Biology subjects

Zehbe, I.

Publications and source records attributed to Zehbe, I..

8 recordsLinked to original sources

bioRxiv2022

Targeting the Human Papillomavirus 16 E6 Oncoprotein with Antibodies

The human papillomavirus (HPV) 16 genome encodes two oncoproteins, E6 and E7, which are essential for viral carcinogenesis. While E7 promotes cell proliferation, E6 abolishes the resulting p53-dependent apoptotic response. Due to this specific function, E6 is considered a suitable target for the development of a variety of therapeutic agents such as antibodies. Here, we review anti-E6 antibodies/antibody fragments generated by us and others, as well as present our latest results with Camelidae-derived single-domain antibodies (sdAbs). We had previously isolated a pool of anti-E6 sdAbs to identify E6 binders with the potential to be used clinically and in research. While our previous work has focused on recombinant E6 proteins, here we evaluated these sdAbs binding capacity to the endogenous E6 protein using co-immunoprecipitation and immunofluorescence. We obtained reproducible results in these applications with two sdAbs, filling a gap in HPV research. Despite their apparent E6 binding ability, these sdAbs do not raise p53 levels or induce apoptosis. Thus, while these reagents are valuable diagnostic and detection tools, identifying their therapeutic potential will require further development and testing.

cancer biology↗

bioRxiv2020

Virus-host protein-protein interactions between human papillomavirus 16 E6 A1 and D2/D3 sub-lineages: variances and similarities

High-risk strains of human papillomavirus are causative agents for cervical and other mucosal cancers with type 16 being the most frequent. Compared to the European Prototype (A1, denoted "EP"), the Asian-American (D2/D3, denoted "AA") sub-lineage or "variant" is reported to have increased abilities to promote carcinogenesis. Few global interactome studies have looked at protein-protein interactions (PPIs) between host proteins and variants of the key transforming E6 protein. We applied a primary human foreskin keratinocyte model transduced with EP and AA variant E6 genes and co-immunoprecipitated expressed E6 proteins along with interacting cellular proteins to detect virus-host binding partners. We reasoned that, due to single nucleotide polymorphisms, AAE6 and EPE6 may have unique PPIs with host cellular proteins--conferring gain or loss of function--resulting in varied abilities to promote carcinogenesis. Using liquid chromatography-mass spectrometry and stringent interactor selection criteria based on the number of peptides, we identified 25 candidates: 6 unique to each of AAE6 and EPE6, along with 13 E6 targets common to both AAE6 and EPE6. We also applied a more inclusive process based on pathway selection and discovered 171 target proteins: 90 unique AAE6 and 61 unique EPE6 along with 20 common E6 targets between the two sub-lineages. Interpretations for both approaches were made using databases such as UniProt, BioGRID and Reactome. Detected E6 targets are implicated in important hallmarks of cancer: deregulating Notch and other signaling, energetics and hypoxia, DNA replication and repair, and immune response. Validation experiments, such as reverse co-immunoprecipitation and RNA interference, are required to substantiate these findings. Here, we provide an unprecedented resource for new research questions in HR HPV biology. The current data also underline our labs driving hypothesis that E6, being a "master regulator" in HPV-positive cancers, is an excellent candidate for anti-cancer treatment strategies. Author SummaryChronic infection with high-risk human papillomavirus (HPV) type 16 is the most prevalent cause of cervical and other mucosal cancers. The E6 oncoproteins of the European Prototype (EP) and the Asian-American (AA) HPV variants differentially promote carcinogenesis. We looked at protein-protein interactions between host proteins and two key HPV variant E6 proteins of these strains to reveal how high risk HPVs cause cancer, based on the proteins they bind to in infected cells. Our methodology combined molecular biology and data mining techniques using widely available databases. We confirmed and discovered novel virus-host associations that explained how HPV AA and EP variants differ in their carcinogenic capabilities, and confirmed the candidacy of the E6 protein as a viable target for HPV therapies.

molecular biology↗

bioRxiv2020

Human papillomavirus type 16 sub-lineages and integration in cancer

Our lab has been intrigued by the fact that viral genomes often take on the role of mobile elements to perpetuate their existence in a complex organisms genome. Multiple DNA viruses such as Epstein-Barr virus, hepatitis B virus, and human papillomavirus (HPV) can invade their host genome, as "genomic parasites". We have been investigating HPV type 16 (HPV16), which is a prominent human tumour virus. In our recent in vitro work using 3D organoids, a common variant of HPV16s coding region elicited early integration into the host genome compared to the HPV16 prototype sequence. Next-generation sequencing (NGS) data confirmed a transcriptomic profile of increased proliferation and chromosomal instability--both hallmarks of cancer. Epidemiologically, this variant is associated with a high cervical cancer incidence. To take inquiries a step further, we investigated variant-specific integration across HPV16-related cancers using NGS data from population-derived clinical samples in The Cancer Genome Atlas (TCGA)-curated database. Data were analyzed for HPV16 positivity, sub-lineage, and viral-host integration using a bioinformatic pipeline of open-source tools, including HPVDetector. Here, we report the analysis of 120 cervical cancer cases comprising HPV16 positive and negative samples as well as their different sub-lineage and integration status. The integration signature between variant and prototype did not differ quantitatively but qualitatively: that of the variant being related to hypoxia/energetics (Warburg effect) and that of the prototype being much more varied to include host immune abrogation and cancer pathways activation. We conclude by discussing challenges and future directions for expanding these analyses.

cancer biology↗

bioRxiv2019

The potentials and pitfalls of a human cervical organoid model including Langerhans cells

