RESUMEN
A two-component recombinant fusion protein antigen was re-engineered and tested as a medical counter measure against the possible biological threat of aerosolized Yersinia pestis. The active component of the proposed subunit vaccine combines the F1 capsular protein and V virulence antigen of Y. pestis and improves upon the design of an earlier histidine-tagged fusion protein. In the current study, different production strains were screened for suitable expression and a purification process was optimized to isolate an F1-V fusion protein absent extraneous coding sequences. Soluble F1-V protein was isolated to 99% purity by sequential liquid chromatography including capture and refolding of urea-denatured protein via anion exchange, followed by hydrophobic interaction, concentration, and then transfer into buffered saline for direct use after frozen storage. Protein identity and primary structure were verified by mass spectrometry and Edman sequencing, confirming a purified product of 477 amino acids and removal of the N-terminal methionine. Purity, quality, and higher-order structure were compared between lots using RP-HPLC, intrinsic fluorescence, CD spectroscopy, and multi-angle light scattering spectroscopy, all of which indicated a consistent and properly folded product. As formulated with aluminum hydroxide adjuvant and administered in a single subcutaneous dose, this new F1-V protein also protected mice from wild-type and non-encapsulated Y. pestis challenge strains, modeling prophylaxis against pneumonic and bubonic plague. These findings confirm that the fusion protein architecture provides superior protection over the former licensed product, establish a foundation from which to create a robust production process, and set forth assays for the development of F1-V as the active pharmaceutical ingredient of the next plague vaccine.
Asunto(s)
Antígenos Bacterianos/administración & dosificación , Proteínas Bacterianas/administración & dosificación , Diseño de Fármacos , Peste/microbiología , Peste/prevención & control , Ingeniería de Proteínas/métodos , Vacunas de Subunidad/administración & dosificación , Yersinia pestis/efectos de los fármacos , Animales , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Evaluación Preclínica de Medicamentos , Etiquetas de Secuencia Expresada , Femenino , Humanos , Ratones , Proteínas Citotóxicas Formadoras de Poros , Proteínas Recombinantes de Fusión/administración & dosificación , Resultado del Tratamiento , Vacunas Sintéticas/administración & dosificación , Yersinia pestis/citologíaRESUMEN
F1-V is a recombinant plague antigen comprising the capsular (F1) and virulence-associated (V) proteins. Given intramuscularly with Alhydrogel, it protects mice against challenge, but is less effective in non-human primates against high-dose aerosolized Yersinia pestis challenge, perhaps because it fails to induce respiratory immunity. Intranasal immunization of mice with F1-V formulated with a Proteosome-based adjuvant (Protollin), elicited high titers of specific IgA in lungs whereas intranasal F1-V alone or intramuscular Alhydrogel-adsorbed F1-V did not. The Protollin-adjuvanted F1-V vaccine also induced high serum titers of specific IgG, comparable to those induced by intramuscular Alhydrogel-adsorbed F1-V. Mice immunized intranasally with Protollin-F1-V were 100% protected against aerosol challenge with 170 LD50 of Y. pestis and 80% against 255 LD50.