Biology subjects

Boettler, T.

Publications and source records attributed to Boettler, T..

4 recordsLinked to original sources

bioRxiv2026

A Modular Chromosomally Integrated Yeast Surface Display Platform for Quantitative Serology and Immune Profiling

Precise quantification of antigen-specific antibodies is essential for assessing immune status and fundamental to both clinical and research settings. However, current serological platforms remain limited in their ability to combine quantitative performance, flexibility, and scalability. Here, we present a chromosomally integrated yeast surface display (YSD) system engineered for stable, quantitative, and scalable antibody profiling. Genomic integration of the YSD cassette supports uniform antigen presentation without continuous selective pressure, reducing variability associated with plasmid-based systems. Two distinct cell-wall anchoring strategies are implemented within a modular synthetic framework that enables rapid, cloning-free strain construction. Coupled with flow-cytometric readout, the system provides sensitive detection of antigen-specific antibodies with single-cell normalization for antigen expression. In addition, the combination of barcoded YSD strain libraries with fluorescence-activated cell sorting and next-generation sequencing enables multiplexed serological profiling across multiple samples and antigens. As a proof of concept, we demonstrate quantitative antibody detection against SARS-CoV-2 and extend the approach to hepatitis virus antigens, highlighting the utility of this platform as a flexible framework for serological assay development and immune profiling.

bioengineering↗

bioRxiv2023

A high-content RNA-based imaging assay reveals integrin beta 1 as a cofactor for cell entry of non-enveloped hepatitis E virus

Hepatitis E virus (HEV) is a major cause of acute hepatitis and mainly transmitted faecal-orally. HEV particles in faeces are non-enveloped, while those in the blood possess a cell-derived lipid envelope. Despite being a global health concern, there is limited understanding of the steps in the HEV life cycle, particularly cell entry. A previous study proposed integrin alpha 3 (ITGA3) as a potential host factor for nHEV entry, but the {beta}-integrin partner that co-mediates HEV entry has not been described. To address this knowledge gap and resolve the existing controversies surrounding HEV cell entry, we developed an RNA-FISH-based high-content imaging assay alllowing investigation of the entry pathways of both naked and enveloped HEV particles. Our observations indicate that naked HEV particles interact with the surface receptor integrin beta 1 (ITGB1), which likely facilitates their trafficking through the recycling endosome. In contrast, enveloped HEV particles do not interact with ITGB1 and instead use the classical endocytic pathway via the early endosome. Importantly, both forms of HEV require endosomal acidification and proteolytic cleavage by lysosomal cathepsins, which ultimately results in delivery of the HEV genome to the cytoplasm.

microbiology↗

bioRxiv2023

Efficient formation and maintenance of humoral and CD4 T cell immunity targeting the viral capsid in acute-resolving hepatitis E infection

Background and aimsCD4 T cells shape the neutralizing antibody (nAb) response and facilitate viral clearance in various infections. Knowledge of their phenotype, specificity and dynamics in hepatitis E virus (HEV) infection is limited. HEV is enterically transmitted as a naked virus (nHEV) but acquires a host-derived quasi-envelope (eHEV) when budding from cells. While nHEV is composed of the open-reading-frame (ORF)-2-derived capsid, eHEV particles also contain ORF3-derived proteins. We aimed to longitudinally characterize the HEV-specific CD4 T cells and neutralizing antibodies that target either nHEV or eHEV particles in immunocompetent individuals with acute and resolved HEV infection. MethodsHEV-specific CD4 T cells were analyzed by intracellular cytokine staining after stimulation with in silico predicted ORF1- and ORF2-derived epitopes and overlapping peptides spanning the ORF3 region. Ex vivo multi-parametric characterization of capsid-specific CD4 T cells was performed using customized MHC class II tetramers. Total and neutralizing antibodies targeting nHEV or eHEV particles were determined. ResultsHEV-specific CD4 T cell frequencies and antibody titers are highest in individuals with acute infection and decline in a time-dependent process with an antigen hierarchy. HEV-specific CD4 T cells primarily target the ORF2-derived capsid, which correlates with the presence of nAbs targeting nHEV. In contrast, ORF3-specific CD4 T cells are hardly detectable and eHEV is less efficiently neutralized. Capsid-specific CD4 T cells undergo memory formation and stepwise contraction, accompanied by dynamic phenotypical and transcriptional changes over time. ConclusionThe viral capsid is the main target of HEV-specific CD4 T cells and antibodies in acute resolving infection, correlating with efficient neutralization of nHEV. Capsid-specific immunity rapidly emerges followed by a stepwise contraction for several years after infection. Impact and implicationsThe interplay of CD4 T cells and neutralizing antibody responses is critical in the host defense against viral infections, yet little is known about their characteristics in hepatitis E virus (HEV) infection. We conducted a longitudinal study of immunocompetent individuals with acute and resolved HEV infection to understand the characteristics of HEV-specific CD4 T cells and neutralizing antibodies targeting different viral proteins and particles. We found that HEV-specific CD4 T cells mainly target the viral capsid, leading to efficient neutralization of the naked virus (nHEV) while the quasi-envelope (eHEV) particles are less susceptible to neutralization. As individuals with pre-existing liver disease and immunocompromised individuals are at risk for fulminant or chronic courses of HEV infection, these individuals might benefit from the development of vaccination strategies which require a detailed knowledge of HEV-specific CD4 T cell and antibody immunity.

immunology↗

bioRxiv2020

Ex vivo detection of SARS-CoV-2-specific CD8+ T cells: rapid induction, prolonged contraction, and formation of functional memory

CD8+ T cells are critical for the elimination and long-lasting protection of many viral infections, but their role in the current SARS-CoV-2 pandemic is unclear. Emerging data indicates that SARS-CoV-2-specific CD8+ T cells are detectable in the majority of individuals recovering from SARS-CoV-2 infection. However, optimal virus-specific epitopes, the role of pre-existing heterologous immunity as well as their kinetics and differentiation program during disease control have not been defined in detail. Here, we show that both pre-existing and newly induced SARS-CoV-2-specific CD8+ T-cell responses are potentially important determinants of immune protection in mild SARS-CoV-2 infection. In particular, our results can be summarized as follows: First, immunodominant SARS-CoV-2-specific CD8+ T-cell epitopes are targeted in the majority of individuals with convalescent SARS-CoV-2 infection. Second, MHC class I tetramer analyses revealed the emergence of phenotypically diverse and functionally competent pre-existing and newly induced SARS-CoV-2-specific memory CD8+ T cells that showed similar characteristics compared to influenza-specific CD8+ T cells. Third, SARS-CoV-2-specific CD8+ T-cell responses are more robustly detectable than antibodies against the SARS-CoV-2-spike protein. This was confirmed in a longitudinal analysis of acute-resolving infection that demonstrated rapid induction of the SARS-CoV-2-specific CD8+ T cells within a week followed by a prolonged contraction phase that outlasted the waning humoral immune response indicating that CD8+ T-cell responses might serve as a more precise correlate of antiviral immunity than antibody measurements after convalescence. Collectively, these data provide new insights into the fine specificity, heterogeneity, and dynamics of SARS-CoV-2-specific memory CD8+ T cells, potentially informing the rational development of a protective vaccine against SARS-CoV-2.

immunology↗