RESUMEN

Although the World Health Organization recommends the use of in vitro techniques to qualify rabies vaccine lot release, very limited proposals have been made to arrive at a harmonized approach for wide scale usage. The present study proposed and evaluated the use of a novel avidin-biotin ELISA as an alternative to these in vivo tests in rabies vaccine manufacture. This assay utilized a neutralizing pan reactive monoclonal antibody (mAb) reactive with the conserved site-II of the natively folded rabies glycoprotein. Linear regression analysis of the in vitro glycoprotein estimates with the in vivo potency values, showed a good correlation (r(2)=0.8) with veterinary vaccines, but a poor correlation (r(2)=0.2) with human vaccines. However, we could qualitatively arrive at cut-off glycoprotein estimates from the ELISA, above which all the vaccines were declared to be protective by mouse challenge studies (>2.5IU/dose).

Asunto(s)

Ensayo de Inmunoadsorción Enzimática/métodos , Glicoproteínas/análisis , Vacunas Antirrábicas/análisis , Proteínas Virales/análisis , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Humanos , Ratones , Rabia/prevención & control

RESUMEN

Rabies is a fatal zoonotic disease of serious public health and economic significance worldwide. The rabies virus glycoprotein (RVG) has been the major target for subunit vaccine development, since it harbors domains responsible for induction of virus-neutralizing antibodies, infectivity, and neurovirulence. The glycoprotein (G) was cloned using the baculovirus expression vector system (BEVS) and expressed in Spodoptera frugiperda (Sf-9) cells. In order to obtain a soluble form of G suitable for experimentation in mice, 18 different combinations of buffers and detergents were evaluated for their ability to solubilize the insect cell membrane-associated G. The combination that involved 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) detergent in lysis buffer 1, formulated with Tris, NaCl, 10% dimethyl sulfoxide (DMSO), and EDTA, gave the highest yield of soluble G, as evidenced by the experimental data. Subsequently, several other parameters, such as the concentration of CHAPS and the duration and temperature of the treatment for the effective solubilization of G, were optimized. The CHAPS detergent, buffered at a concentration of 0.4% to 0.7% (wt/vol) at room temperature (23 to 25°C) for 30 min to 1 h using buffer 1, containing 10% DMSO, resulted in consistently high yields. The G solubilized using CHAPS detergent was found to be immunogenic when tested in mice, as evidenced by high virus-neutralizing antibody titers in sera and 100% protection upon virulent intracerebral challenge with the challenge virus standard (CVS) strain of rabies virus. The results of the mice study indicated that G solubilized with CHAPS detergent retained the immunologically relevant domains in the native conformation, thereby paving the way for producing a cell-free and efficacious subunit vaccine.

Asunto(s)

Antígenos Virales/inmunología , Glicoproteínas/inmunología , Vacunas Antirrábicas/inmunología , Virus de la Rabia/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Antígenos Virales/genética , Antígenos Virales/aislamiento & purificación , Baculoviridae , Tampones (Química) , Línea Celular , Clonación Molecular , Detergentes , Expresión Génica , Vectores Genéticos , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Insectos , Ratones , Rabia/inmunología , Rabia/prevención & control , Vacunas Antirrábicas/administración & dosificación , Vacunas Antirrábicas/química , Vacunas Antirrábicas/aislamiento & purificación , Virus de la Rabia/genética , Solubilidad , Spodoptera , Análisis de Supervivencia , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/química , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/aislamiento & purificación , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/aislamiento & purificación

RESUMEN

Animal cell lines have become very popular substrates for the production of vaccines and biopharmaceuticals. Characterization of candidate production cell lines is central to ensure product safety and maintenance of consistency in the manufacture of biologicals. Nested PCR and isoenzyme analysis have been used widely to prove the identity and purity of various cell lines and primary cells individually and also after deliberate cross-contamination. The nested PCR based on the Cytochrome b (Cyt b) gene of mitochondrial DNA (Mt DNA) was found to be more sensitive than isoenzyme analysis in detecting low levels of contaminants (as low as 1%). Interestingly, competition between different co-cultured cell lines has shown in one case that cross-contamination need not always results in a mixed cell population. The nested PCR technique for the Cyt b gene described in this study appears to be a potential replacement for isoenzyme analysis and here we demonstrate the PCR method used is sensitive and reliable for cell line authentication in a simple, rapid and reliable format to help assure the authenticity of cell substrates for the production of safe vaccines and biopharmaceuticals.