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Ebola virus (EBOV), a filovirus family member, is a highly pathogenic virus that causes Ebola hemorrhagic fever (EHF) resulting in documented mortality rates in humans as high as 50%. Currently, the basic EBOV virus-like particle (VLP) vaccine contains the Ebola virus (EBOV) matrix VP40 and attachment glycoprotein (GP). VLPs are morphologically and biochemically similar to parental virus, yet because they lack a genome and cannot replicate, are safe enough to be used as vaccines. We hypothesize that addition of a constitutionally active retinoic acid-inducible gene 1 (RIG-I) would enhance the ability of the vaccine to induce interferon-dependent immune functions yielding an improved vaccine. Expression of EBOV VP40 in 293T cells induces the spontaneous production of VLPs into the media supernatant and if expressed with EBOV GP, will produce VLPs studded with the attachment GP. Recombinant chimeric constitutively active (ca)RIG-I-VP40 matrix and a nonfunctional mutant L58A (mu)RIG-I-VP40 matrix genes were constructed to produce VLPs containing constitutively active and nonfunctional RIG-I. Supernatant from 293Ts transfected with caRIG-I-VP40, muRIG-I-VP40 or VP40 along with GP expression plasmids were tested for the presence of VLPs. Western blotting of purified VLPs confirmed the presence of RIG-I in caRIG-I-VP40 and muRIG-I-VP40, but not VP40 containing VLPs. Monocyte-like and PMA-differentiated macrophage-like THP-1 Dual cells were treated with nothing, VP40+GP, caRIG-I-VP40+GP, muRIG-I-VP40+GP VLPs as well as LPS and a Vaccinia virus (VACV-70) positive controls and tested for induction of interferon (IFN) signaling. CaRIG-I containing, but not muRIG-I containing VLPs induced interferon signaling from both macrophages and monocytes. These results lead us to conclude that supplemented CaRIG-I would be ideal for robust induction of interferon-dependent immune functions, which may improve vaccine efficacy.

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Final Report Form, Research Report

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First Advisor

Osvaldo Martinez



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