Demonstration of cross-protective vaccine immunity against an emerging pathogenic Ebolavirus Species

Abstract

A major challenge in developing vaccines for emerging pathogens is their continued evolution and ability to escape human immunity. Therefore, an important goal of vaccine research is to advance vaccine candidates with sufficient breadth to respond to new outbreaks of previously undetected viruses. Ebolavirus (EBOV) vaccines have demonstrated protection against EBOV infection in nonhuman primates (NHP) and show promise in human clinical trials but immune protection occurs only with vaccines whose antigens are matched to the infectious challenge species. A 2007 hemorrhagic fever outbreak in Uganda demonstrated the existence of a new EBOV species, Bundibugyo (BEBOV), that differed from viruses covered by current vaccine candidates by up to 43% in genome sequence. To address the question of whether cross-protective immunity can be generated against this novel species, cynomolgus macaques were immunized with DNA/rAd5 vaccines expressing ZEBOV and SEBOV glycoprotein (GP) prior to lethal challenge with BEBOV. Vaccinated subjects developed robust, antigen-specific humoral and cellular immune responses against the GP from ZEBOV as well as cellular immunity against BEBOV GP, and immunized macaques were uniformly protected against lethal challenge with BEBOV. This report provides the first demonstration of vaccine-induced protective immunity against challenge with a heterologous EBOV species, and shows that Ebola vaccines capable of eliciting potent cellular immunity may provide the best strategy for eliciting cross-protection against newly emerging heterologous EBOV species.

Figure 1. Vaccination and challenge schedule.

(A) Vaccine group NHP were injected with plasmid DNA and rAd5 vectors encoding GP from SEBOV and/or ZEBOV. Individual DNA immunizations were spaced by 4–6 week intervals and the rAd boost was given one year after the final DNA prime. Animals were exposed to a lethal dose of BEBOV 7 weeks after the rAd5 GP boost. (B) Distance matrix-based phylogenetic tree for EBOV GP. Numbers at the nodes are boostrap percentages. Vertical branches are for graphic representation; horizontal branch lengths are measured as substitutions per site (scale bar  = 0.07). The infectious challenge species is shown in blue. Vaccine strains are indicated in bold.

Figure 2. Development of vaccine-induced antibody responses.

(A) The quantity of anti-Ebola GP IgG in plasma samples from vaccinated cynomolgus macaques was determined by ELISA as described in Methods. Results are shown for samples obtained after the final DNA prime and prior to immunization with rAd (pre-boost, light blue), and for samples obtained 3 weeks after boosting with rAd-GP (post-boost, dark blue). Plasma antibody titers are presented as EC90 reciprocal dilution titers. (B) ELISA IgG against ZEBOV (blue) and BEBOV (red) antigens. Plasma dilution series are shown for each immunized subject. The averages for negative control samples (n = 4) are shown for ZEBOV (black dotted line) and BEBOV (black dashed line).

Figure 3. T-cell immune responses measured by intracellular cytokine staining.

PBMC were stimulated with overlapping peptides spanning the GP protein, and antigen-specific CD4⁺ and CD8⁺ cells were enumerated in the memory cell gates (A) by the detection of cytokine production. The percentages of cytokine-positive CD4⁺ (panel B) and CD8⁺ (panel C) T cell memory cells specific for ZEBOV (blue) and BEBOV (red) were determined at 4 weeks post rAd-GP boost. Responses are shown for each individual cytokine measured as the total proportion of cells positive for TNFα (blue), IFNγ (gray), IL-2 (red), expressed as a percentage of the memory cell population after background subtraction of control, unstimulated, samples run in parallel.

Figure 4. Bundibugyo EBOV challenge.

Control and vaccinated animals were exposed to a target dose of ∼1,000 TCID₅₀ of BEBOV. Blood samples were collected before and after infection for the determination of hepatic enzyme levels. ALT (panel A) and AST (panel B) were measured using a General chemistry 12 reagent disk for the Piccolo Analyzer (Abaxis). Viral load (panel C) was determined by quantitative RT-PCR of plasma RNA using primers specific for BEBOV. Control and vaccinated animal values are shown in red and blue bars, respectively. Kaplan Meier survival curves (panel D) were drawn using GraphPad Prism.