RESUMEN
The major capsid protein (L1) of human papillomaviruses (HPV) expressed in heterologous systems assembles into virus-like particles (VLPs). We report cloning and expression of codon optimized HPV L1 genes of the two high-risk HPV types 16 and 18 in methylotropic yeast, Pichia pastoris. The VLPs produced in P. pastoris were subjected to three step purification method involving density gradient centrifugations and size exclusion chromatography. The enriched VLPs were characterized using conformation-specific monoclonal antibodies in ELISA and by transmission electron microscopy. Mice immunized with a bivalent HPV16 and HPV18 VLPs developed high serum antibody titers to both HPV types that persisted for 190 days post vaccination. Serum of mice immunized with the HPV-VLP preparations could neutralize homologous pseudoviruses in an in vitro assays. Our results demonstrate that the L1 proteins expressed in P. pastoris fold properly as evidenced by assembly into VLPs and induction of type-specific neutralizing antibody response in mice. This work constitutes a step towards developing an alternate production platform for generating an affordable HPV vaccine to meet the needs of developing countries.
Asunto(s)
Proteínas de la Cápside , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Proteínas Oncogénicas Virales , Vacunas contra Papillomavirus/inmunología , Pichia/genética , Vacunas de Partículas Similares a Virus/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Clonación Molecular , Células HEK293 , Papillomavirus Humano 16/inmunología , Papillomavirus Humano 18/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/inmunología , Infecciones por Papillomavirus/prevención & controlRESUMEN
The epidemiology of canine parvovirus (CPV) infections in dogs in India was examined using 27 isolates collected during a two-year period. The VP2 genes of 22 isolates were sequenced, and the deduced amino acid sequences were compared. The results indicated that the isolates belonged to CPV type 2a except four, which belonged to CPV type 2b. Comparison of the VP2 gene sequences revealed that the Indian isolates formed separate lineages distinct from the South East Asian isolates. The canine parvovirus isolates in India appear to evolve independently, and distinct geographical patterns of evolution could not be discerned in the isolates examined.