A thermostable, chromatographically purified Ebola nano-VLP vaccine.

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.

Cross-protection conferred by filovirus virus-like particles containing trimeric hybrid glycoprotein.

Filoviruses are causative agents of hemorrhagic fever, and to date no effective vaccine or therapeutic has been approved to combat infection. Filovirus glycoprotein (GP) is the critical immunogenic component of filovirus vaccines, eliciting high levels of antibody after successful vaccination. Previous work has shown that protection against both Ebola virus (EBOV) and Marburg virus (MARV) can be achieved by vaccinating with a mixture of virus-like particles (VLPs) expressing either EBOV GP or MARV GP. In this study, the potential for eliciting effective immune responses against EBOV, Sudan virus, and MARV with a single GP construct was tested. Trimeric hybrid GPs were produced that expressed the sequence of Marburg GP2 in conjunction with a hybrid GP1 composed EBOV and Sudan virus GP sequences. VLPs expressing these constructs, along with EBOV VP40, provided comparable protection against MARV challenge, resulting in 75 or 100% protection. Protection from EBOV challenge differed depending upon the hybrid used, however, with one conferring 75% protection and one conferring no protection. By comparing the overall antibody titers and the neutralizing antibody titers specific for each virus, it is shown that higher antibody responses were elicited by the C terminal region of GP1 than by the N terminal region, and this correlated with protection. These data collectively suggest that GP2 and the C terminal region of GP1 are highly immunogenic, and they advance progress toward the development of a pan-filovirus vaccine.

Improved formulation of a recombinant ricin A-chain vaccine increases its stability and effective antigenicity.

Ricin is a potent toxin associated with bioterrorism for which no vaccine or specific countermeasures are currently available. A stable, non-toxic and immunogenic recombinant ricin A-chain vaccine (RTA 1-33/44-198) has been developed by protein engineering. We identified optimal formulation conditions for this vaccine under which it remained stable and potent in storage for up to 18 months, and resisted multiple rounds of freeze-thawing without stabilizing co-solvents. Reformulation from phosphate buffer to succinate buffer increased adherence of the protein to aluminum hydroxide adjuvant from 15 to 91%, with a concomitant increase of nearly threefold in effective antigenicity in a mouse model. Using Fourier-transform infrared spectroscopy, we examined the secondary structure of the protein while it was adhered to aluminum hydroxide. Adjuvant adsorption produced only a small apparent change in secondary structure, while significantly stabilizing the protein to thermal denaturation. The vaccine therefore may be safely stored in the presence of adjuvant. Our results suggest that optimization of adherence of a protein antigen to aluminum adjuvant can be a useful route to increasing both stability and effectiveness, and support a role for a "depot effect" of adjuvant.

Mutational effects on protein folding stability and antigenicity: the case of streptococcal pyrogenic exotoxin A.

The influence of mutationally induced changes in protein folding on development of effective neutralizing antibodies during vaccination remains largely unexplored. In this study, we probed how mutational substitutions of streptococcal pyrogenic exotoxin A (SPEA), a model bacterial superantigen, affect native conformational stability and antigenicity. Stability changes for the toxin variants were determined using circular dichroism and fluorescence measurements, and scanning calorimetry. Self-association was assayed by dynamic light scattering. Inactivated SPEA proteins containing particular combinations of mutations elicited antibodies in HLA-DQ8 transgenic mice that neutralized SPEA superantigenicity in vitro, and protected animals from lethal toxin challenge. However, a highly destabilized cysteine-free mutant of SPEA did not provide effective immunity, nor did an irreversibly denatured version of an otherwise effective mutant protein. These results suggest that protein conformation plays a significant role in generating effective neutralizing antibodies to this toxin, and may be an important factor to consider in vaccine design.