Figure 1: Construction and characterization of RhCMV vectors engineered to express EBOV GP (designated RhCMV/EBOV-GP). | Scientific Reports

Figure 1: Construction and characterization of RhCMV vectors engineered to express EBOV GP (designated RhCMV/EBOV-GP).

From: Cytomegalovirus-based vaccine expressing Ebola virus glycoprotein protects nonhuman primates from Ebola virus infection

Figure 1

(A) Schematic representation of RhCMV/EBOV-GP. Codon-optimized full-length EBOV GP was inserted within the RhCMV genome (68.1) to replace the endogenous Rh112 (pp65b) ORF. This approach places GP under the control of the endogenous RhCMV Rh112 promoter. (B) Multi-step growth analysis of RhCMV/EBOV-GP. RFs were infected at a multiplicity of infection (MOI) of 0.01 with either RhCMV/WT, RhCMV/EBOV-GP[2–8] or RhCMV/EBOV-GP[6–1]. Supernatant was collected at days indicated post infection and titered using a TCID50 assay. The assay was performed in triplicate and standard deviation is shown. (C) Western analysis of RhCMV/EBOV-GP infected RF cell lysates showing stable expression of EBOV GP until at least passage 7. The EBOV GP was tagged at the carboxyl terminus with a V5 epitope to facilitate detection, and V5 epitope tag-specific monoclonal antibody (mAb) or a GP EBOV-specific mAb were used for detection. V5 activity was observed against 3 bands as predicted [26kDa GP2, 110kDa preGPer (full length endoplasmic reticulum form; not shown) and 160kDa preGP (full length Golgi form; not shown)]55. The GP-specific mAb was used to detect GP1 (140kDa) as the V5 tag is localized at the carboxyl-terminus of GP. Endogenous RhCMV IE-1 was used as an infection level control.

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