Preparation And Preliminary Long Term Toxicity Studies Of Tat Fusion Protein

Objective: To prepare TatE-pET32a fusion protein, study the immunogenicity and immunotoxicity in Sprague-Dawley(SD)rats induced by TatE-pET32a fusion protein emulsified with Freund's adjuvant, and to identify the toxicity, its severity and reversibility by so as to perform a safety evaluation for HIV-1 vaccine of Tat fusion protein . Methods: (1) Protein Preparation: The E.coli BL21(DE3) containing pET32a-TatE(1-101aa)was cultured in a large scale. Then Isopropyl-β-D-Thiogalactoside (IPTG) was added to induce the expression of the aimed protein. The bacteria, which were collected through centrifugation, were schizolysed by ultrasounding following resuspended Lysic buffer. Refrigerated centrifugation was adopted to collect the supernatant containing the aimed protein. After filtration with filter paper, the filtrate was purified with the Ni-NTA column (Ni-column) to collect the aimed protein (Tat fusion protein). SDS-PAGE experiment was carried out to validate the size of the aimed protein. Bradford was to analyses the concentration of the collected refolded Tat fusion protein. Indirect ELISA assay was used to detect the immunogenicity of Tat fusion protein. (2) The preliminary longterm toxicity study: Seventy SD rats were divided into seven groups randomly: Tat fusion protein iv group (200μg / rat), Tat fusion protein sc group (200μg / rat), Tat fusion protein (20μg / rat) emulsionized with Freund's adjuvant sc low dose group, Tat fusion protein (200μg / rat) emulsionized with Freund's adjuvant sc high dose group, Freund's adjuvant sc control group, menstruum emulsionized with Freund's adjuvant sc control group, physiological saline control group. The rats were administrated once a week for 8 weeks. All the animals were treated subcutaneously except ten rats of the Tat fusion protein iv group. The protein was emulsionized with Freund's adjuvant in the same volume. The rats were weighted each week before the treatment. One week after the termination of administration (W9) and the recovered period (W12), 6 and 4 rats were anesthetized and dissected respectively. Blood samples were collected through abdominal aorta for the hemotology, blood biochemistry, immunogenicity and immunotoxicity. Meanwhile, the major organs were observed histopathologically. At the third, fifth, seventh, ninth and twelfth week, blood samples were collected through venous sinus of orbit for the antibody assay by enzyme linked immunosorbent assay (ELISA). Hemotology detection include Red Blood Cell(RBC), Hemoglobin (HGB), Haematocrit (Hct), Mean Cell Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelets (PLAT), White Blood Cell (WBC), Neutrophil (NEUT), Lymphocyte (LYMPH), Monocytes (MONO), Eosinophil (EOS), Basophil (BASO), Large Unstained Cell (LUC), Reticulocyte (RETIC), Prothrombin Time (PT) and so on. The biochemistry detection include Alanine Transarninase (ALT), Aspartate Aminotransferase (AST), Alkaline Phosphatase (ALP), Lactate Dehydrogenase (LDH), Total Bilirubin (TBIL), Blood Urea Nitrogen (BUN), Creatinine (Cr), Total Protein (TP), Albumin (ALB), Glucose (Glu), Total Cholesterol (TCH), Triglyeride (TG), Creatine Phosphate Kinase (CPK), Ca, P, K, Na, Cl and so on. Immunology detection included the antibody assay and CD4~+, CD8~+ lymphocyte phenotype analyses. IgM, IgA, IgG, C3, IL-1, IL-4, IL-6, IL-10, TNF-αand IFN-γall had been detected. Results: (1) All the animals, except those in the Tat fusion protein iv group and physiological saline sc group, had a scratch on the sites of drug administration. All the animals in the subcutaneous injection groups, except those in the physiological saline control group were observed with different degrees of swelling, skin ulceration and crusting. Histopathologic examination suggested that the lump was filled with inflammatory materiae. The possilbe reason may be the inflammatory reaction induced by the delayed absorption of the protein emulsified with adjuvant. (2) Compared with the menstruum+Freun's adjuvant control group and saline control group, the SD rats of withdrawal stage were observed with significantly higher NEUT ratio, lower LYMPH ratio and WBC in rising trend ( No statisctical difference analysis) when administrated with high dose of Tat-FP+Freund's adjuvant, which might be relavant to severe local inflammation. During recovery stage, the SD rats were observed with significantly higher NEUT ratio, lower LYMPH ratio when administrated with high dose of Tat-FP+Freund's adjuvant, which might be due to incomplete recovery of local inflammation. (3) Compared with the adjuvant control group, solvent+Freund's adjuvant control group and saline control group, no significant differences were observed in the values of blood biochemistry of Tat-FP groups during withdrawal and recovery stages. (4) The results of bone marrow slides showed there was an inflammatory reaction in the bone marrow. (5) There was no signifiant change in the absolute and relative weights of organs. No phthological change in the parenchyma organs and no histological change induced by immunotoxicity were observed in all the groups. However, there was local inflammation in rats after administration with adjuvant ingredients. (6) The ratio of CD4~+/CD8~+ of Tat fusion protein sc group and Tat fusion protein emulsified with adjuvant sc high dose group showed a significant raise compared with the control groups, but the %CD8+ cells showed a decrease, at the same time, IFN-γwas increased compared with the control groups. As we know, IFN-γwas positive correlate with CTL (CD8~+CD28~+) and negative correlate with regulatory T cell (CD8~+CD28~-). These results indicate that Tat-FP may mainly decrease the ratio of regulatory T cell (CD8~+CD28~-). (7) The antibody results indicated that Tat fusion protein emulsionized with Freund's adjuvant might produce rapid, continuous and long-lasting antibodies in a high titer (1:12800). These results indicated that Tat fusion protein had a well immunogenicity. IgG and C3 in Tat fusion protein emulsified with adjuvant sc high dose group had increased significantly, indicate that effective immune responese had been induced. The result of IL-4 and IFN-γhad been showed that Tat fusion protein emulsified with adjuvant sc in high dose induced effective humoral immunity and cellular immunity. Conclusions: The SD rats were administrated subcutaneously once a week with Tat fusion protein emulsified with adjuvant for 8 weeks, which excitated high-titer antibody in SD rats. But it could also induce locally inflammatory reaction. However, the toxicity was reversible. As for the drug's administration, the researchers should chose the optimal dosage proportion between the compound and adjuvant so as to prevent severe local inflammation induced by combined use of high dose of the compound and adjuvant. More efforts are needed to evaluate the therapeutic effect and safety of Tat fusion protein as HIV vaccine.