Preliminary Evaluation Of Rotavirus Type G9 (ZTR18) Inactivated Vaccine Immunological Response

Background:rotavirus (RV) is the most important pathogen of infant diarrhea in the fall and winter, the world each year about 100 million infants and young children the RV infection, with nearly 500000 people infected with severe death. Discovered so far based on features of RV VP6, rotavirus A ～G,A total of seven groups, including infection of infants and young children mainly for group A rotavirus. The RV infection and no obvious difference of economic and health conditions, countries around the world all have happened, developed countries and developing countries, infection rates are similar, mortality is higher in developing countries. The RV infection disease harm to human and economic burden. There is currently no effective drug treatment rotavirus, vaccination is to prevent and control the RV infection, effective way to reduce mortality. The world health organization (world health organization, the WHO) also actively promote governments rotavirus vaccine research and development.There currently listed Rotarix (genotype is G1P[8]), RotaTeq (genotypes are G1, G2, G3, G4, G6 and P[8]) and Rotorway (genotype is G10P[12]) three kinds attenuated vaccine. These three attenuated vaccines infections play an important role in the prevention of RV. Attenuated vaccine itself, there are some common problems, such as might occur virulence throwback; exogenous factors pollution during the preparation of the vaccine, the impact of pre-existing antibodies on vaccine effectiveness; not for immunodeficiency or immunosuppressive therapy groups and the like. Therefore, the development of other forms of rotavirus vaccine is a new way of thinking, including inactivated vaccines and subunit vaccines.No matter what form of vaccine, disease epidemiology research dynamic and pathogen characteristics are very important. Since the 1970 s the RV was found, on the basis of group A rotavirus virus VP7 glycoprotein antigens (G) features at least 14 type, VP4 protein hydrolase (p-type) features at least 19 type, genotype and serotype. Epidemiological studies have shown that caused the RV epidemic strains in different areas and different time is different genotypes. Initially popular main poisonous plant type for G1 type, for example, followed by the popularity of G2 and G3 and other strains. In many parts of China recently epidemiological data suggest that type G9 has gradually become a major epidemic strains. Although the studies of the RV virus showed that overlapping between different type identification and a certain level of cross protection, but different type of cross protection exist significant difference. In general, the same type of protection for the highest. Therefore, select the current or future major epidemic strain over a period of time as the material of vaccine development, carry out the RV inactivated vaccine of related basic research is of great significance.Methods:1) with the keywords "rotavirus" and "rotavirus" respectively in China hownet (CNKI) and Pubmed to literature of 2003～2013 were retrospectively retrieved, and the collected literature analysis of epidemiological data; 2) with the medical biology institute of molecular biology laboratory of separation ZTR.18 RV (G9) wild strain, the extend the rhesus monkey embryonic kidney cells (MA104 cells) to extend to the ninth generation, and then on a large scale training preparation poison disease; 3) detect via PAGE electrophoresis banding type and genome stability; 4) by rt-pcr-type VP7 gene sequencing detection; 5) by immunofluorescence method (FFA) virus infectious droplets degrees; 6) by ultrafiltration and QFF, G-25 chromatography steps such as purified virus; 7) with the final concentration of 0.00925% formaldehyde,37℃ for 72 hours inactivated; 8) by the cell lesion and fluorescence method to detect oven residual live virus; 9) ELISA determination during the preparation of vaccine virus antigen (EU) enzyme units; 10) ultrafiltrate was determined by Lowry method and protein content change after the vaccine inactivated; 11) with negative staining electron microscope before and after the inactivated RV morphology change; 12) choose SPF ICR male mice, according to the different immune dose EU starting from 320, times than the decline, which is divided into seven a PBS control group and experimental group, each group of six, intramuscular injection, a total of immune twice, each time interval of 14 days; 13) using ZTR18 (G9) inactivated vaccine and ZTR68 (G1) inactivated vaccine, bivalent inactivated vaccines, bivalent vaccine in two monovalent inactivated vaccines are 160 EU, use of experimental animals and immunization program with 12); 14) by ELISA analysis of two monovalent inactivated vaccines and bivalent inactivated vaccine immune mice serum antibody response; 15) focal inhibition (FFIT) with fluorescence detection serum antibody induced by three kinds of vaccines for ZTR68 (G1), Wa (G1), S2 (G2), SA11 (G3), Gottfried (G4) and ZTR18 titer(G9).Results:1) the number of positive cases of infection in the first three provinces of Guangdong (186,587 cases), Zhejiang (133,390 cases) and Hubei (11,104 cases); 2) The positive rate of infection in the first three provinces most (75.20%), Henan, Jilin (64.94%) and Ningxia Hui Autonomous Region (60.16%); 3) G-type top three is G3 (8509例), G1 (6490 cases) and G2 (1601例); 4) G type G9 type RV showing the number of cases in recent years rapid growth; 5) P-type first three are the most P8 (8483 cases), P4 (2017 cases) and P6 (740 cases); 6) treatment in China is mainly used in the symptomatic treatment vaccine clinical way; 7) PAGE prove passaged virus genome bands during the original strains of the same; 8) RT-PCR-sequencing and sequence alignment confirmed the virus passaging VP7 gene is not mutated; measuring 9) FFA law virus titer 10517CCID50/ml; 10) ELISA detection ZTR18 (G9 type) rotavirus vaccine inactivated by ultrafiltration, purified, inactivated vaccine antigen content was 1600EU/ml) Lowry assay, virus harvest ultrafiltration, purification, off Live from the protein content of the harvest solution 0.8 mg/ml reduced inactivation of 0.16 mg/ml; 12) before and after the negative staining observed inactivated RV morphology, complete virus particles were visible; 13) Animal experiments show, ZTR18 (G9 type) inactivated vaccine to 160EU /100(0.1 best immunization dose.14) ELISA measured bivalent inactivated vaccine in mice serum ELISA titer ZTR68 (G1 type) and ZTR18 (G9 type) effect two monovalent inactivated vaccine prices; 15) of the same type and cross-neutralization results It showed bivalent rotavirus vaccine and inactivated serum and ZTR68 two monovalent inactivated vaccine in mice (G1 type), Wa (G1 type), S2 (G2 type), SA11 (G3 type), Gottfried (G4 type) and ZTR18 (G9 type) six RV serotypes have a certain cross-neutralization.Conclusions:1) Epidemiological studies suggest, G9 type RV might be the next major epidemic type.2) The current preparation having good experimental vaccine immunogenicity 3) bivalent inactivated vaccine induced by ELISA antibody titers higher than the unit price; 5) Anti ZTR68 (G1 type) and ZTR18 (G9 type) antibodies can neutralize ZTR68 (G1 type), Wa (G1 type), S2 (G2 type), SA11 (G3 type), Gottfried (G4 type) and ZTR18 (G9 type) six kinds of serotype RV, but the degree of neutralization (potency) in the presence of some differences between different types of the same type and potency higher than profiled.