1. Demonstration of Cross-Protective Vaccine Immunity against an Emerging Pathogenic Ebolavirus Species May 20, 2010 PLoS Pathogens Volume 6 Issue 5

2. 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 crossprotective 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.

3. Methodology Immunization of cynomolgus macaques with two plasmid vectors encoding for GP(Z) and GP(S/G) Infection with BEBOV Determination of liver enzyme levels for serum alanine aminotransferase and aspartate aminotransferase (Blood chemistry) Anti-Ebola GP IgG ELISA T cell intracellular cytokine secretion analysis RNA isolation and quantitative real- time RT-PCR (qRT-PCR) Computational analysis of GP sequences Statistical analysis

4. Immunization and Infection Biojector IM injection, bilateral deltoid, of cynomolgus macaques (Macaca fascicularis) and infection with 1,000 TCID 50 BEBOV

5. Blood Chemistry Determination of liver enzyme levels for serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST)

6. Anti-Ebola GP IgG ELISA Polyvinyl chloride ELISA plates coated with EBOLA GP Incubated with subject sera or plasma Bound IgG detected using goat anti-human IgG conjugated to horseradish peroxidase and Sigma Fast o-phenylenediamine dihydrochloride

7. T cell Intracellular Cytokine Secretion Analysis PBMC separated from whole blood samples over Ficoll Stained with antibodies against lineage markers and intracellularly with antibodies against cytokines Analyzed by flow cytometry

8. RNA isolation and qRT-PCR RNA extracted with RNAqueous kit Iscript One-step qRT- PCR kit

9. Significance of the Study Vaccine development against Ebola viruses = high priority Aggressive nature of Ebola virus hemorrhagic fever (EHF) symptoms Rapid spread of infection High mortality rate Threat of bioterrorism Frequency of EBOV outbreaks in Africa has increased

10. Significance of the Study Four known species: Zaire (ZEBOV) Sudan (SEBOV) Reston (REBOV) Cote d’Ivoire (CIEBOV) Bundibugyo (BEBOV) = fifth species discovered in late 2007 in Western Uganda rAd5 vaccines = encode GP from SEBOV and ZEBOV sequences differ from BEBOV by 38–47% at the amino acid level

11. Innovation Needle-free injection system (Biojector) special technique of forcing liquid medication at high speed through a tiny orifice that is held against the skin

12. Innovation DNA/rAd prime-boost vaccination Targets structural proteins from ZEBOV and SEBOV Cross-protective immunity against BEBOV rAd vectors = boosted antigen-specific immune response even a year after the final DNA prime CD4 + and CD8 + T-cell effector immunity

13. Contribution to the Field of Virology BEBOV less pathogenic more sensitive to host immunity Conservation of immunogenic regions exists between different species of Ebola virus New design for next-generation vaccines a mechanistic basis for vaccine-induced immune protection against Ebola virus infection Possible protection against Ebola virus species whose antigens are not present in the vaccine formulation Eliciting robust T-cell immunity greatest potential to protect against other newly emerging pathogenic Ebola virus species