Tetanus antigen modulates the gene expression profile of aluminum phosphate adjuvant in spleen lymphocytes in vivo

Abstract

Adjuvants play an important role in stimulation of the immune response to antigens. Very little is known about the molecular mechanisms of this stimulation. Here we address this issue by studying gene expression profiles from spleen lymphocytes after in vivo immunization of mice with a clinically relevant vaccine, tetanus toxoid formulated with aluminum phosphate as adjuvant (TT(ADJ)), or the adjuvant alone (ADJ). The Th1/Th2 response to TT(ADJ) was obtained from a combination of up- and downstream markers to conventional cytokines, which were in good agreement with cytokine protein levels. A clustering algorithm revealed that ADJ elicited expression of 47 genes active in cytotoxic lymphocytes, inflammation, oncogenesis, stress, toxicity and cell cycle regulation. In TT(ADJ) these adjuvant-elicited genes were expressed at lower levels and a compensatory onset of protective and inhibitory genes was observed. We conclude that the antigen, to a larger extent than previously recognized, modulates the molecular mechanism of the aluminum phosphate adjuvant and that the identified genes may serve as predictive biomarkers in the development of new adjuvants and vaccines.

PEI – a potent, but not harmless, mucosal immuno-stimulator of mixed T-helper cell response and FasL-mediated cell death in mice

“…This showed that the main contributor to the difference in the total number of genes between the two time points is PEIÀ, while the gene numbers for PEI+ vaccine did not change significantly. This is in contrast to our earlier observations derived from the analyses of the systemic immune response in spleen lymphocytes from mice after immunization with tetanus toxoid or diphtheria toxoid vaccines, 14,16 where an early burst of gene expression during the immune response has been shown. However, only a smaller part of these genes were found to be involved in immune response reactions at 24 h in PEIÀ, while the major part of these genes were found to be involved in cellular processes other than immune response.…”

“…17 In order to identify the optimal administration dose for the systemic immune response, PEI+ samples obtained from administration of the lower (33.75 mg) and the higher (67.5 mg) dose were compared concerning the expression levels of genes identified earlier to be important in the systemic immune response of the tetanus and diphtheria toxoid vaccines. 14,16 In summary, gene expression levels from the higher dose PEI+ samples were much lower as compared to lower dose PEI+ samples (data not shown). To name the most important genes: the markers for antigen-specific CD8 + T cells, SATB1 and lung Kruppel-like factor (LKLF), the costimulatory signal B7-2 (CD86) on antigen-presenting cells, the early T-cell activation signal Syk, the T-cell activation antigens CD2 and LFA-1 a (CD11a) and CD4 receptor as well as genes involved in the cytokine regulation for both the Th1 and Th2 responses (data not shown).…”

“…14,16 This might be a characteristic of DNA vaccines in general or account for this specific antigen-adjuvant combination.…”

“…These time points have been verified earlier as ideal for the study of cytokine responses on the gene expression level. 14,16 Spleen lymphocytes of PEIÀ mice and the non-immunized mice were cultured for the same time periods, but not in vitro restimulated with antigen in order to avoid both potential non-specific and specific effects of the LacZ antigen. Also, we had observed earlier that in vitro restimulation of lymphocytes from non-immunized mice with tetanus toxoid antigen did not result in expression changes of genes other than those that are involved in mitotic effects.…”

“…After a booster dose, spleen cells were harvested and pooled for each formulation (n¼3-4 mice) to diminish interindividual variation effects. [14][15][16] The spleen lymphocytes were isolated and restimulated in vitro with LacZ antigen peptide for 6 days and a second time for 4 and 24 h before RNA purification. These time points have been verified earlier as ideal for the study of cytokine responses on the gene expression level.…”