Abstract
BackgroundEbola hemorrhagic fever is caused by the Ebola filovirus (EBOV), which is one of the most aggressive infectious agents known worldwide. The EBOV pathogenesis starts with uncontrolled viral replication and subversion of both the innate and adaptive host immune response. The multifunctional viral VP35 protein is involved in this process by exerting an antagonistic action against the early antiviral alpha/beta interferon (IFN-α/β) response, and represents a suitable target for the development of strategies to control EBOV infection.Phage display technology permits to select antibodies as single chain Fragment variable (scFv) from an artificial immune system, due to their ability to specifically recognize the antigen of interest. ScFv is ideal for genetic manipulation and to obtain antibody constructs useful for targeting either antigens expressed on cell surface or intracellular antigens if the scFv is expressed as intracellular antibody (intrabody) or delivered into the cells.ResultsMonoclonal antibodies (mAb) in scFv format specific for the EBOV VP35 were isolated from the ETH-2 library of human recombinant antibodies by phage display technology. Five different clones were identified by sequencing, produced in E.coli and expressed in CHO mammalian cells to be characterized in vitro. All the selected scFvs were able to react with recombinant VP35 protein in ELISA, one of the scFvs being also able to react in Western Blot assay (WB). In addition, all scFvs were expressed in cell cytoplasm as intrabodies; a luciferase reporter gene inhibition assay performed in A549 cells showed that two of the scFvs can significantly hamper the inhibition of the IFN-β-induced RIG-I signaling cascade mediated by EBOV VP35.ConclusionFive antibodies in scFv format recognize an active form of EBOV VP35 in ELISA, while one antibody also recognizes VP35 in WB. Two of these scFvs were also able to interfere with the intracellular activity of VP35 in a cell system in vitro. These findings suggest that such antibodies in scFv format might be employed to develop therapeutic molecules able to hamper EBOV infections.
Highlights
Ebola hemorrhagic fever is caused by the Ebola filovirus (EBOV), which is one of the most aggressive infectious agents known worldwide
Isolation and characterization of EBOV Viral protein 35 (VP35)-specific antibodies In order to isolate antibodies specific for the recombinant VP35 expressed in E.coli and purified in an active form [24, 27], an approach based on the phage display technology was used
The antiVP35 reactivity in enzymelinked immunosorbent assay (ELISA) was confirmed for all B10, A10, E1, F9 and H7 single chain Fragment variable (scFv), while in Western Blot assay (WB) the positivity was only observed for scFv A10, which reacted with a 37 kDa protein identified as the recombinant His-tagged VP35 protein detected by the anti-His Monoclonal antibodies (mAb)
Summary
Ebola hemorrhagic fever is caused by the Ebola filovirus (EBOV), which is one of the most aggressive infectious agents known worldwide. The EBOV pathogenesis starts with uncontrolled viral replication and subversion of both the innate and adaptive host immune response. Taï Forest, and Zaire EBOV are responsible for outbreaks in humans, whereas Reston EBOV infects non-human primates, and Bombali virus was recently discovered in bats [2, 3]. Fatal EBOV infections are characterized by rapid viral replication combined with an inadequate antiviral response. The EBOV pathogenesis starts with the subversion of both innate and adaptive immune response and the consequent induction of harmful inflammatory responses and tissue necrosis due to disseminated infections [4, 5]
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