Development And Efficacy Evaluation Of A New TB Subunit Vaccine And Research On Evaluation Of Type â…  Anaphylaxis In Vaccines

Caused by Mycobacterium tuberculosis (MTB), tuberculosis (TB) is a kind of serious infectious disease with a long history. As the second leading cause of mortality in the world, tuberculosis is a heavy burden to most developing countries around the world. According the data published by World Health Organization (WHO) in 2009, 9.27 million new cases of TB occurred in 2007 and 1.32 million HIV-negative people died from TB worldwide. Most of the new cases were in Asia (55%) and Africa (31%), and China ranked second (1.3 million) in terms of total numbers of cases, which meant a serious condition for the control of tuberculosis in our country.Mycobacterium bovis bacillus Calmette-Guerin (BCG) is a unique vaccine against TB that is currently available. BCG efficiently protects children against severe disease, but this vaccine does not offer consistent protection for adults, and it does not prevent reactivation of latent TB. Development of new TB vaccines is badly needed not only to prevent the infants and uninfected people against TB, but also to enhance the cellular immunity of those who had been infected but not active patients and eliminate the MTB faster.In this study, we constructed two cloning and expression vectors-pET30a-Ag85b and pET30a-HspX by molecular biological technology, expressed the protective antigen Ag85B and latent antigen HspX of H37Rv. Those two purified antigens were combined with other two secreted antigens CFP-10 and ESAT-6 (in the form of fusion protein CFP-10:ESAT-6(C/E)), then mixed with the adjuvants CpG DNA and aluminum hydroxide. This subunit vaccine was first reported in the world. Evaluations of immugenicity and protection efficacy were carried out on mice and H37Rv-challenged guinea pigs; guinea pigs were immunized with immonogen comprised of OVA and Al(OH)3 to establish a modle of typeⅠanaphylaxis and some quantative indicators were also discovered to evaluate typeⅠanaphylaxis caused by vaccines quantatively.1. Preparation of the new TB subunit vaccineTwo cloning and expression vectors-pET30a-Ag85b and pET30a-HspX were constructed by molecular biological technology, transfered into the host Escherichia coli, and expressed two proteins induced by IPTG. After the identification, Ag85b was proved to be in the form of inclusion body and HspX was in the supernatant. After a series of preparation, sample of Ag85b was purified on the anon exchange column Source30 Q and HspX on anon exchange column QHP and hydrophobic chromato-graphy column, and the two proteins were of high purity. Further experiments like SDS-PAGE, protein sequencing and WB were also carried out to identify the physico-chemical property like molecular weitht, purity, isoelectric point and biologic activity of the two proteins, which set the fountion for further evaluation of immunogenicity and protection efficacy.The two proteins were mixed with another fusion protein CFP-10:ESAT-6 in a certain proportion, and combined with the adjuvants of CpG and Al(OH)3 in a proper order.2. Immunogenicity evaluation of the new TB subunit vaccineMice were vaccinated with the vaccine every one week on the back sub-cutaneously, three times totally. One week after the last injection, mice were sacrificed by removing the eye balls, blood and lymphocytes of spleen were collected; level of specific IgG in sera was determined; lymphocytes of spleen were stimulated in vitro by three antigens, level of cells proliferation and counts of lymphocytes capable of secrecting antigen-specific IFN-y were determined; peritoneal mast cells (PMC) of mice were collected and stimulated in vitro, level of IL-12 secreted by PMC was determined. Results showed that the level of specific IgG, lymphocytes proliferation, counts of lymphocytes capable of secrecting antigen-specific IFN-γand level of IL-12 in the (Ag+Al+CpG) group were all significantly higher than that of control group, and even higher than that of Ag group. These results indicate that the new vaccine can induced the cell mediated immunity and humoral immunity at a high level, and also mean that only combined with the adjuvants can the recombination protein possess a high level of immunogenicity, and that's why all of the subunit vaccines which entered clinical evaluation in recent years contained strong adjuvants systems.3. Protection efficacy evaluation of the new TB subunit vaccineGuinea pigs were challenged by injection of H37Rv subcutaneously on the inner side of thigh and the post-exposure model was established. Then the guinea pigs were immunized with the vaccine every two weeks, three times totally. Two weeks after the last injection, guinea pigs were sacrificed by injection of pentobarbital sodium peritoneally and liver, spleen and lung were obtained to be examined and total lesion scores were calculated; the homogenates of spleen were inoculated the modified LG medium base, four weeks later the cfu were counted and bacterial loads were calculated. Though the results of bacterial loads in the spleen and mean total liver, lung and spleen lesion scores showed a better efficacy in vaccine groups than in normal saline groups or single antigens groups, no significant difference was discovered which meant the protective efficacy was limited. The reasons for the limited efficacy may be the immature technics in vaccine development including the proportion of each component and the order of mixing adjuvants. In addition, the immature post exposure model may be another reason, the model used in this study was constructed by injecting H37Rv subcutaneously on the inner side of hind leg and that should be optimized in the future. In this study, we didn't set a positive control(BCG group) because the model in this study is post exposure model and BCG had poor protection efficacy especially for the population which has been infected by tuberculosis.4. Establishment of the guinea pig model of type I anaphylaxis Safety evaluation is an important part in the preclinical evaluation of vaccines. To construct a positive model for evaluation of typeⅠanaphylaxis in immunotoxicity caused by vaccines, guinea pigs were sensitized by OVA and Al(OH)3, the first immunization was through injection subcutaneous on the back and the followed booster immunizations were through injections intramuscular on the hind legs. One week after the last immunization, sensitized guinea pigs were challenged by intravenous injection of OVA, syndromes of typeⅠanaphylaxis were observed, bronchoalveolar lavage fluid (BALF) was collected and histamine and leukotriene in BALF were determined; peritoneal mast cells (PMC) were collected from peritoneal lavage of unchallenged guinea pigs and stimulated in vitro, histamine and leukotriene in supernatant were determined.Results showed that obvious syndromes of typeⅠanaphylaxis were observed after the sensitized guinea pigs were challenged; levels of histamine and leukotriene in BALF were significantly higher than that of control group; the levels of histamine and leukotriene secreted by PMC were also significantly higher than that of control group. Those results indicate that the guinea pig model of typeⅠanaphylaxis has been established successfully and some quantative indicators have been discovered. Addition to the observation of syndromes of typeⅠanaphylaxis which is a qualitive indicator, the levels of histamine and leukotriene either in BALF or secreted by PMC may become quantitative indicators for evaluation of typeⅠanaphylaxis caused by vaccine.5. ConclusionIn this study, we combined the protective antigen of Mtb Ag85b, latent antigen HspX and other two secreted antigen CFP10 and ESAT6, and then mixed with two adjuvants CpG DNA and Al(OH)3, to prepare a new TB subunit vaccine.We evaluated the immunogenicity and protection efficacy of this new vaccine, results showed that the vaccine can induced strong cell immunity and humoral immunity in mice but limited protection in challenged guinea pigs. We plan to optimize the expressing form of the two antigens and proportion of the vaccine to achieve a better protection and supply a ideal therapeutic vaccine against TB. Meanwhile, the early secreted antigens included in the new vaccine also make it proper to be a prophylactic vaccine instead of BCG or a booster vaccine to BCG.We preliminarily establish a guinea pig model of typeⅠanaphylaxis, established some quantative indicators like levels of histamine and leukotriene in BALF and levels of histamine and leukotriene secreted by PMC. These indicators may be applied to evaluate the typeⅠanaphylaxis quantatively.