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A thermostable, chromatographically purified Ebola nano-VLP vaccine.

Carra JH, Martins KA, Schokman RD, Robinson CG, Steffens JT, Bavari S - J Transl Med (2015)

Bottom Line: Filovirus virus-like particles (VLP) are strong immunogens with the potential for development into a safe, non-infectious vaccine.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

a Nanopore event duration (ordinate) and blockade magnitude (abscissa) of non-lyophilized (blue circles) and lyophilized (green triangles) nano-VLP. b ELISA to probe the conformational integrity of GP in nano-VLP that were not lyophilized, lyophilized, and lyophilized and heated to 75°C for 1 h. Blue bars linear epitope antibody 6D8, red bars conformational antibody 6D3, green bars conformational antibody 13C6.
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Fig7: a Nanopore event duration (ordinate) and blockade magnitude (abscissa) of non-lyophilized (blue circles) and lyophilized (green triangles) nano-VLP. b ELISA to probe the conformational integrity of GP in nano-VLP that were not lyophilized, lyophilized, and lyophilized and heated to 75°C for 1 h. Blue bars linear epitope antibody 6D8, red bars conformational antibody 6D3, green bars conformational antibody 13C6.

Mentions: Transmission electron microscopy of the nano-VLP with PTA staining showed linear, branched, spherical and “6” shaped particles (Figure 6a). Linear filaments were as long as 1.5 microns, but most particles were shorter filaments or spheres of ~200 nm diameter. Particle size was also examined using a qViro device (Figure 7a). With the nano-VLP sample, 7.1% of observed events had a passage duration of >0.2 ms. These events may represent passage of the shorter filaments found in Figure 6a. Results from the qViro nanopore analysis appeared to be consistent with the EM data. The nano-VLP were not observed to possess lengths of several microns as is commonly found in sucrose-gradient purified VLP and authentic virions. The mean particle diameter was calculated to be 230 nm using a spherical bead standard; however, this method does not take into account the shape of the filament fragments. Although some of the fragments of filaments present were longer than the 0.45 micron pore size filter used in this preparation, their width was less than 100 nm.Figure 6


A thermostable, chromatographically purified Ebola nano-VLP vaccine.

Carra JH, Martins KA, Schokman RD, Robinson CG, Steffens JT, Bavari S - J Transl Med (2015)

a Nanopore event duration (ordinate) and blockade magnitude (abscissa) of non-lyophilized (blue circles) and lyophilized (green triangles) nano-VLP. b ELISA to probe the conformational integrity of GP in nano-VLP that were not lyophilized, lyophilized, and lyophilized and heated to 75°C for 1 h. Blue bars linear epitope antibody 6D8, red bars conformational antibody 6D3, green bars conformational antibody 13C6.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4502941&req=5

Fig7: a Nanopore event duration (ordinate) and blockade magnitude (abscissa) of non-lyophilized (blue circles) and lyophilized (green triangles) nano-VLP. b ELISA to probe the conformational integrity of GP in nano-VLP that were not lyophilized, lyophilized, and lyophilized and heated to 75°C for 1 h. Blue bars linear epitope antibody 6D8, red bars conformational antibody 6D3, green bars conformational antibody 13C6.
Mentions: Transmission electron microscopy of the nano-VLP with PTA staining showed linear, branched, spherical and “6” shaped particles (Figure 6a). Linear filaments were as long as 1.5 microns, but most particles were shorter filaments or spheres of ~200 nm diameter. Particle size was also examined using a qViro device (Figure 7a). With the nano-VLP sample, 7.1% of observed events had a passage duration of >0.2 ms. These events may represent passage of the shorter filaments found in Figure 6a. Results from the qViro nanopore analysis appeared to be consistent with the EM data. The nano-VLP were not observed to possess lengths of several microns as is commonly found in sucrose-gradient purified VLP and authentic virions. The mean particle diameter was calculated to be 230 nm using a spherical bead standard; however, this method does not take into account the shape of the filament fragments. Although some of the fragments of filaments present were longer than the 0.45 micron pore size filter used in this preparation, their width was less than 100 nm.Figure 6

Bottom Line: Filovirus virus-like particles (VLP) are strong immunogens with the potential for development into a safe, non-infectious vaccine.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus