Study On Concentrated Purified And Inactivated Vaccine Of Rabies

Rabies is an important zoonoses, mainly caused lesions of the central nervous system, and the mortality of which is almost 100%. China is rabies-prone area, where cases of human rabies have been climbing to the second in the world. In China, domesticate dogs are important transmission host of rabies, and 85% -95% cases of the rabies are caused through bites by dogs, 50% ~70 % of which have occurred incountryside. Due to the lack of safe, effective, inexpensive and convenient-to-use animal rabies vaccine, and indifferents awareness of the masses, so fraction of immune coverage among demesticate dogs is very low in China, and even no more rabies immune plan among wild animals. Those had result in difficulty one after another of prevention and cure of human rabies.As statistics noted by CDC, in 2007, the incidence of human rabies cases broke 3000 for the first time since 1990, which is forming the third popular peak. Epidemiological survey showed that more than 95% of human rabies cases are caused directly or indirectly through contacting with disease or infect dogs and cats. Therefore, in order to restrain human rabies effectively, we must develop safe and effective animal rabies vaccine, to carry out immunifaction forced the dogs, cats in rabies epidemic areas. While the quality of vaccines, the immunogenicity and safety of the vaccine, is the key to the prevention of rabies. Therefore, we have carried out the experimental study on concentration and purification of a rabies vaccine.We first identified to the culture conditions, physical and chemical characteristics, morphologic characteristics, specificity, genetic stability, cultural characteristics as well as viral contont, immunogenicity, safety and other aspects of the Rb/E3-15 (the 15 generation substitute in Vero cell cultures of ERA strain of Rabies Virus in canine 5-mixed vaccine). Rb/E3-15 was identified that it has the typical form of rabies virus, and is sensitive to heat, acid, aether, while insensitive to 5– IUDR. It can combind with anti-rabies monoclonal antibody, and accord with the general biology and chemistry characteristics of the rabies virus. Virulence of the strains to mice is stable at the LD50 of 10-5.50~10-5.60 / 0.03ml. The strain is pure, safe to canine and has good immunogenicity, stable genetic characteristics, which conforms to the standards of preparation of rabies vaccine. Through the identification, the biological characteristics, toxicity, immunogenicity, and purity of the strain are ensured to achieve the standards.Vero cells were used to proliferate Rb/E3-15 virus, and its virulence was measured by direct immunofluorescence and identified its morphology and nucleic acid. Rb/E3-15 antigen we obtained was concentrated and precipitated by zinc acetate, then purified virus particles with Sepharose 4FF chromatography column, and confirmed it by transmission electron microscope. 1/4000 of BPL and 0.02% of formaldehyde were used to inactivate virus. The inactivated virus was inoculated in 5-6 grams mice and Vero cells to compare the inactivated effect. The purified inactivated virus particles were determined by protein content, aseptic examination and antigen content, then coupled with liposome and Al(OH)3 adjuvant, to prepare concentration, purification, inactivated adjuvant vaccine, and do NIH efficacy experiment. By the direct immunofluorescence experiments, the TCID50 of Rb/E3-15 virus is 10-7; while the purified protein content is 0.2 mg / ml; by electron microscope you can see a large number of virus particles; two inactivated methods have good effect to the virus; results of NIH efficacy experiment showed that the titer of vaccine is greater than 2.5 IU.In order to explore a simple, quick, specific, sensitive methods to detect antibody of rabies, to provide a reliable basis for clinical application, we adopted fluorescent antibody virus neutralization test (FAVN), rapid fluorescent focus inhibition test and IgG antibody detection kit of canine rabies (ELISA) established in my laboratory and do comparative study of three methods. Use these three methods to detect individual serum samples immunized by rabies vaccine (Vero cells), results showed that the sensitivity and specificity of FAVN established in my laboratory is similar with RFFIT and ELISA.In order to interpret the immune effect of the vaccine, our qualified rabies vaccines were used to do animal test, and compared to aluminum adjuvant vaccine of pre-purification and concentration, and detect the cell immunity levels by lymphocyte conversion ratio test. Levels of neutralizing antibody is the determinative factor of the evaluation whether animal can resist attacking of rabies virus. In this experiment, fluorescent antibody virus neutralization test was used to detect sera of mice and dogs before and after immunized by vaccine. Results showed that it can induce a high level of antibodies in mice and dogs after three immunization, and mice obtained completely immunoprotection. The immune effect of concentrated purified vaccine in dogs was superior to the vaccine never concentrate and purify. Through lymphocyte conversion ratio test, the skeptophylaxis effect of liposomes adjuvant is superior to the effect of aluminum adjuvant. This concentrated purified inactivated vaccine immunized animals without adverse reaction, and had good safe.In this experiment, through the identification to the virus Rb/E3-15, we obtained the vaccine virus strain and purified antigen, prepared concentrated purified inactivated vaccine and carried out the immune study of animals with the vaccine. FAVN method established in our laboratory was used to detect rabies antibodies after vaccination. The experimental results showed that the concentrated, purified inactivated adjuvant vaccine has a good security and immunization effect.