Ebola Virus-Like Particle-Based Vaccine Protects Nonhuman Primates against Lethal Ebola Virus Challenge

SUPPLEMENT ARTICLE Ebola Virus-Like Particle–Based Vaccine Protects Nonhuman Primates against Lethal Ebola Virus Challenge Kelly L. Warfield,a Dana L. Swenson,a Gene G. Olinger, Warren V. Kalina, M. Javad Aman,b and Sina Bavari US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland During the past 20 years, owing to advances in molecular biology and in the understanding of basic virology, scientists have been able to develop subunit vaccines based on virus-like particles (VLPs). VLPs for many viruses have been developed and are based on the knowledge that expression of specific viral structural proteins results in the self-assembly of particles that morphologically resemble the authentic virus [1]. Some Potential conflicts of interest: K.L.W., D.L.S., M.J.A., and S.B. hold patent rights to Ebola and Marburg virus-like particle–based vaccines. G.G.O. and W.V.K.: none reported. Presented in part: Filoviruses: Recent Advances and Future Challenges, International Centre for Infectious Diseases Symposium, Winnipeg, Manitoba, Canada, 17–19 September 2006. Financial support: Defense Threat Reduction Agency. Supplement sponsorship is detailed in the Acknowledgments. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the US Army. a K.L.W. and D.L.S. contributed equally to this work. b Present affiliation: Integrated BioTherapeutics Inc., Frederick, Maryland. Reprints or correspondence: Dr. Sina Bavari or Dr. Kelly Warfield, US Army Medical Research Institute of Infectious Diseases, 1425 Porter St., Fort Detrick, MD 21702 ( or ). The Journal of Infectious Diseases 2007; 196:S430–7 This article is in the public domain, and no copyright is claimed. 0022-1899/2007/19610S2-0043 DOI: 10.1086/520583 S430 • JID 2007:196 (Suppl 2) • Warfield et al. of the many advantages of using VLPs as vaccines include (1) their similar morphology to the live enveloped or nonenveloped viruses from which they are derived; (2) a strong safety profile, since they are nonreplicating; (3) no concerns regarding viral vector or preexisting antivector immunity; (4) the fact that they can be generated in large quantities by use of mammalian or insect cell lines; (5) their ability to generate innate, humoral, and cellular immunity; and (6) the fact that they have been safely and effectively administered in humans [1–7]. We and others have demonstrated previously the generation of enveloped Ebola VLPs (eVLPs) in mammalian and insect cell-expression systems [8–13]. VLPs containing glycoprotein (GP) and VP40 derived from Ebola virus (EBOV) have been used successfully to vaccinate rodents [2, 13–17]. Both BALB/c and C57BL/6 mice have been protected against a range of challenge doses (∼10–1,000 pfu or ∼300–30,000 LD50) by means of dose-dependent eVLP vaccination in the presence or absence of adjuvant [2, 14, 15]. Addition of saponinderived QS-21 or RIBI adjuvant to the eVLP-vaccine regimen allows administration of a decreased dose of the vaccine and completely protects mice and guinea Background. Currently, there are no licensed vaccines or therapeutics for the prevention or treatment of infection by the highly lethal filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), in humans. We previously had demonstrated the protective efficacy of virus-like particle (VLP)–based vaccines against EBOV and MARV infection in rodents. Methods. To determine the efficacy of vaccination with Ebola VLPs (eVLPs) in nonhuman primates, we vaccinated cynomolgus macaques with eVLPs containing EBOV glycoprotein (GP), nucleoprotein (NP), and VP40 matrix protein and challenged the macaques with 1000 pfu of EBOV. Results. Serum samples from the eVLP-vaccinated nonhuman primates demonstrated EBOV-specific antibody titers, as measured by enzyme-linked immunosorbent assay, complement-mediated lysis assay, and antibodydependent cell-mediated cytotoxicity assay. CD44+ T cells from eVLP-vaccinated macaques but not from a naive macaque responded with vigorous production of tumor necrosis factor–a after EBOV-peptide stimulation. All 5 eVLP-vaccinated monkeys survived challenge without clinical or laboratory signs of EBOV infection, whereas the control animal died of infection. Conclusion. On the basis of safety and efficacy, eVLPs represent a promising filovirus vaccine for use in humans. pigs against challenge, even after only 1 inoculation (authors’ unpublished data) [18]. eVLP vaccination completely prevents viremia and clinical symptoms after EBOV challenge in rodents but does not induce sterile immunity, as evidenced by an expansion of immune responses to viral proteins not present in the vaccine, after challenge [2, 14, 15, 18]. However, the question of whether eVLPs would be able to protect nonhuman primates against EBOV infection has remained. Therefore, the goal of the current study was to determine whether eVLPs would be viable vaccine candidates for the protection of primates against lethal EBOV challenge. MATERIALS AND METHODS VLP-Based Vaccine in Nonhuman Primates • JID 2007:196 (Suppl 2) • S431 Generation and characterization of eVLPs. 293T-derived eVLPs containing EBOV GP, nucleoprotein (NP), and VP40 were prepared essentially as described elsewhere [9, 14, 16, 17]. Total protein concentrations in the vaccine preparations were determined in the presence of detergent, by use of a Bradford protein assay (BioRad). Expression of GP, NP, and VP40 in each vaccine preparation was verified by Western blot analysis [9, 17, 18]. eVLPs were processed and imaged via electron microscopy, as described elsewhere [9, 14, 16, 17]. Endotoxin levels in all eVLP preparations used in this study were !0.03 endotoxin units/mg, as determined by the Limulus amebocyte lysate test (Invitrogen). Animals. In testing before the start of this study, the cynomolgus macaques used were found to be antibody negative for filovirus, simian T cell leukemia virus–1, simian immunodeficiency virus, and herpes B virus. The eVLP-vaccinated monkeys received 3 intramuscular injections, at 42-day intervals, of ∼1.0 mL of sterile saline containing 250 mg of eVLPs and 0.5 mL of RIBI adjuvant (Corixa). Blood samples were obtained from the femoral vein of monkeys under anesthesia. Female cynomolgus macaques of 3–4 kg in weight were challenged by intramuscular injection of ∼1000 pfu of Zaire EBOV (ZEBOV; 1995 outbreak strain) [19]. Viremia was determined by means of a traditional plaque assay [20]. Hematology and kidney- and liver-associated enzymes were assessed as described elsewhere [21]. For ethical reasons, the use of relevant historical control animals was required by the Laboratory Animal Care and Use Committee of the US Army Medical Research Institute of Infectious Diseases (Fort Detrick, MD), to reduce the number of nonhuman primates needed in these studies. For this reason, the control monkey in the current study was not treated, so that, for the data analysis, (...truncated)