumoniae was performed. It was confirmed as an active Nε-acetyl-lysine deacetylase of peptide substrates, and its activity can be efficiently inhibited by the mammalian HDAC inhibitors SAHA and trichostatin A. Substrate preference assays detected its high specificity towards acetylated lysine residues adjacent to a positively charged residue. The protein forms a tetramer in solution. Crystal structures reveal that the L1 loop of the protein plays a central role in tetramerization. Deletion of the L1 loop leads to a dimeric complex and reduced lysine deacetylase activity, suggesting a potential cooperative catalysis of the tetrameric complex. The 'head-to-head' interaction of KpHdaH complex subunits is relatively independent of the L1 loop and restricts the entrance of the substrate-binding pocket. The substrate-binding pocket is deep and branched, consisting of a central tunnel and two side pockets. Its complicated structure probably contributing to the substrate specificity of the deacetylase. Physiological investigations indicated that deletion of hdah gene leads to marginal increase of biofilm mass, and mild but significant growth acceleration in minimal medium supplemented with glucose and acetate. Through genomic context and interactor assays, groups of proteins possibly related to KpHdaH’s function were addressed.
This study provides structure-function insight of HdaH protein in K. pneunomiae and a suggestion of developing specific inhibitor. Althrough a definitive conclusion regarding the native substrate and physiological role of deacetylase have not been reached, this study has provided suggestions and laid a robust foundation for future investigations.
Charakterisierung von Membranvesikeln aus Gram-negativen und Gram-positiven pathogenen Bakterien
(2024)
Bakterielle Pathogene können in ihren Wirten Infektionen auslösen, die gesundheitsgefährdend sind oder sogar zum Tod führen können. Zu diesen Pathogenen zählen auch das Gram-negative Bakterium Aeromonas salmonicida (A. salmonicida) und die Gram-positiven Bakterien Renibacterium salmoninarum (R. salmoninarum) und Streptococcus pneumoniae (S. pneumoniae). Während R. salmoninarum und A. salmonicida Fischpathogene sind, die in Salmoniden die bakterielle Nierenkrankheit (bacterial kidney disease) (BKD) (R. salmoninarum) bzw. die Fischfurunkulose (A. salmonicida) auslösen können, ist S. pneumoniae ein humaner Pathogen, der den oberen Atemwegstrakt und andere Gewebe kolonisieren und Krankheiten wie Sepsis, Meningitidis und Pneumonie auslösen kann. Gegen alle diese Pathogene existieren Vakzin-Strategien bestehend aus Kapselpolysacchariden oder inaktivierten Bakterien, um einer Infektion und deren Folgen vorzubeugen. Allerdings wirken diese Vakzine oft nur gegen bestimmte Serotypen (S. pneumoniae) oder sind mit hohem Stress und Nebenwirkungen für den Wirt verbunden (A. salmonicida, R. salmoninarum). Aus diesem Grund wird aktiv an alternativen Strategien zur Vakzinierung geforscht. Eine mögliche Route sind bakterielle Membranvesikel (MV), die sich von der bakteriellen Oberfläche Gram-positiver und Gram-negativer Bakterien abschnüren und viele potenziell immunogene Proteine auf ihrer Oberfläche tragen.
In dieser Arbeit wurde das Proteinrepertoire der bakteriellen MV von R. salmoninarum, A. salmonicida und S. pneumoniae mittels Massenspektrometrie untersucht. In der aufgereinigten MV Fraktion von R. salmoninarum wurden die immunsupprimierenden Proteine P57/Msa und P22, die wichtige Virulenzfaktoren von R. salmoninarum sind, in hoher Abundanz detektiert. Weiterhin wurden neues Lipoprotein C/Protein von 60 kDa (NlpC/P60) Hydrolasen und weitere Zellwand-modifizierende Proteine in der MV Fraktion gefunden, was einen Hinweis darauf darstellen könnte, dass diese Proteinklasse eine Relevanz bei der Entstehung von MV in Gram-positiven Bakterien hat. Bei der Untersuchung der MV von A. salmonicida wurde analysiert, inwieweit sich das MV Proteinrepertoire durch Kultivierungsbedingungen beeinflussen lässt. Die MV, die unter Bedingungen mit geringer Eisenverfügbarkeit aufgereinigt wurden, hatten eine ähnliche Größe und Konzentration im Vergleich zu der Kontrollbedingung. Allerdings wurden zahlreiche Proteine in der MV Fraktion detektiert, die bei geringer Eisenverfügbarkeit signifikant in ihrer Abundanz erhöht waren. Hierzu zählten vor allem TonB-abhängige Eisen- und Siderophoretransporter, aber auch Hemolysin und Lipasen. Eine erhöhte Kultivierungstemperatur resultierte in einer geringeren Vesikelkonzentration verglichen mit der Kontrollbedingung. Allerdings führte die erhöhte Kultivierungstemperatur zu einer signifikant gesteigerten Hemolysin Abundanz. Bei der Kultivierung von A. salmonicida mit dem Antibiotika Florfenicol waren die MV in ihrer Größe deutlich verringert. Weiterhin wurden viele ribosomale Proteine in der MV Fraktion gefunden, was auf bakterielle Lyse hinweisen könnte. Der Vergleich zwischen dem Erntezeitpunkt der MV in der stationären Wachstumsphase und der Sterbephase von S. pneumoniae zeigte, dass Vesikel aus der Sterbephase im Durchschnitt leicht vergrößert und in der Konzentration 10-fach erhöht waren. Zusätzlich war Autolysin in den MV, die in der Sterbephase geerntet wurden, signifikant in der Abundanz erhöht.
