| Human rotavirus was first discovered in 1973 and has since been one of the major pathogens causing infantile diarrhea worldwide.Although it has been several decades since the discovery,there is still no effective treatment at this stage.Therefore,the use of rotavirus vaccine to prevent and reduce the proportion of infection.The social and economic burden caused by rotavirus infection has become a serious global public health problem,which has also aroused great concern of WHO.Therefore,in the development of vaccines,global resources have been mobilized to achieve a high degree of coordination.Rotavirus vaccines have been researched and prepared since the 1980s,and live attenuated vaccines have become the main trend of rotavirus.The masses obtained a certain protective effect after being vaccinated with attenuated rotavirus vaccine.However,live attenuated vaccines have higher requirements for early production and preparation,and there are more safety hazards after vaccination,including the risk of virulence reversion and intussusception.In addition,antibodies in breast milk may affect the effectiveness of the vaccine,especially the use of live vaccines by immunodeficiency people will have more complex and serious consequences.Therefore,the development of inactivated rotavirus vaccine is of great significance to prevent rotavirus infection and reduce severe mortality.It is worth noting that,no matter what form of vaccine is developed,it is very important to study the epidemiological dynamics and pathogen characteristics of the disease.Early-stage epidemiological studies on rotavirus infection have shown that the genotypes of the strains causing rotavirus epidemic are different in different regions and at different times.Based on the antigenicity of glycoprotein VP7 and structural protein VP4,27 G genotypes and 35 P genotypes have been discovered so far.The main epidemic strain was G1 at the beginning,and recent epidemiological data in many areas of China suggest that G9 has gradually become the main epidemic.According to the results of the immune cross test,it was found that the serum neutralizing antibodies produced by the G1 and G9 virus immunization cross-recognize different types of rotavirus,but there are obvious differences in the cross-protection capabilities of different types.Among them,the analysis found that the sera obtained by immunization with G1 rotavirus had weak neutralizing ability to G9 virus,and the sera immunized with G9 rotavirus were not ideal for the protection of G1 rotavirus.Objectives:In this experiment,we used two human rotavirus strains ZTR-68(G1P[8])and ZTR-18(G9P[8])isolated from the laboratory.G9)The inactivated rotavirus vaccine was developed,and its immunogenicity and protection were studied and observed.Methods:The rotavirus ZTR-18(G9P[8])strain isolated in the laboratory was subcultured,the titer was determined by the plaque method,the morphology was observed by transmission electron microscopy and then concentrated and purified by chromatography.The G9 rotavirus inactivated vaccine was prepared by inactivating the purified solution with formaldehyde,and the ED50 study of the G1 rotavirus inactivated vaccine and the G9 rotavirus inactivated vaccine was conducted.After determining the effective immunization dose of the G9 type,conduct a matching immunogenicity study with the G1 rotavirus inactivated vaccine prepared by the laboratory to determine the effective immunization dose of the bivalent rotavirus inactivated vaccine.Using 5-day-old Balb/c mice,a model of diarrhea in suckling rats was established by gavage and challenge.After confirming that the model was established,a bivalent(G1+G9)rotavirus inactivated vaccine(80EU+160EU)was prepared for vaccine protection research.A bivalent(G1+G9)rotavirus inactivated vaccine(80EU+160EU)was used to immunize Balb/c female mice.Through intraperitoneal immunization,the total number of immunizations is two,with a time interval of two weeks.After the immunization is completed,the female and the male are in the same cage,waiting for the immunized female to give birth to the suckling.Five-day-old suckling rats were selected for the challenge protection test.Stomach was collected by squeezing the abdomen at 24h,48h and 72h after challenge.The suckling rats were euthanized at 24h,48h and 72h,and their intestinal tissues were taken for pathological examination.At the same time,suckling rats were randomly dissected,collecting suckling rat sera,collecting the required tissues and storing them in two pieces.One piece was later made into paraffin sections for HE staining and morphological changes were observed.Another tissue is used to extract RNA and measure the virus copy number.An ELISA experiment was performed using suckling mouse serum to measure the antibody content in the serum.Results:The isolated and cultured human rotavirus ZTR-18(G9P[8])strain can achieve a titer of 106.0 PFU/mL after passage,and after the preparation conditions are mature,the inactivated rotavirus vaccine prepared in the later stage has been tested and has good results Immunogenicity.The results of the half effective test showed that the ED50 of the inactivated G1 rotavirus vaccine prepared using rotavirus ZTR-68 strain was 20EU,so 80EU was selected as the dose for immunization evaluation.The ED50 of the inactivated G9 rotavirus vaccine prepared using the rotavirus ZTR-18 strain is 40EU,so 160EU was selected as the dose for immunization evaluation.After the immunized mother mouse gives birth to the suckling mouse in the same cage,the attack protection test can be carried out.The serum of suckling mice was collected and detected by ELISA.The antibody in suckling mice was at a high level,and the titer of neutralizing antibody could reach 1:1024.The results of diarrhea score and percentage statistics show that compared with the PBS control group,the diarrhea symptoms of the vaccine-immunized group are reduced or even not diarrhea,which can effectively prevent diarrhea caused by rotavirus infection.Suckling rat tissues(jejunum,ileum,kidney)were collected,and later made into paraffin sections for HE staining for observation.Pathological observation showed that compared with the PBS control group,the small intestinal epithelial cells of the immunized group had reduced vacuolization,swelling and The damage is significantly reduced.Conclusions:The bivalent(G1+G9)rotavirus inactivated vaccine(80EU+160EU)has good immunogenicity and can effectively prevent or reduce diarrhea caused by rotavirus infection in the suckling mouse model... |
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