Norovirus infections belong to the most common causes of human gastroenteritis worldwide and epidemic outbreaks are responsible for hundreds of thousands of deaths annually. In humans, noroviruses are known to bind to gastrointestinal epithelia via recognition of blood-group active mucin-type O-glycans. Considering the involvement of l-α-fucose residues in these glycans, their high valency on epithelial surfaces far surpasses the low affinity, though specific interactions of monovalent milk oligosaccharides. Based on these findings, we attempted to identify polyfucoses (fucans) with the capacity to block binding of the currently most prevalent norovirus strain GII.4 (Sydney, 2012, JX459908) to human and animal gastrointestinal mucins. We provide evidence that inhibitory effects on capsid binding are exerted in a competitive manner by α-fucosyl residues on Fucus vesiculosus fucoidan, but also on the galacto-fucan from Undaria pinnatifida and their oligo-fucose processing products. Insight into novel structural aspects of fucoidan and derived oligosaccharides from low-mass Undaria pinnatifida were revealed by GCMS and MALDI mass spectrometry. In targeting noroviral spread attenuation, this study provides first steps towards a prophylactic food additive that is produced from algal species.

Efficient delivery of therapeutics to the central nervous system (CNS) is one of the major challenges in treating neurological diseases due to brain barriers, which prevent entry of almost all potential therapeutic agents into the CNS. Targeting receptors that induce receptor-mediated transcytosis (RMT) across brain barriers has long been heralded as a potential solution to this problem, but this approach has yet to deliver clinical improvements for patients. Here, we set out to identify and characterize bivalent antibodies against the transferrin receptor 1 (TfR) as mediators of RMT. We identified the antibody YU904-F06 (hereafter referred to as F06) that showed efficient transcytosis as a bivalent IgG in two independent in vitro models of brain barriers. Despite its high affinity at extracellular pH levels, we determined that F06's binding to TfR was greatly reduced at lower pH levels expected during endocytic acidification. We postulated, with the support of a validated predictive mathematical model of RMT, that the pH-sensitivity of F06 allowed it to overcome the lysosomal degradation that has been previously reported for high affinity bivalent binders of TfR. Finally, we demonstrated that F06 could mediate the transcytosis of scFvs that target TREM2 or EGFRvIII as potential therapeutic cargos. In conclusion, we present a proof-of-concept antibody and rationale for the design of high affinity bivalent anti-TfR antibodies that effectively induce RMT by exploiting pH-sensitivity in binding.

Non-typeable Haemophilus influenzae (NTHI) is a pathogen of the human respiratory tract causing the majority of invasive H. influenzae infections. Severe invasive infections such as septicemia and meningitis occur rarely, but the lack of a protecting vaccine and the increasing antibiotic resistance of NTHI impede treatment and emphasize its relevance as a potential meningitis causing pathogen. Meningitis results from pathogens crossing blood–brain barriers and invading the immune privileged central nervous system (CNS). In this study, we addressed the potential of NTHI to enter the brain by invading cells of the choroid plexus (CP) prior to meningeal inflammation to enlighten NTHI pathophysiological mechanisms. A cell culture model of human CP epithelial cells, which form the blood–cerebrospinal fluid barrier (BCSFB) in vivo, was used to analyze adhesion and invasion by immunofluorescence and electron microscopy. NTHI invade CP cells in vitro in a polar fashion from the blood-facing side. Furthermore, NTHI invasion rates are increased compared to encapsulated HiB and HiF strains. Fimbriae occurrence attenuated adhesion and invasion. Thus, our findings underline the role of the BCSFB as a potential entry port for NTHI into the brain and provide strong evidence for a function of the CP during NTHI invasion into the CNS during the course of meningitis.

Enterovirus A 71 (EV-A71) has caused major epidemics worldwide. While it is most commonly associated with Hand-Foot-Mouth Disease (HFMD) or nonspecific presentations as upper or lower respiratory tract infection, it can also lead to severe neurologic manifestations especially in young children. Detailed understanding of the pathogenesis of EV-A71 CNS invasion as well as of the neurotropic subgenotypes is currently lacking. EV-A71 infection at the level of the brain barriers was assessed with human in vitro models of the Blood-Cerebrospinal Fluid Barrier (BCSFB) using Human choroid plexus papilloma (HIBCPP) cells and the Blood-Brain Barrier (BBB) using Human brain microvascular endothelial cells (HBMEC). Six clinically relevant genotypes, namely BrCr, B5, C1, C1-like, C2 and C4 were compared. Besides BrCr, the reference strain for EV-A71 and C2, which is known for its worldwide presence, C1 and C1-like were chosen as they are mainly circulating in Europe whereas C4 and B5 are mainly known from the Asian-Pacific region. Infection was analyzed qualitatively via cytopathic effect (CPE) with light microscopy and immunofluorescence staining. Quantification of viral genome copies was performed via qPCR. In parallel, barrier integrity of the BCSFB was monitored via measurement of transepithelial resistance. LIVE/DEAD assays were applied for viability controls. HIBCPP cells and HBMEC differed in their susceptibility to infection with different genotypes of EV-A71 namely BrCr, B5, C1, C1-like, C2 and C4. None of the strains caused a lytic infection in either model, in contrast to infection in RD cells. In HIBCPP cells as a model for the BCSFB, B5 led to the highest infection rates. Significant differences in the polar release of viral progeny to the CSF side were detected for B5 (56.42 ± 7.13 %) compared to BrCR (30.39 ± 8.11 %), C1 (8.73 ± 8.89 %), C1-like (23.90 ± 17.28 %), C2 (16.89 ± 8.95 %) and C4 (21.46 ± 3.16 %). In HBMEC as a model for the BBB, BrCr showed significantly higher apical release compared to the other genotypes. The minimum p-value (0.0019) was recorded for p_BrCrvs.B5 at 24 h and 48 h, while the maximum p-value (0.0379) was observed for p_BrCrvs.B5 at 72 h. Our findings reveal that different genotypes of EV-A71 exhibit distinct tropisms at the level of the brain barriers, which may correlate with variations in the clinical course of infection. Clinical and molecular surveillance of EV infections beyond those currently established in the Asia-Pacific region is warranted.

The human choroid plexus (CP) is the location of the blood–cerebrospinal fluid (CSF) barrier (BCSFB). Whereas the epithelial cells of the CP mainly contribute to the formation of the BCSFB, the vessels of the CP are built by fenestrated endothelial cells. Still, the CP endothelium can contribute to barrier function. By ectopic expression of human telomerase reverse transcriptase (hTERT) in primary human CP endothelial cells (HCPEnCs), we recently generated and characterized immortalized HCPEnCs (iHCPEnCs). Here, we compared primary cells of the sixth passage (HCPEnCs p6) with a lower (p20) and a higher passage (p50) of iHCPEnCs by transcriptome analysis. A high concordance of HCPEnCs and both passages of iHCPEnCs was observed, as only small proportions of the transcripts examined were significantly altered. Differentially expressed genes (DEGs) were identified and assigned to potentially affected biological processes by gene set enrichment analysis (GSEA). Various components of the endothelial barrier-relevant Wnt signaling were detected in HCPEnCs and iHCPEnCs. Functional analysis of HCPEnCs and iHCPEnCs showed equal marginal activation of Wnt signaling, supporting the downregulation of β-catenin (CTNNB) signaling in CP endothelial cells, and a contribution to the barrier function by the CP endothelium was retained until passage 100 (p100) of iHCPEnCs. Overall, our data support the suitability of iHCPEnCs as an in vitro model of the CP endothelium over extended passages.