RESUMO
West Nile virus (WNV) is a mosquito-borne neurotrophic flavivirus causing mild febrile illness to severe encephalitis and acute flaccid paralysis with long-term or permanent neurological disorders. Due to the absence of targeted therapy or vaccines, there is a growing need to develop effective anti-WNV therapy. In this study, single-domain antibodies (sdAbs) were developed against the domain III (DIII) of WNV's envelope glycoprotein to interrupt the interaction between DIII and the human brain microvascular endothelial cells (hBMEC). The peripheral blood mononuclear cells of the llama immunized with recombinant DIIIL297-S403 (rDIII) were used to generate a variable heavy chain only (VHH)-Escherichia coli library, and phage display was performed using the M13K07ΔpIII Hyperphages system. Phages displaying sdAbs against rDIII were panned with the synthetic analogs of the DIII receptor binding motifs, DIII-1G299-K307 and DIII-2V371-R388, and the VHH gene from the eluted phages was subcloned into E. coli SHuffle. Soluble sdAbs purified from 96 E. coli SHuffle clones were screened to identify 20 candidates strongly binding to the synthetic analogs of DIII-1G299-K307 and DIII-2V371-R388 on a dot blot assay. Among them, sdAbA1, sdAbA6, sdAbA9, and sdAbA10 blocked the interaction between rDIII and human brain microvascular endothelial cells (hBMECs) on Western blot and cell ELISA. However, optimum stability during the overexpression was noticed only for sdAbA10 and it also neutralized the WNV-like particles (WNV-VLP) in the Luciferase assay with an half maximal effective concentration (EC50) of 1.48 nm. Furthermore, the hemocompatibility and cytotoxicity of sdAbA10 were assessed by a hemolytic assay and XTT-based hBMEC proliferation assay resulting in 0.1% of hemolytic activity and 82% hBMEC viability, respectively. Therefore, the sdAbA10 targeting DIII-2V371-R388 of the WNV envelope glycoprotein is observed to be suitable for in vivo trials as a specific therapy for WNV-induced neuropathogenesis.
RESUMO
Borrelia bavariensis can invade the central nervous system (CNS) by crossing the blood-brain barrier (BBB). It is predicted that B. bavariensis evokes numerous signaling cascades in the human brain microvascular endothelial cells (hBMECs) and exploits them to traverse across the BBB. The complete picture of signaling events in hBMECs induced by B. bavariensis remains uncovered. Using RNA sequencing, we mapped 11,398 genes and identified 295 differentially expressed genes (DEGs, 251 upregulated genes and 44 downregulated genes) in B. bavariensis challenged hBMECs. The results obtained from RNA-seq were validated with qPCR. Gene ontology analysis revealed the participation of DEGs in a number of biological processes like cell communication, organization of the extracellular matrix, vesicle-mediated transport, cell response triggered by pattern recognition receptors, antigen processing via MHC class I, cellular stress, metabolism, signal transduction, etc. The expression of several non-protein coding genes was also evoked. In this manuscript, we discuss in detail the correlation between several signaling cascades elicited and the translocation of BBB by B. bavariensis. The data revealed here may contribute to a better understanding of the mechanisms employed by B. bavariensis to cross the BBB.
RESUMO
West Nile virus (WNV), re-emerging neurotropic flavivirus, can cross the blood-brain barrier (BBB) and cause fatal encephalitis and meningitis. Infection of the human brain microvascular endothelial cells (hBMECs), building blocks of the BBB, represents the pivotal step in neuroinvasion. Domain III (DIII) of the envelope (E) glycoprotein is a key receptor-binding domain, thus, it is an attractive target for anti-flavivirus strategies. Here, two combinatorial phage display peptide libraries, Ph.D.-C7C and Ph.D.-12, were panned against receptor-binding site (RBS) on DIII to isolate peptides that could block DIII. From series of pannings, nine peptides (seven 7-mer cyclic and two 12-mer linear) were selected and overexpressed in E. coli SHuffle T5. Presence of disulfide bond in 7-mer peptides was confirmed with thiol-reactive maleimide labeling. Except for linear peptide 19 (HYSWSWIAYSPG), all peptides proved to be DIII binders. Among all peptides, 4 cyclic peptides (CTKTDVHFC, CIHSSTRAC, CTYENHRTC, and CLAQSHPLC) showed significant blocking of the interaction between DIII and hBMECs, and ability to neutralize infection in cultured cells. None of these peptides showed toxic or hemolytic activity. Peptides identified in this study may serve as potential candidates for the development of novel antiviral therapeutics against WNV.
