Ebola Virus Glycoprotein Domains Associated with Protective Efficacy

Abstract

Ebola virus (EBOV) is the cause of sporadic outbreaks of human hemorrhagic disease in Africa, and the best-characterized virus in the filovirus family. The West African epidemic accelerated the clinical development of vaccines and therapeutics, leading to licensure of vaccines and antibody-based therapeutics for human use in recent years. The most widely used vaccine is based on vesicular stomatitis virus (VSV) expressing the EBOV glycoprotein (GP) (VSV-EBOV). Due to its favorable immune cell targeting, this vaccine has also been used as a base vector for the development of second generation VSV-based vaccines against Influenza, Nipah, and Zika viruses. However, in these situations, it may be beneficial if the immunogenicity against EBOV GP is minimized to induce a better protective immune response against the other foreign immunogen. Here, we analyzed if EBOV GP can be truncated to be less immunogenic, yet still able to drive replication of the vaccine vector. We found that the EBOV GP glycan cap and the mucin-like domain are both dispensable for VSV-EBOV replication. The glycan cap, however, appears critical for mediating a protective immune response against lethal EBOV challenge in mice.

Keywords: EBOV; VSV; filovirus; glycan cap; mucin-like domain; vesicular stomatitis virus.

Figure 1

Vaccine construction and in vitro characterization. (A) Schematic of the EBOV glycoprotein (GP). SP, signal peptide; RBD, receptor-binding domain; GC, glycan cap; MLD, mucin-like domain; FP, fusion peptide; CD, coiled-coil domain; TM, transmembrane domain. Arrow indicates furin-cleavage site. (B) The VSV-EBOV vector was modified to express a GP containing a deletion of the MLD (VSV-EBOVΔMLD) or the GC plus MLD (VSV-EBOVΔGCΔMLD). After successful recovery of the viruses from plasmid transfections, protein expression was confirmed by Western blot analysis using monoclonal antibodies specific for the EBOV GP (left panel) or VSV matrix (M) protein (right panel). Lane 1, VSV-EBOV; lane 2, VSV-EBOVΔMLD; lane 3, VSV-EBOVΔGCΔMLD; lane 4, VSV wildtype (wt); lane 5, uninfected control. (C) Growth kinetics were performed in triplicate on Vero E6 cells at a multiplicity of infection of 0.01. Geometric mean and SD are depicted. Statistically significant differences are indicated as follows: p < 0.0001 (****).

Figure 2

Study outline and protective efficacy in mice. (A) Study outline. Groups of CD1 mice were intraperitoneally (IP) vaccinated with a single dose of 1 × 10⁴ PFU of the vaccines 28 days prior to lethal challenge. (B) Body weight changes and (C) survival curves are shown. (D) On day 4 after challenge, 4 mice in each group were euthanized for sample collection, and virus titers were determined. Geometric mean and SD are depicted. Statistical significance is indicated as follows p < 0.0001 (****), p < 0.001 (***), p < 0.01 (**), and p < 0.05 (*).

Figure 3

Humoral immune responses in vaccinated and challenged mice. (A) EBOV GP-specific IgG was assessed in serum samples from mice on day 0 (n = 4; 28 days after vaccination), day 4 (n = 4) and day 42 (all survivors). Geometric mean and SD are depicted. Statistical significance is indicated, p < 0.05 (*). (B) Neutralizing activity in the serum of vaccinated mice (day 0, n = 4 per group) and (C) challenge survivors (day 42; n = 6 for VSV-EBOV and VSV-EBOV∆MLD or n = 1 for VSV-EBOV∆GC∆MLD) was determined using the EBOV trVLP system expressing eGFP. Mean and SD are depicted.