Stoilov, P. G. | bioRxiv Science

bioRxiv2022

The 13A4 monoclonal antibody to the mouse PROM1 protein recognizes a structural epitope

PurposeWe endeavored to map the epitope of the rat monoclonal antibody mAB 13A4 to the mouse PROM1 (CD133, AC133) protein. mAB 13A4 is the main reagent used to detect the mouse PROM1 protein. PROM1 is required for the maintenance of primary cilia and mutations in the Prom1 gene are associated with retinal degeneration. MethodsEpitope tagged clones of splice variants and tiled deletion mutants were used to map the mAB 13A4 epitope and test the predicted tertiary structure of PROM1. The proteins were expressed in Neuro 2a cells and analyzed by Western blot with antibodies to PROM1 and the epitope tag. ResultsDeletions in the second and third extracellular domains of the PROM1 protein disrupted the mAB 13A4 epitope. Furthermore, the affinity of mAB 13A4 to the major PROM1 isoform in photoreceptor cells is significantly reduced due to the inclusion of a photoreceptor-specific alternative exon in the third extracellular domain. Interestingly, a deletion in the photoreceptor specific isoform of six amino acids adjacent to the alternative exon restored the affinity of mAB 13A4 to PROM1. ConclusionmAB 13A4 recognizes a structural epitope that is stabilized by two of the extracellular domains of PROM1. The results of our mutagenesis are consistent with the computationally predicted helical bundle structure of PROM1 and point to the utility of mAB 13A4 for evaluating the effect of mutations on the PROM1 structure. We show that the PROM1 isoform composition needs to be considered when interpreting tissue and developmental expression data produced by mAB 13A4.

immunology↗

bioRxiv2022

SARS-CoV-2 Delta variant induces enhanced pathology and inflammatory responses in K18-hACE2 mice

The COVID-19 pandemic has been fueled by novel variants of concern (VOC) that have increased transmissibility, receptor binding affinity, and other properties that enhance disease. The goal of this study is to characterize unique pathogenesis of the Delta VOC strain in the K18-hACE2-mouse challenge model. Challenge studies suggested that the lethal dose of Delta was higher than Alpha or Beta strains. To characterize the differences in the Delta strains pathogenesis, a time-course experiment was performed to evaluate the overall host response to Alpha or Delta variant challenge. qRT-PCR analysis of Alpha- or Delta- challenged mice revealed no significant difference between viral RNA burden in the lung, nasal wash or brain. However, histopathological analysis revealed high lung tissue inflammation and cell infiltration following Delta- but not Alpha-challenge at day 6. Additionally, pro-inflammatory cytokines were highest at day 6 in Delta-challenged mice suggesting enhanced pneumonia. Total RNA-sequencing analysis of lungs comparing infected to uninfected mice revealed that Alpha-challenged mice have more total genes differentially activated, conversely, Delta-challenged mice have a higher magnitude of differential gene expression. Delta-challenged mice have increased interferon-dependent gene expression and IFN-{gamma} production compared to Alpha. Analysis of TCR clonotypes suggested that Delta challenged mice have increased T-cell infiltration compared to Alpha challenged. Our data suggest that Delta has evolved to engage interferon responses in a manner that may enhance pathogenesis. The in vivo and in silico observations of this study underscore the need to conduct experiments with VOC strains to best model COVID-19 when evaluating therapeutics and vaccines. ImportanceThe Delta variant of SARS-CoV-2 is known to be more transmissible and cause severe disease in human hosts due to mutations in its genome that are divergent from previous variants of concern (VOC). Our study evaluates the pathogenesis of Delta in the K18-hACE2 mouse model compared to the Alpha VOC. We observed that relative to Alpha, Delta challenge results in enhanced inflammation and tissue damage with stronger antiviral responses. These observations provide insight into Deltas unique pathogenesis.

immunology↗