Production of immunogenic human papillomavirus-16 major capsid protein derived virus like particles.

Background & objective: Recombinant DNA technology allows expression of the human papillomavirus (HPV) major capsid protein (L1) in heterologous expression systems and the recombinant protein self assembles to virus-like particles (VLP). We took up this study to produce recombinant HPV-16 L1 in yeast, establish the process of recombinant L1 derived VLP preparation and develop an ELISA using VLP as the antigen for serological evaluation of anti HPV-16 L1 antibody status. Methods: Complete HPV-16 L1 was amplified from genomic DNA of an esophageal cancer biopsy, cloned and the protein was expressed in a galactose-inducible Saccharomyces cerevisiae expression system. Self assembled VLP was purified by a two-step density gradient centrifugation process and the VLP preparation used to test its suitability in developing an ELISA. Results: The recombinant protein was predominantly a ~55 KD species with distinct immunoreactivity and formed VLP as confirmed by electron microscopy. An ELISA using the VLP showed its efficacy in appropriate immunoreactivity to serum/plasma IgG. Interpretation & conclusions: Recombinant HPV-16 capsid protein derived VLP was produced and the VLP antigen based ELISA can be used to probe serological association of HPV with different clinical conditions. The VLP technology can be improved further and harnessed for future vaccine development efforts in the country.

Studies on the efficiency of measles virus antigen production using VERO cell culture in a microcarrier system

1. A large amount of antigen is required to conduct seroepidemiologic surveys of measles. Thus, a process to obtain measles virus antigen using a bioreactor was standardized. 2. The virus was grown in a 3.7-1 culture of VERO cells using a Celligen cell culture system containing 2 mg/ml of microcarriers (cytodex I) at 37 degrees C and 60 rpm. The cultures infected with 0.5 m.o.i. of measles virus were harvested after the appearance of the cytopathic effect. The virus suspension was clarified and concentrated by ultracentrifugation. Intracellular and extracellular virus titers were determined by hemagglutination (HA) and by induction of a cytopathic effect in cell culture (TCID50). 3. Intracellular virus presented 5-7 x 10(6) TCID50/0.1 ml, HA activity per 50 microliters equal to 32, with a total HA activity of 4,480 HA units (HAU) and specific activity of 116 HAU/mg. In the concentrated supernatants, the HA titer of extracellular virus was 64, with a total HA activity of 1,024 HAU and a specific activity of 1,600 HAU/mg. 4. The antigen obtained was suitable for the detection of antibodies against measles virus in assays such as ELISA and DOT-ELISA (using 1 micrograms/well to ELISA and 2 micrograms/DOT). 5. The microcarrier system produced antigen sufficient for 26 ELISAs/ml compared to 5.7 ELISAs/ml obtained for the static culture system.