Study Of Different Kinds Of Adjuvants On Immune System In BALB/c Mice

As a drug targeted to immune system, the serious adverse effect caused byTGN1412in the first-in-man Phase I clinical trial in2006demonstrated the need for aperfect immunotoxicity assays and animal models in standard pre-clinical safety tests, aswell as immunologic toxic biomarkers that are sensitive enough to be predictive of clinicaloutcome in human.Currently, there are no small molecular immuno-enhancing drugs that have a cleareffect except cytokines applied in clinical and adjuvant is a substance that can enhanceimmune response. Thus, as immunopotentiators, three kinds of adjuvants namelyCpG-ODN, Poly I:C and alum adjuvant were selected for the research. Aiming to identifysignificant differently expressed genes relative to immuno-enhancing effects, microarraywas conducted to determine the effects of different adjuvants on gene expression profilesin immune system in mice. Meanwhile, immune state was assessed comprehensively indifferent levels of immune systems for potential immunologic toxic biomarkers.Andalternative model in vitro was further performed for the assessment and validation ofimmunotoxicity. The mechanism of different adjuvants investigated this study will alsoprovide a basis for adjuvant selection in vaccine research and development.a) Methods:In vivo study: Female BALB/c mice aged8-10weeks were injected byintramuscular in both quadriceps with50μl per quadricep of PBS, alum adjuvant(10mg/kg), different doses of Poly I:C(0.25,2.5,10mg/kg) or CpG-ODN(0.1,1,5mg/kg)diluted in PBS，respectively. Blood was taken and mice were sacrificed at1h,3h,6h,24hand72h after treatment. Immune organs were weighed and examined by histopathologicalexamination. TDAR test was conducted to evaluate immune function. Phenotypes inmurine spleen cells were measured by flow cytometry, cytokine concentrations in plasmaafter stimulated in vitro for5hours were determined by Lunimex. Also, spleens wereremoved and stored in RNAlater for further whole-mouse genome microarray analysis.In vitro study: Spleens were removed from female mice aged8-10weeks understerile conditions and splenic cells were stimulated with PBS, alum adjuvant(0.04mg/ml), diverse concentration of Poly I:C(0.05,0.25mg/ml) or CpG-ODN(0.005,0.025mg/ml)for1h,3h,6h,12h,24h and72h. Survival rate of cells was detected at different timepoints after stimulation. After stimulation for6hours, cell RNA was extracted formicroarray analysis.b) Results:In vivo study: Spleen weight increased after stimulated with Poly I:C or CpG-ODNat72hour after dosing. Tangible body macrophages elevated in splenic white pulp andparacortex of lymph node after Poly I:C or CpG-ODN administration at24h. TDAR testindicated that anti-KLH specific IgG antibody titer was higher in mice grouped in Poly I:Cor CpG-ODN than vehicle group. After Poly I:C or CpG-ODN treatment for6h, a roughlyincreased proportion of CD86, CD69positive cells in spleen was observed and levels ofpro-inflammatory cytokine and chemokine in plasma improved. Also, the concentration ofsome cytokine and chemokine increased in alum adjuvant stimulated mice. After6hours’treatment, Poly I:C up-regulated IFN-related genes, MHC class I genes, promoted antigenprocessing and presentation of peptide antigen via MHC class I, regulated T cell mediatedcytotoxicity positively and induced T-helper1type immune response. At higher doses,CpG-ODN induced IFN-related genes expression, activated cytokine-mediated signalingpathway and B cell receptor signaling pathway. CpG-ODN could promote T and Blymphocyte proliferation. Howere, fewer genes changed after alum administration andthese genes have little relationship with alum adjuvanticity.In vitro study: CpG-ODN had a strong ability to promote proliferation of spleniccells in later phase post stimulation and the effect of Poly I:C on proliferating was gentlerelatively. As the same as the result of in vivo study, IFN-related genes and MHC class Igenes were up-regulated after stimulation with Poly I:C at6hour in vitro, and T cellproliferation was regulated positively. At higher doses, CpG-ODN promoted expression ofIFN-related genes, activation of B cells and proliferation of T cells. Significant expressedgenes stimulated with alum had a weak correlation with immunopotentiation.c) Conclusions:Alum adjuvant, Poly I:C and CpG-ODN all had strong immuno-enhancing effect onmice. Changes in transcript profiling had a good accordance to the results of TDAR,determination of concentration of cytokines and chemokines, analysis of immune cellphenotypes and histopathological examination. Furthermore, Poly I:C and CpG-ODNwere better suited to bias immune reaction towards cell immune response than alumadjuvant. Gene changes in spleen cells stimulated directly with different adjuvants in vitro were in according with the microarry result of spleen in vivo study. This study showed agood attempt for the application of microarray to the evaluation of immunological toxicityand the search for latent biomarkers. Mechnism of different asjuvants investigated in thissudy would propose a basis for selection of perfect adjuvant in vaccine design.