Biology subjects

Panning, M.

Publications and source records attributed to Panning, M..

4 recordsLinked to original sources

bioRxiv2024

varVAMP: automated pan-specific primer design for tiled full genome sequencing and qPCR of highly diverse viral pathogens.

Time- and cost-saving surveillance of viral pathogens is achieved by tiled sequencing in which a viral genome is amplified in overlapping PCR amplicons and qPCR. However, designing pan-specific primers for viral pathogens that have high genomic variability represents a major challenge. Here, we present a bioinformatics command-line tool, called varVAMP (variable virus amplicons). It relies on multiple sequence alignments of highly variable virus sequences and enables automatic pan-specific primer design for qPCR or tiled amplicon whole genome sequencing. The varVAMP software guarantees pan-specificity by two means: it designs primers in regions with minimal variability and introduces degenerate nucleotides into primer sequences to compensate for common sequence variations. We demonstrate varVAMPs utility by designing and evaluating novel pan-specific primer schemes suitable for sequencing the genomes of SARS-CoV-2, Hepatitis E virus, rat Hepatitis E virus, Hepatitis A virus, Borna-disease-virus-1, and Poliovirus. Moreover, we established highly sensitive and specific Poliovirus qPCR assays that could potentially simplify current Poliovirus surveillance. Importantly, wet-lab and bioinformatic techniques established for SARS-CoV-2 tiled amplicon sequencing were readily transferable to these new primer schemes and will allow sequencing laboratories to extend their established methodology to other human pathogens.

bioinformatics↗

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↗

bioRxiv2020

The landscape of SARS-CoV-2 RNA modifications

In 2019 the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the first documented cases of severe lung disease COVID-19. Since then, SARS-CoV-2 has been spreading around the globe resulting in a severe pandemic with over 500.000 fatalities and large economical and social disruptions in human societies. Gaining knowledge on how SARS-Cov-2 interacts with its host cells and causes COVID-19 is crucial for the intervention of novel therapeutic strategies. SARS-CoV-2, like other coronaviruses, is a positive-strand RNA virus. The viral RNA is modified by RNA-modifying enzymes provided by the host cell. Direct RNA sequencing (DRS) using nanopores enables unbiased sensing of canonical and modified RNA bases of the viral transcripts. In this work, we used DRS to precisely annotate the open reading frames and the landscape of SARS-CoV-2 RNA modifications. We provide the first DRS data of SARS-CoV-2 in infected human lung epithelial cells. From sequencing three isolates, we derive a robust identification of SARS-CoV-2 modification sites within a physiologically relevant host cell type. A comparison of our data with the DRS data from a previous SARS-CoV-2 isolate, both raised in monkey renal cells, reveals consistent RNA modifications across the viral genome. Conservation of the RNA modification pattern during progression of the current pandemic suggests that this pattern is likely essential for the life cycle of SARS-CoV-2 and represents a possible target for drug interventions.

genetics↗