Zusammenfassend konnte gezeigt werden, dass sich das Proteinrepertoire der MV durch Faktoren wie die Wachstumsphase oder Kultivierungsbedingungen drastisch beeinflussen lässt. Es wurden putativ immunogene Proteine (Eisentransporter, Lipoproteine) in den MV aller untersuchten Pathogenen gefunden, was das Potenzial der MV als Vakzin-Plattform zeigt. Besonders eine Kultivierung bei Bedingungen mit geringer Eisenverfügbarkeit könnte durch die hohe Anzahl an Eisen-regulierten Membranproteinen bei einer Vakzinentwicklung von Vorteil sein.
Investigating protective immune responses against SARS-CoV-2 infections in small animal models
(2025)
During the SARS-CoV-2 pandemic, different viral variants emerged with distinct
transmissibility, pathogenicity, and immune evasion features. Although these traits
were reflected in the pandemic course, knowledge about immunity against the
emerging variants remained limited and lagged behind. Information about early
antiviral immunity mainly came from blood or tissue samples of individuals who died
from COVID-19, which cannot fully reflect local protective immune responses during
acute SARS-CoV-2 infections. This thesis utilized small animals to model COVID-19 in
humans and used it afterward to test mRNA vaccine efficacy. The aim was to gain
insights into early immune responses after SARS-CoV-2 infection and mRNA-induced
protective immunity in various tissues, which is challenging to accomplish in humans.
Publication I demonstrates that infection of K18-hACE2 mice with ancestral SARS-CoV-
2, Beta, or Delta VOC results in unique patterns of viral dissemination into the lungs
and distinct inflammatory responses within the first 5 days post-infection (DPI). The
Beta variant spread to the lungs earlier and caused higher levels of inflammatory
cytokines and more significant infiltration of innate immune cells than the ancestral
SARS-CoV-2. Adaptive immune responses were detected within 7 DPI and were
associated with lower viral burden, suggesting a potential role in clearing the infection.
The depletion of B and T cells allowed correlating viral clearance with virus-specific
antibody levels and T cell responses. While innate immune responses differed among
SARS-CoV-2 variants, adaptive responses developed similarly, suggesting a potential
target for pan-variant countermeasures. Publication II indicates that low-dose mixed
mRNA vaccines, containing half the amount of each monovalent vaccine, produce
similar levels of neutralizing antibodies and virus-specific T cells in the lungs, protecting
K18-hACE2 mice from lethal disease. The vaccination of Wistar rats showed that
bivalent vaccination can elicit neutralizing antibodies against different variants (e.g.,
BA1 and BA.5) that are not included in the vaccine formulation. The study suggests that
alternative vaccination strategies could generate cross-protective immunity, helping to
overcome immune evasion by SARS-CoV-2 variants. Publication III investigates
protective immunity against SARS-CoV-2 upon mRNA vaccination in a mouse model
with restricted B cell responses, commonly seen in vaccinated individuals with
immunodeficiencies. It demonstrates that T cells can compensate for the lack of serum
antibodies to prevent sublethal disease in mice. Serum antibody levels correlated with
reduced disease severity and mRNA vaccine-induced IgG limited viral replication in the
nasal conchae, indicating a potential role in protecting from virus transmission.
This thesis provides valuable insights into protective immune responses against
infection with various SARS-CoV-2 variants and upon mRNA vaccination and informs
about vaccination strategies to overcome immune escape. The numerous
experimental studies within this thesis are essential for characterizing emerging viral
variants and developing effective vaccines. These findings offer valuable insights that
can guide the development of effective countermeasures to combat immune evasion
by future SARS-CoV-2 variants.
