Safety evaluation of repeated intramuscular injection of CpG 684-HBV vaccine in rats

Abstract：Objective:To investigate the nonclinical safety of the novel CpG 684-HBV vaccine in SD rats after repeated dose administration,and provide reference for the dose level design and side effects monitoring in clinical trials.Methods:The rats were divided into three treatment groups:vehicle control,50 μg CpG 684 + 10 μg HBsAg,500 μg CpG 684 + 10 μg HBsAg groups.Each group was composed of 40 rats,30 rats in main toxicity group and 10 rats in satellite group,with half males and half females in each group.The animals were dosed by intramuscular injection at week 0,2,4 and 6.During the study,each animal was observed for clinical signs and injection site irritations.Body weights,body temperature,and food consumption were measured.Serum cytokines of IL-6,TNF-α and IFN-γ,and anti-HBV antibody were determined.Urinalysis,hematology,coagulation,serum chemistry and anti-nuclear antibody examinations were conducted respectively prior to terminal and recovery necropsies.A full necropsy was then conducted on the animals,macroscopic examination performed,and tissues were weighed and processed for microscopic examination.Results:The vaccine injection induced a transient decrease in food consumption in the treatment groups.Low-and high-dose CpG 684-HBV vaccine treatment induced marked increases in WBC and neutrophil counts and slight decrease in hemoglobin in male and female rats.High-dose vaccine caused slight decrease in HCT in rats of both sexes,slight decrease in RBC count in female rats,and slight decrease in MCV and MCH in male rats.Low-dose vaccine also caused slight decrease in HCT in females.Both dose levels of vaccine caused decrease in serum albumin and A/G ratio in males and females.High-dose vaccine induced decrease in female serum total protein and increase in ALT in rats of both sexes.Increases in serum K +,phosphorus,ALT,AST and LDH,and decrease in Na + and Cl-were observed in two male rats in the high-dose group at the terminal necropsy,with the histopathological findings of expansion and congestion in hepatic sinusoid and interstitial vasodilation in some other parenchymal organs.The spleen weight and spleen/body weight ratio were increased in the male rats of the low-and high-dose groups and female rats of the high-dose group at the terminal necropsy.The above changes were all markedly recovered at the end of recovery period.Treatment-related microscopic findings included inflammatory cell infiltration,hyperplasia of fibrous tissue and necrosis of muscular tissue at the injection site,sciatic nerve and adjacent muscle,and increase in tingible body macrophages and broadening of paracortex zone in lymphoid nodule of inguinal lymph nodes,which obviously vanished at the end of the recovery period.CpG 684-HBV vaccine induced anti-HBV antibody in rats.No other obvious abnormalities were found.Conclusion:Repeated intramuscular injection of CpG 684-HBV vaccine induced increases in peripheral neutrophil and spleen weight and slight decreases in food consumption,hemoglobin,HCT,and albumin.High-dose vaccine caused increase in serum ALT.Treatment-related microscopic findings were inflammatory reaction lesions at injection site and inguinal lymph nodes.All the above changes were reversible and the vaccine did not induce other obvious abnormalities.