| Recombinant Adsorbed Hepatitis B Vaccine (prepared from Chinese hamster ovary cells) is a liquid product containing "hepatitis B surface antigen" which is prepared from the transformed Chinese hamster ovary cells (CHO) and rendered insoluble by adding aluminum salt, which is used as adjuvant to boost the immune response of vaccine. Although hepatitis B vaccine prevents infection, previous studies have shown that it has several side effects, the most frequent local side effects that may occur include induration, pain and pruritus. The systemic side effects that persist or become troublesome when using recombinant hepatitis B vaccine include diarrhea, dizziness, fatigue, headache, irritability, loss of appetite and mild fever. The most harmful effects of vaccines are caused by their chemical constituents, such as aluminum that is used as an adjuvant in hepatitis B vaccine. The early detection of unexpected adverse events is very important in vaccine safety. However, Pharmaceutical companies usually perform safety testing of vaccines, but all requirements of the World Health Organization (WHO) and drug pharmacopoeias depend on general toxicity testing, and the gene expression study of hepatitis B vaccine is not done routinely to test vaccine quality. Therefore we applied more sensitive and more scientifically well-grounded methods for the quality control of vaccine safety.In the first part, In vivo study, we applied a new technique of gene expression analysis to detect the inflammation and metabolism genes that might be affected by hepatitis B vaccine in mouse liver. Twenty-four mice were used and divided into three groups:the first and second groups contained nine mice and were treated with one or two human doses of vaccine, respectively, and the third group was used as a control. A microarray test showed that expression of144genes in the liver was significantly changed after1day of vaccination.. Seven of these genes which related to inflammation (S100A8, S100A9, Saa2, and Fgll) and metabolism (Acaala, Acaalb, Pckl) were chosen and confirmed by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) at1,4and7days. The expression level of S100A8was significantly increased in the group with two doses of vaccine at all three time points. In the group with one dose of vaccine, S100A8expression was only significantly increased at1and4days. This means that the vaccine had a prolonged effect when two doses were administered. Similar results were obtained for Saa2, S100A9and Acaalb, which were significantly, increased at all three time points in second group, whereas Fgll, Acaa1a and Pck1were only significantly increased at1day in both groups. The expression level of these genes can be considered as a biomarker for the effects of the vaccine. This study tested the hypothesis that gene expression profiling can reveal indicators of subtle liver injury that is induced by a dose of a substance that does not cause overt toxicity as defined by conventional criteria of toxicology.The objective of the second part, In vitro and In vivo studies, is to define the form of cell death that might be induced by hepatitis B vaccine and to establish an in vitro model system amenable to mechanistic investigations of cytotoxicity induced by and to investigate the mechanisms of vaccine-induce cell death. Hepa1-6(mouse liver hepatoma cell line) was treated with two doses of adjuvanted hepatitis B vaccine (0.5and1μg protein per ml) and cell integrity was measured after24,48and72h. Hepatitis B vaccine exposure increased cell apoptosis as detected by flow cytometry and TUNEL assay. Vaccine exposure was accompanied by significant increases in the levels of activated caspase3, a key effector caspase in the apoptosis cascade. Alterations in apoptosis-related gene expression levels were studied by quantitative RT-PCR. A pattern of gene expression appeared that caspase dependent and intrinsic apoptosis was found in which release of cytochrome c from the mitochondria triggers the assembly of a caspase activation complex, that was evident by increase of Apaf-1and caspase9expressions to assemble the apoptosome. Activation of caspase3, then caspase7and trigger hallmarks of apoptosis such as DNA fragmentation and cell membrane shrinkage were resulted from the activation of ICAD and ROCK-1respectively. The results were supported by study apoptosis in mice liver after injected with20μg/ml for24,48,72h and tested for activated caspase3and DNA fragmentation.In conclusion, this study has demonstrated that the hepatitis B vaccine containing aluminum adjuvant has effect to change the epression level of inflammation and metabolism genes which reflected subtoxic/adverse effects of the vaccine, especially subtle liver injury and is a potent inducer of apoptotic cell death even with low dose in Hepal-6cells as a model for an in vitro study in addition to induce apoptosis in mice liver.
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