Studies On Preparation Of Hepatitis A Vaccine By Microcarrier Culture Technique

Inactivated hepatitis A vaccine is highly immunogenic, safe and efficacious in preventing HAV infection. It is estimated that the persistence of anti-HAV neutralizing antibody following vaccination may be more than 20 years. Therefore it has practical application and marketing value. However, production of HAV by traditional process is difficult to obtain enough HAV antigen for killed HAV vaccine. In order to solve this problem, we did a preliminary study on the process of microcarrier culture method which used to be automatic and lare-scale technique for preparing inactivated hepatitis A vaccine.This study is divided into 4 parts: (1)Isolation, adaptation and identification of HAV, (2)Production of HAV by microcarrier culture technique, (3)Preparation of inactivated hepatitis A vaccine and test of its immunogenicity, (4)Genotype of HAV isolate and preliminary study of HAV adaptation mechanism.First, we need to adapt HAV to Vero cells which is a good cell substrate for microcarrier culture. Rhesus monkey embryo kidney (MEK) cells were used to isolate HAV from clinical fecal specimens, as a transitional step for adaptation of Vero cells. Wild-type isolates were demonstrated to be HAV by blocking experiments, neutralising experiments, indirect immunofluorescence (IF), immune electron microscopy (IEM) and nucleotide sequence analysis.Antigenic titers gradually increased as HAV was serially passaged in MEK cells with HAV isolates W, X from MEK cell and isolate Ch6 from KMB17 cell. Maximum HAV antigen titers of strain Ch6 at passage 9 on day 28, strain W at passage 7 on day 21, strain X at passage 7 on day 28 were 1:1024, 1:512, 1:1024, respectively and infective titers (logTCID₅₀/ml) were 8. 33, 8.17, 8. 50, respectively.Passage 4 of HAV strains W, X, Ch6 adapted to MEK cells was passaged in Vero cells. Strains Ch6 and W were adapted to Vero cells after three blind subculture, and viral antigenic titers continuously rised with succeeding passages in Vero cells. Highest antigenic titers obtained with strains Ch6 and W at passage 6 on day 21 both were 1:256, and infective titers (logTCID₅₀/ml) were 8. 17 and 8.00, respectively. Antigenic titer of...