Single-dose attenuated Vesiculovax vaccines protect primates against Ebola Makona virus

Abstract

The family Filoviridae contains three genera, Ebolavirus (EBOV), Marburg virus, and Cuevavirus. Some members of the EBOV genus, including Zaire ebolavirus (ZEBOV), can cause lethal haemorrhagic fever in humans. During 2014 an unprecedented ZEBOV outbreak occurred in West Africa and is still ongoing, resulting in over 10,000 deaths, and causing global concern of uncontrolled disease. To meet this challenge a rapid-acting vaccine is needed. Many vaccine approaches have shown promise in being able to protect nonhuman primates against ZEBOV. In response to the current ZEBOV outbreak several of these vaccines have been fast tracked for human use. However, it is not known whether any of these vaccines can provide protection against the new outbreak Makona strain of ZEBOV. One of these approaches is a first-generation recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing the ZEBOV glycoprotein (GP) (rVSV/ZEBOV). To address safety concerns associated with this vector, we developed two candidate, further-attenuated rVSV/ZEBOV vaccines. Both attenuated vaccines produced an approximately tenfold lower vaccine-associated viraemia compared to the first-generation vaccine and both provided complete, single-dose protection of macaques from lethal challenge with the Makona outbreak strain of ZEBOV.

Extended Data Figure 1. Relative immunogenicity of rVSV/ZEBOV vectors in cynomolgus macaques

At study day −28, cynomolgus macaques were immunized IM with 2 × 10⁷ PFU of either N4 or N1 vectors. Ten days after a single immunization, PBMCs were prepared and ZEBOV GP specific T cell responses were quantitated by IFN-γ ELISpot assay. a. ZEBOV GP-specific IFN-γ ELISpot responses in individual macaques. b. Average ZEBOV GP-specific IFN-γ ELISpot responses with standard error of the means indicated.

Figure 1. rVSV/ZEBOV vector design, growth kinetics and vaccine study strategy

a. Genome organization comparing ZEBOV GP (Mayinga strain) expressing rVSV vectors as described in methods. The rVSV/ZEBOVΔG (ΔG) vector had the natural VSV G gene replaced with the ZEBOV GP at position 4 within the genome. rVSVN1CT1GP3 (N1) vector retained the position of VSV N in position 1 (red box), insertion of ZEBOV GP at position 3 and a truncated form of VSV G containing the CT1 truncation was inserted at position 6. The rVSVN4CT1GP1 (N4) vector had the insertion of ZEBOV GP in position 1, attenuating N gene translocation (N4) (black box) and truncated G protein cytoplasmic tail (CT1). Numbers above vector constructs designate genome positions. Virus leader (Le), trailer (Tr), and intergenic regions are shown in black. Shaded regions represent deleted amino acid regions. b. Single-cycle growth kinetics comparing the ΔG, N1, and N4 vectors depicted in (a). Data shown are mean ± SD from two biological replicates titrated by plaque assay in triplicate. Titer differences between ΔG and N1 vectors were statistically significant at 4 (p = 0.0001, 12 (p = 0.0055), and 24 hours post infection (p = 0.0001). Likewise, ΔG and N4 vector titers were significantly different at 4 (p = 0.0001), 12 (p = 0.0005), 24 (p = 0.0001), and 48 hours post infection (p = 0.0068). Unpaired t-test, p = 0.05. c. Crystal violet stained Vero cell monolayers showing plaques generated by the ΔG, N1, and N4 vectors at 48 hours post infection. d. Flow chart showing the days of vaccination (triangles), days of sampling (arrows), day of challenge (*). Blue triangle, unvaccinated cohort; orange triangle, N1 vaccinated cohort; black triangle, N4 vaccinated cohort.

Figure 2. N1 and N4 vaccination results in circulating anti-ZEBOV GP IgG and protection in cynomolgus macaques

a. Reciprocal endpoint dilution titers for circulating IgG against ZEBOV GP for control (blue), N1 (orange), and N4 cohorts (black-grey) on day of vaccination (−28), 10 days post vaccination (−18), and on day of challenge (0). Red dashed line depicts limit of detection for ELISA assay. Error bars represent the standard error of the means. b. Kaplan-Meier survival curve for each cohort post ZEBOV challenge. c. Circulating infectious virus load displayed as plaque forming units per ml (PFU/ml). Data shown are from individual animals. Lower limit of detection is 25 PFU/ml.

Figure 3. Comparison of ZEBOV antigen in tissues of cynomolgus macaques either vaccinated or unvaccinated

a. and c. Liver, diffuse cytoplasmic immmunolabeling (brown) of sinusoidal lining cells in both ZEBOV-infected control animals. b. and d. Spleen, diffuse cytoplasmic immunolabeling of dendriform mononuclear cells in the red and white pulp of ZEBOV-infected control animals. e. and f. Liver and spleen respectively with a lack of immunolabeling from N1 cohort animal 0910078. g. and h. Liver and spleen respectively with a lack of immunolabeling from N4 cohort animal 0807174.