Three-dimensional cell culturing to capture a life-like experimental environment has become a versatile tool for basic and clinical research. Mucosal and skin tissues can be grown as \"organoids\" in a petri dish and serve a wide variety of research questions. Here, we report our experience with human cervical organoids which could also include an immune component, e.g. Langerhans cells. We employ commercially available human cervical keratinocytes and fibroblasts as well as a myeloid cell line matured and purified into langerin-positive Langerhans cells. These are then seeded on a layer of keratinocytes with underlying dermal equivalent. Using about 10-fold more than the reported number in healthy cervical tissue (1-3%), we obtain differentiated cervical epithelium after 14 days with ~ 1% being Langerhans cells. We provide a detailed protocol for interested researchers to apply the described \"aseptic\" organoid model for all sorts of investigations--with or without Langerhans cells.

cell biology↗

bioRxiv2018

Single-domain antibodies represent novel alternatives to monoclonal antibodies as targeting agents against the human papillomavirus 16 E6 protein

Approximately one-fifth of all malignancies worldwide are etiologically-associated with a persistent viral or bacterial infection. Thus, there is particular interest in therapeutic molecules which utilize components of a natural immune response to specifically inhibit oncogenic microbial proteins, as it is anticipated they will elicit fewer off-target effects than conventional treatments. This concept has been explored in the context of human papillomavirus type 16 (HPV16)-related cancers, through the development of monoclonal antibodies and fragments thereof against the viral E6 oncoprotein. However, challenges related to the biology of E6 as well as the functional properties of the antibodies themselves appear to have precluded their clinical translation. In this study, we attempted to address these issues by exploring the utility of the variable domains of camelid heavy-chain-only antibodies (denoted as VHHs). Through the construction and panning of two llama immune VHH phage display libraries, a pool of potential VHHs was isolated. The interactions of these VHHs with recombinant E6 protein were further characterized using ELISA, Western blotting under both denaturing and native conditions, as well as surface plasmon resonance, and three antibodies were identified that bound recombinant E6 with affinities in the nanomolar range. Our results now lead the way for subsequent studies into the ability of these novel molecules to inhibit HPV16-infected cells in vitro and in vivo.

cancer biology↗

bioRxiv2018

Human Cervical Keratinocyte-Derived Monolayer and Organoid Cultures for Disease Modelling and Drug Screening

The successful isolation and propagation of patient-derived keratinocytes from cervical lesions constitute a more appropriate model of cervical disease than traditional cervical cancer-derived cell lines such as SiHa and CaSki. Our aim was to streamline the growth of patient-obtained, cervical keratinocytes into a reproducible process. We performed an observational case series study with 60 women referred to colposcopy for a diagnostic biopsy. Main outcome measures were how many samples could be passaged at least once, and where enough cells could be established, to precisely define their proliferation profile over time. Altering cell culture conditions over those reported by other groups markedly improved outcomes. We were also successful in making freeze backs which could be resuscitated for additional experiments. For best results, biopsy-intrinsic factors such as size and tissue digestion appear to be major variables. This seems to be the first systematic report with a well characterized and defined sample size, detailed protocol, carefully assessed cell yield and performance, and to successfully grow multi-layered, organoid cultures from cervical keratinocytes. This research is particularly impactful for constituting a sample repository-on-demand for appropriate disease modelling and drug screening under the umbrella of personalized health.

cancer biology↗

bioRxiv2017

Epithelial stratification shapes infection dynamics

Infections of stratified epithelia collectively represent a large burden on global health. Experimental models provide a means to understand how the cell dynamics themselves influence the outcomes of these infections. Mathematical approaches are needed to improve quantification and theoretical advancement of these complex systems. Here, we develop a general ecology-inspired model for stratified epithelial dynamics, which allows us to simulate infections and to estimate parameters that are difficult to measure with organotypic cell cultures. To explore how epithelial cell dynamics affect infection dynamics, we focus on two contrasting pathogens of the cervicovaginal epithelium: Chlamydia trachomatis and Human papillomaviruses. We find that key infection symptoms stem from differential interactions with the layers, while clearance and pathogen burden are bottom-up processes. Cell protective responses to infections (e.g. increased cell proliferation) generally lowered pathogen load but there were specific effects based on infection strategies. These generic responses by the epithelium, then, will have varying results depending on the pathogens infection strategy. Our modeling approach opens new perspectives for 3D tissue culture experimental systems of infections and, more generally, for developing and testing hypotheses related to infections of stratified epithelia.

systems biology↗

bioRxiv2017

Pathogen-Host Analysis Tool (PHAT): an Integrative Platform to Analyze Pathogen-Host Relationships in Next-Generation Sequencing Data

SummaryThe Pathogen-Host Analysis Tool (PHAT) is an application for processing and analyzing next-generation sequencing (NGS) data as it relates to relationships between pathogen and host organisms. Unlike custom scripts and tedious pipeline programming, PHAT provides an integrative platform encompassing raw and aligned sequence and reference file input, quality control (QC) reporting, alignment and variant calling, linear and circular alignment viewing, and graphical and tabular output. This novel tool aims to be user-friendly for life scientists studying diverse pathogen-host relationships.\n\nAvailability and ImplementationThe project is publicly available on GitHub (https://github.com/chgibb/PHAT) and includes convenient installers, as well as portable and source versions, for both Windows and Linux (Debian and RedHat). Up-to-date documentation for PHAT, including user guides and development notes, can be found at https://chgibb.github.io/PHATDocs/. We encourage users and developers to provide feedback (error reporting, suggestions, and comments) using GitHub Issues.\n\nContactLead software developer: chris.gibb@outlook.com

bioinformatics↗