Invasion by the malaria merozoite depends on recognition of specific erythrocyte surface receptors by parasite ligands. Plasmodium falciparum uses multiple ligands, including at least two gene families, reticulocyte binding protein homologues (RBLs) and erythrocyte binding proteins/ligands (EBLs). The combination of different RBLs and EBLs expressed in a merozoite defines the invasion pathway utilized and could also play a role in parasite virulence. The binding regions of EBLs lie in a conserved cysteine-rich domain while the binding domain of RBL is still not well characterized. Here, we identify the erythrocyte binding region of the P. falciparum reticulocyte binding protein homologue 1 (PfRH1) and show that antibodies raised against the functional binding region efficiently inhibit invasion. In addition, we directly demonstrate that changes in the expression of RBLs can constitute an immune evasion mechanism of the malaria merozoite.
The envelope glycoprotein located at the outermost surface of the flavivirus particle mediates entry of virus into host cells. In this study, the involvement of domain III of West Nile virus (WNV-DIII) envelope protein in binding to host cell surface was investigated. WNV-DIII was first expressed as a recombinant protein and purified after a solubilization and refolding procedure. The refolded WNV-DIII protein displays a content of b-sheets consistent with known homologous structures of other flavivirus envelope DIII, shown by using circular dichroism analysis. Purified recombinant WNV-DIII protein was able to inhibit WNV entry into Vero cells and C6/36 mosquito cells. Recombinant WNV-DIII only partially blocked the entry of dengue-2 (Den 2) virus into Vero cells. However, entry of Den 2 virus into C6/36 was blocked effectively by recombinant WNV-DIII. Murine polyclonal serum produced against recombinant WNV-DIII protein inhibited infection with WNV and to a much lesser extent with Den 2 virus, as demonstrated by plaque neutralization assays. Together these results provided strong evidence that immunoglobulin-like DIII of WNV envelope protein is responsible for binding to receptor on the surface of host cells. The data also suggest that similar attachment molecule(s) or receptor(s) were used by WNV and Den 2 virus for entry into C6/36 mosquito cells.
The flavivirus envelope glycoprotein (E) is responsible for viral attachment and entry by membrane fusion. Its ectodomain is the primary target of the humoral immune response. In particular, the Cterminal Ig-like domain III of E, which is exposed at the surface of the viral particle, forms an attractive antigen for raising protective monoclonal antibodies (mAb). 9F12, a mouse mAb raised against a dengue virus (DENV) serotype 2 recombinant domain III, cross-reacts with corresponding domains from the other three DENV serotypes and also with West Nile virus. mAb 9F12 binds with nanomolar affinity to a conserved epitope that maps to the viral surface comprising residues 305, 307, 310 and 330 of the E protein. mAb 9F12 neutralizes all four DENV serotypes in plaque reduction assays. We expressed a single-chain Fv from 9F12 that retains the binding activity of the parent mAb. Adsorption and fusion inhibition assays indicate that mAb 9F12 prevents early steps of viral entry. Its virus inhibition activity and broad cross-reactivity makes mAb 9F12 a suitable candidate for optimization and humanization into a therapeutic antibody to treat severe infections by dengue.
Domain III of the dengue virus envelope protein (EDIII, aa295-395) has an immunoglobulin fold and is the proposed receptor-binding domain of the virus. Previous studies have shown that monoclonal antibodies against EDIII can be neutralizing and have therapeutic potential. Here, cloned Fab-phage libraries of human and mouse origin were screened for DENV specific antibodies. Firstly, bacterially expressed EDIII or whole virus particles were used as bait in biopanning against a large naïve human Fab-phage library (>10 billion independent clones). Multiple panning strategies were employed, and in excess of 1000 clones were screened, but all of the antibodies identified bound the envelope in regions outside EDIII suggesting EDIII antibodies are virtually absent from the naïve human repertoire. Next, a chimeric Fab-phage library was constructed from a panel of EDIII specific mouse hybridomas by pooling the VH and VL chain sequences from the hybridomas and cloning these into the pComb3X phagemid vector with human CH and CL encoding sequences. Biopanning against EDIII identified a unique antibody (C9) that cross-reacts with EDIII from DENV1-3 and, in the IgG format, binds and neutralizes DENV2 in cell-based assays. Sequence analysis and saturation mutagenesis of complementary determining regions (CDR) in the C9 light chain suggest an antigen recognition model in which the LCDR3 is a key determinant of EDIII specificity, while modifications in LCDR1 and LCDR2 affect DENV serotype cross-reactivity. Overall, this study supports the current prevailing opinion that neutralizing anti-EDIII monoclonal antibodies can be readily generated in murine systems, but in humans the anti-DENV immune response is directed away from domain III.
The poultry industry is the largest source of meat and eggs for human consumption worldwide. However, viral outbreaks in farmed stock are a common occurrence and a major source of concern for the industry. Mortality and morbidity resulting from an outbreak can cause significant economic losses with subsequent detrimental impacts on the global food supply chain. Mass vaccination is one of the main strategies for controlling and preventing viral infection in poultry. The development of broadly protective vaccines against avian viral diseases will alleviate selection pressure on field virus strains and simplify vaccination regimens for commercial farms with overall savings in husbandry costs. With the increasing number of emerging and re-emerging viral infectious diseases in the poultry industry, there is an urgent need to understand the strategies for broadening the protective efficacy of the vaccines against distinct viral strains. The current review provides an overview of viral vaccines and vaccination regimens available for common avian viral infections, and strategies for developing safer and more efficacious viral vaccines for poultry.
The Envelope protein domain 111 (Ed3) 24 1.5 Structure of flavivirus 1.6 Interaction with host cell surface receptors 1.6.1 Low pH induced endosomalfusion by flavivirus 32 1.6.2 Flavivirus maturation and Budding 38 1.7 Viral entry inhibitors 40 1.7.1 Anti flaviviral antibodies 1.8 Aim of the study 2: MATERIALS AND METHODS 2.1 Molecular cloning 2.1.1 Overexpression ofrEd3 2.1.2 Denaturation and refolding of rEd3 50 2.1.3 Purification ofrEd3 by gel filtration chromatography 53 2.1.4 Physicochemical characterization ofrEd3 2.1.5 Enzymatic cleavage ofTrx and His tags from rEd3 2.1.6 Binding of anti-His or anti-E antibody to rEd3 2.1.7 Inhibition of viral entry by rEd3 2.1.8 Characterization ofmAb 9F12 binding to rEd3 and virus 2.2 Maintenance of cell cultures and hybridomas 2.2.1 Virus propagation, purification and quantification 2.2.2 Identification of virus containing fractions from gradients by SDS-IV
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