A thermostable, chromatographically purified Ebola nano-VLP vaccine

Journal of Translational Medicine

Sina Bavari¹  Jesse T Steffens¹  Camenzind G Robinson²  Rowena D Schokman¹  Karen A O Martins¹  John H Carra¹ 

[1] Molecular and Translational Sciences, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick 21702-9211, MD, USA;Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn 20147, VA, USA

关键词: Thermostability;    Immunogenicity;    Virus-like particle;    Ebola;    vaccine;    Filovirus;   

Others  :  1221460
DOI  :  10.1186/s12967-015-0593-y

 received in 2015-04-06, accepted in 2015-07-02,  发布年份 2015

【 摘 要 】

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.

【 授权许可】

   
2015 Carra et al.

【 图 表 】

Figure1.

Figure2.

Figure3.

Figure4.

Figure5.

Figure6.

Figure7.

Figure8.

【 参考文献 】

• [1]Feldmann H, Jones S, Klenk HD, Schnittler HJ. Ebola virus: from discovery to vaccine. Nat Rev Immunol. 2003; 3:677-685.
• [2]Beniac DR, Melito PL, Devarennes SL, Hiebert SL, Rabb MJ, Lamboo LL et al.. The organisation of Ebola virus reveals a capacity for extensive, modular polyploidy. PLoS One. 2012; 7:e29608.
• [3]Warfield KL, Bosio CM, Welcher BC, Deal EM, Mohamadzadeh M, Schmaljohn A et al.. Ebola virus-like particles protect from lethal Ebola virus infection. Proc Natl Acad Sci USA. 2003; 100:15889-15894.
• [4]Warfield KL, Posten NA, Swenson DL, Olinger GG, Esposito D, Gillette WK et al.. Filovirus-like particles produced in insect cells: immunogenicity and protection in rodents. J Infect Dis. 2007; 196 Suppl 2:S421-S429.
• [5]Warfield KL, Swenson DL, Demmin G, Bavari S. Filovirus-like particles as vaccines and discovery tools. Expert Rev Vaccines. 2005; 4:429-440.
• [6]Swenson DL, Warfield KL, Negley DL, Schmaljohn A, Aman MJ, Bavari S. Virus-like particles exhibit potential as a pan-filovirus vaccine for both Ebola and Marburg viral infections. Vaccine. 2005; 23:3033-3042.
• [7]Martins KA, Warren TK, Bavari S. Characterization of a putative filovirus vaccine: virus-like particles. Virol Sin. 2013; 28:65-70.
• [8]Warfield KL, Swenson DL, Olinger GG, Kalina WV, Aman MJ, Bavari S. Ebola virus-like particle-based vaccine protects nonhuman primates against lethal Ebola virus challenge. J Infect Dis. 2007; 196 Suppl 2:S430-S437.
• [9]Warfield KL, Dye JM, Wells JB, Unfer RC, Holtsberg FW, Shulenin S et al.. Homologous and heterologous protection of nonhuman primates by Ebola and Sudan virus-like particles. PLoS One. 2015; 10:e0118881.
• [10]Wahl-Jensen V, Kurz SK, Hazelton PR, Schnittler HJ, Stroher U, Burton DR et al.. Role of Ebola virus secreted glycoproteins and virus-like particles in activation of human macrophages. J Virol. 2005; 79:2413-2419.
• [11]Bachmann MF, Jennings GT. Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns. Nat Rev Immunol. 2010; 10:787-796.
• [12]Zhao L, Seth A, Wibowo N, Zhao CX, Mitter N, Yu C et al.. Nanoparticle vaccines. Vaccine. 2014; 32:327-337.
• [13]Ungaro F, Conte C, Quaglia F, Tornesello ML, Buonaguro FM, Buonaguro L. VLPs and particle strategies for cancer vaccines. Expert Rev Vaccines. 2013; 12:1173-1193.
• [14]Silva JM, Videira M, Gaspar R, Preat V, Florindo HF. Immune system targeting by biodegradable nanoparticles for cancer vaccines. J Control Release. 2013; 168:179-199.
• [15]Manolova V, Flace A, Bauer M, Schwarz K, Saudan P, Bachmann MF. Nanoparticles target distinct dendritic cell populations according to their size. Eur J Immunol. 2008; 38:1404-1413.
• [16]Champion JA, Mitragotri S. Shape induced inhibition of phagocytosis of polymer particles. Pharm Res. 2009; 26:244-249.
• [17]Roberts GS, Yu S, Zeng Q, Chan LC, Anderson W, Colby AH et al.. Tunable pores for measuring concentrations of synthetic and biological nanoparticle dispersions. Biosens Bioelectron. 2012; 31:17-25.
• [18]Bray M, Davis K, Geisbert T, Schmaljohn C, Huggins J. A mouse model for evaluation of prophylaxis and therapy of Ebola hemorrhagic fever. J Infect Dis. 1998; 178:651-661.
• [19]Bavari S, Bosio CM, Wiegand E, Ruthel G, Will AB, Geisbert TW et al.. Lipid raft microdomains: a gateway for compartmentalized trafficking of Ebola and Marburg viruses. J Exp Med. 2002; 195:593-602.
• [20]Kozak D, Anderson W, Vogel R, Trau M. Advances in Resistive Pulse Sensors: devices bridging the void between molecular and microscopic detection. Nano Today. 2011; 6:531-545.
• [21]Lee JE, Saphire EO. Neutralizing ebolavirus: structural insights into the envelope glycoprotein and antibodies targeted against it. Curr Opin Struct Biol. 2009; 19:408-417.
• [22]Wilson JA, Hevey M, Bakken R, Guest S, Bray M, Schmaljohn AL et al.. Epitopes involved in antibody-mediated protection from Ebola virus. Science. 2000; 287:1664-1666.
• [23]Murin CD, Fusco ML, Bornholdt ZA, Qiu X, Olinger GG, Zeitlin L et al.. Structures of protective antibodies reveal sites of vulnerability on Ebola virus. Proc Natl Acad Sci USA. 2014; 111:17182-17187.
• [24]Martins KA, Steffens JT, van Tongeren SA, Wells JB, Bergeron AA, Dickson SP et al.. Toll-like receptor agonist augments virus-like particle-mediated protection from Ebola virus with transient immune activation. PLoS One. 2014; 9:e89735.
• [25]Lee JE, Fusco ML, Hessell AJ, Oswald WB, Burton DR, Saphire EO. Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor. Nature. 2008; 454:177-182.
• [26]Hu L, Trefethen JM, Zeng Y, Yee L, Ohtake S, Lechuga-Ballesteros D et al.. Biophysical characterization and conformational stability of Ebola and Marburg virus-like particles. J Pharm Sci. 2011; 100:5156-5173.
• [27]Chen D, Kristensen D. Opportunities and challenges of developing thermostable vaccines. Expert Rev Vaccines. 2009; 8:547-557.