title: A thermostable, chromatographically purified Ebola nano-VLP vaccine.
abstract: BACKGROUND
  Filovirus virus-like particles (VLP) are strong immunogens with the potential for development into a safe, non-infectious vaccine. However, the large size and filamentous structure of this virus has heretofore made production of such a vaccine difficult. Herein, we present new assays and a purification procedure to yield a better characterized and more stable product.
  
  
  METHODS
  Sonication of VLP was used to produce smaller "nano-VLP", which were purified by membrane chromatography. The sizes and lengths of VLP particles were analyzed using electron microscopy and an assay based on transient occlusion of a nanopore. Using conformationally-sensitive antibodies, we developed an in vitro assay for measuring GP conformational integrity in the context of VLP, and used it to profile thermal stability.
  
  
  RESULTS
  We developed a new procedure for rapid isolation of Ebola VLP using membrane chromatography that yields a filterable and immunogenic product. Disruption of VLP filaments by sonication followed by filtration produced smaller particles of more uniform size, having a mean diameter close to 230 nm. These reduced-size VLP retained GP conformation and were protective against mouse-adapted Ebola challenge in mice. The "nano-VLP" consists of GP-coated particles in a mixture of morphologies including circular, branched, "6"-shaped, and filamentous ones up to ~1,500 nm in length. Lyophilization conferred a high level of thermostability on the nano-VLP. Unlike Ebola VLP in solution, which underwent denaturation of GP upon moderate heating, the lyophilized nano-VLP can withstand at least 1 h at 75°C, while retaining conformational integrity of GP and the ability to confer protective immunity in a mouse model.
  
  
  CONCLUSIONS
  We showed that Ebola virus-like particles can be reduced in size to a more amenable range for manipulation, and that these smaller particles retained their temperature stability, the structure of the GP antigen, and the ability to stimulate a protective immune response in mice. We developed a new purification scheme for "nano-VLP" that is more easily scaled up and filterable. The product could also be made thermostable by lyophilization, which is highly significant for vaccines used in tropical countries without a reliable "cold-chain" of refrigeration.
authors.affiliation: Molecular and Translational Sciences, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, 21702-9211, USA. john.h.carra.civ@mail.mil.
authors.forename: John H
authors.initials: JH
authors.lastname: Carra
authors.affiliation: Molecular and Translational Sciences, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, 21702-9211, USA. karen.a.martins2.ctr@mail.mil.
authors.forename: Karen A O
authors.initials: KA
authors.lastname: Martins
authors.affiliation: Molecular and Translational Sciences, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, 21702-9211, USA. rowena.d.schokman.ctr@mail.mil.
authors.forename: Rowena D
authors.initials: RD
authors.lastname: Schokman
authors.affiliation: Pathology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, 21702-9211, USA. robinsonc@janelia.hhmi.org.|Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA, 20147, USA. robinsonc@janelia.hhmi.org.
authors.forename: Camenzind G
authors.initials: CG
authors.lastname: Robinson
authors.affiliation: Molecular and Translational Sciences, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, 21702-9211, USA. jesse.t.steffens.ctr@mail.mil.
authors.forename: Jesse T
authors.initials: JT
authors.lastname: Steffens
authors.affiliation: Molecular and Translational Sciences, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, 21702-9211, USA. sina.bavari.civ@mail.mil.
authors.forename: Sina
authors.initials: S
authors.lastname: Bavari
mesh_terms: D000818:Animals; D002845:Chromatography / Q000379:methods*; D029043:Ebolavirus / Q000276:immunology*; D005260:Female; D005374:Filtration; D006023:Glycoproteins / Q000276:immunology; D057809:HEK293 Cells; D006801:Humans; D008810:Mice, Inbred C57BL; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D058608:Nanopores; D010316:Particle Size; D013010:Sonication; D013696:Temperature; D016896:Treatment Outcome; D014611:Vaccination; D058425:Vaccines, Virus-Like Particle / Q000276:immunology* / Q000648:ultrastructure; D014771:Virion / Q000648:ultrastructure
chemical_list: D006023:Glycoproteins; D058425:Vaccines, Virus-Like Particle