Krebserkrankungen sind nach Herzerkrankungen die zweithäufigste Todesursache
weltweit. Rund 10 Millionen Menschen sterben pro Jahr an Krebserkrankungen und jährlich kommen etwa 19.3 Millionen Krebs-Neuerkrankungen hinzu, wobei die Zahlen weiterhin ansteigen. Trotz innovativer Fortschritte in den Bereichen der diagnostischen Methoden und Kombinationstherapieverfahren hat sich die 5-Jahres-Überlebensrate in den letzten 15 Jahren für einige Tumorentitäten nur unzureichend verbessert. Die hauptsächliche Ursache dafür ist die Entstehung von therapieresistenten Tumoren, die neben dem Therapieversagen zu einem Wiederauftreten des Krebses führen können. Um die Überlebensrate der Patienten zu erhöhen, ist es somit unumgänglich effizientere Therapieansätze zu entwickeln oder bestehende zu optimieren. Physikalisches Gasplasma ist ein medizinisches Tool mit versatilen Eigenschaften und weist unter anderem ein antitumorales Potential auf. Dieses beruht auf der Freisetzung vielfältiger reaktiver Sauerstoff- und Stickstoffspezies (ROS/RNS; Reactive Oxygen Species/Reactive Nitrogen Species) und kann eine Tumorzersetzung durch Induktion von oxidativem Stress initiieren. Durch eine Teil-Ionisation eines Gases mittels Energiezufuhr in Form von starken elektrischen Feldern werden frei bewegliche Elektronen, ionisierte Atome sowie reaktive Spezies generiert, die unterschiedliche biologische Effekte vermitteln können. Neben einer Vielzahl von in vitro und in vivo Untersuchungen suggerieren auch einige klinische Fallstudien tumorinhibierende Effekte von Gasplasmaapplikationen in
Krebspatienten. Allerdings konnte lediglich bei einem Teil der Patienten eine Reduktion der Tumorlast erzielt werden. Ein anderer Teil wiederum zeigte nach einer anfänglich sichtbaren Gasplasma-vermittelten Tumorverringerung im Laufe der Zeit ein verstärktes Tumorwachstum (Therapie-Resistenz). Ziel der vorliegenden Arbeit war es 1) Möglichkeiten zur Optimierung der Behandlungseffizienz von medizinischem Gasplasma zu erforschen und 2) potentielle Wege zur Überwindung entstandener Gasplasma-Behandlungsresistenzen in Tumorzellen zu identifizieren. Die Implementation von 3D-gedruckten Gasplasma-Aufsätzen war dabei ein innovativer Ansatz zur Modulation der Gasplasmaeffekte. Nach Testung von zwei neuartigen Aufsätze zur dynamischen Druckreduktion des Gasplasmas konnte abhängig von der Gasflussstärke und des verwendeten Adapters eine gesteigerte Tumorinhibierung verzeichnet werden. Um das Potenzial der Gasplasmaanwendung in der Onkologie weiter zu explorieren und die Auftrittswahrscheinlichkeit von Resistenzen zu verringern, bietet die
Kombination mit anderen Tumor-Behandlungsverfahren eine weitere Möglichkeit, die therapeutischen Effekte zu verstärken. Untersuchungen hinsichtlich eines synergistischen Wirkmechanismus einer Kombinationsbehandlung von Gasplasma mit neu synthetisierten small molecules konnten vielversprechende Ergebnisse erzielen. Sowohl in vitro als auch in ovo konnte ein erhöhter tumortoxischer Effekt beobachtet werden, welcher in einem Xenograft-Mausmodell validiert werden konnte. Durch Etablierung und Analyse Gasplasma-resistenter Tumorzellen sollten mögliche Adaptationsmechanismen entschlüsselt und neue therapeutische Ansätze zur Umgehung oder Überwindung von Resistenzen entdeckt werden. Physiologische sowie molekularbiologische Untersuchungen enthüllten, dass Gasplasma-insensitive Tumorzellen vor allem durch Aneignung Stammzell-ähnlicher Eigenschaften sowie oxidativer Adaptation dem repetitiven ROS/RNS Stress unbeschadet entkommen können. Der resistente Tumorzell-Phänotyp konnte durch Transkriptomanalysen mit einer signifikant erhöhten Expression des Interleukin-1 receptor, type 2 (IL1R2) korreliert werden. Zusammengefasst konnte in der vorliegenden Arbeit herausgearbeitet werden, dass der Antitumor-Effekt der Gasplasmabehandlung sich durch ein Anpassen der Plasma-Jet-Geometrie sowie durch Kombinationsansätze mit pharmakologischen Substanzen verbessern lässt. Des Weiteren erlaubte die Entwicklung einer Gasplasma-Resistenzmodels von Hautkrebszellen ein tieferes Verständnis der zu Grunde liegenden Mechanismen. Diese Erkenntnisse unterstützen den Ansatz das Potential der Gasplasma-Technologie-Anwendung in der Onkologie durch weitere, zukünftige Untersuchung zu erforschen.