RESUMO
The zoonotic equine encephalitis viruses (EEVs) can cause debilitating and life-threatening disease, leading to ongoing vaccine development efforts for an effective virus-like particle (VLP) vaccine based on 3 strains of EEV (Eastern, Western, and Venezuelan or EEE, WEE and VEE VLPs, respectively). In this work, transmission electron microscopy and light scattering studies showed enveloped, spherical, and â¼70 nm sized VLPs. Biophysical studies demonstrated optimal VLP physical stability in the pH range of 7.5-8.5 and at temperatures below â¼50°C. Interestingly, the individual stability profiles differed notably between the 3 VLPs. Numerous pharmaceutical excipients were screened for their VLP stabilizing effects against thermal stress. Sucrose, sorbitol, sodium chloride, and pluronic F-68 were identified as promising stabilizers and the concentrations and combinations of these additives were optimized. Candidate monovalent VLP bulk formulations were incubated at temperatures ranging from -80°C to 40°C to establish freeze-thaw, long-term (2°C-8°C) and accelerated stability trends. Good VLP stability profiles were observed at each storage temperature, except for a distinct instability observed at -20°C. The interaction of monovalent and trivalent VLP formulations with aluminum adjuvants was examined, both in terms of antigen adsorption and desorption over time. The implications of these findings on future vaccine formulation development of EEV VLPs are discussed.
Assuntos
Vírus da Encefalite/química , Vacinas de Partículas Semelhantes a Vírus/química , Vacinas Virais/química , Adjuvantes Imunológicos/química , Animais , Vírus da Encefalite/imunologia , Encefalomielite Equina/imunologia , Excipientes/química , Cavalos , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/imunologia , Vírion/química , Vírion/imunologiaRESUMO
The coat protein gene from encephalitis virus infecting Dicentrarhus labrax (DIEV) has been cloned by gene amplification, sequenced and expressed in Escherichia coli. DNA sequencing has revealed an open reading frame of 1017 bases encoding a polypeptide of 338 amino acids. The sequence similarities between the DIEV coat protein gene and the same gene in five encephalitis viruses infected other fish species were over 71.5% at the nucleotide level and over 79.5% at the amino acid level. These results indicate that the nodaviruses that cause encephalopathy and retinopathy in fishes are very closed related. E. coli cells harbouring the plasmid containing the DIEV gene can produce the viral coat protein. An efficient purification scheme using a Sepharore-Ni+2 column is presented. This, gives approx. 10 mg of more than 95% pure protein per gr of E. coli culture.
Assuntos
Bass/virologia , Proteínas do Capsídeo , Capsídeo/genética , Capsídeo/metabolismo , Vírus da Encefalite/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Capsídeo/isolamento & purificação , Cromatografia de Afinidade/métodos , Clonagem Molecular , Vírus da Encefalite/genética , Dados de Sequência Molecular , Vírus de RNA/química , Vírus de RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Two-dimensional gel electrophoreses of RNase T1-derived oligonucleotides of the three individual RNA segments of the bunyavirus snowshow hare virus indicate that its three RNA segments possess distinct nucleotide sequences. The fingerprints of the RNA species of snowshoe hare virus differ from those of the antigenically closely related La Crosse virus. Three viral RNA species have been identified in preparations of Melao and Trivittatus as well as snowshoe hare, Lumbo, and La Crosse bunyaviruses.
