GENETIC IMMUNIZATION APPROACHES ON THE CONTROL OF ALLERGIC INFLAMMATION

Abstract

Allergen gene immunization and cytokine gene immunization are novel approaches that have been explored as alternative treatments for allergic diseases. However the major drawback of allergen gene vaccination in human clinical trials is its low immunogenicity. The first part of the study aimed at enhancing and evaluating the immunogenicity and efficacy of DNA vaccines by the incorporation of additional human CpG motifs into the pVAX plasmid backbone. cDNA encoding for a major allergen from the Blomia tropicalis mite, Blo t 5, was cloned into the pVAX vector. CpG-modified and unmodified DNA constructs were designated as pVAXBlot5-DTKT and pVAXBlot5 respectively. The immunogenicity of DNA constructs was evaluated in the Blo t 5-induced murine allergy model. pVAXBlot5-DTKT elicited a higher level of Blo t 5-specific IFNgamma when compared to pVAXBlot5. Both pVAXBlot5-DTKT and pVAXBlot5 attenuated Blo t 5-specific IgE, in addition pVAXBlot5-DTKT elicited higher levels of IgG2c than pVAXBlot5. In addition, allergen gene vaccination also suppressed Blo t 5 specific IFNgamma, IL-17, IL-5, IL-13 and IL-6 induced by intraperitoneal immunization with rBlo t 5 protein adjuvanted with aluminium hydroxide. Co-culture of peripheral blood mononuclear cells (PBMCs) with plasmid DNA revealed that pVAXBlot5-DTKT elicited higher levels of pro-inflammatory cytokines and chemokines when compared to pVAXBlot5 and DTKT oligonucleotides stimulation. Rhesus macaques immunized with both pVAXBlot5 and pVAXBlot5-DTKT showed suppression in Blo t 5-specific IgE when compared to pVAX control group. In addition, monkeys immunized with pVAXBlot5-DTKT also produced higher levels of Blo t 5-specific IgG when compared to pVAXBlot5 group. Furthermore, the frequencies of IFNgamma producing cells in response to stimulation with Blo t 5 peptide pools were higher from monkeys immunized with pVAXBlot5-DTKT as compared to the pVAXBlot5 group. The second part of the thesis aimed to explore the use of the IL-35 encoding gene immunization approach for the treatment of allergic diseases. IL-35 is a novel cytokine that is produced by Foxp3+ regulatory T cells and has been reported to have suppressive functions. However the therapeutic role of IL-35 in Th2 mediated allergic diseases has not been evaluated. Firstly, the effectiveness of pVAX-IL-35 was evaluated in the eosinophilic lung inflammation model induced by a Blo t 5-specific Th2 cell line. Intra-tracheal administration of pVAX-IL-35 was found to be able to attenuate the Blo t 5-specific Th2 cell line induced total cellular infiltration as well as eosinophil, lymphocyte and neutrophil counts in the bronchial alveolar lavage fluids (BALF). In addition, Th2 cytokines as well as chemotactic chemokines were significantly attenuated in the BALF from the experimental group of mice received pVAX-IL-35 as compared to that from the control pVAX group. Systemic administration of pVAX-IL-35 via the intramuscular injection elicited a long-term suppression of Blo t 5-specific humoral responses in mice upon intranasal Blo t 5 instillation. Mice received pVAX-IL-35 injection produced lower titres of Blo t 5-specific IgE and persistently suppressed Blo t 5-specific IgE and IgG1 production. The long-term suppression by the pVAX-IL-35 injection was also found on the production of total serum IgE in mice which received the Blo t 5 specific Th2 cell transfer and intranasal Blo t 5 challenges. Secondly, the effectiveness of pVAX-IL-35 was evaluated in a neutrophilic lung inflammation model induced in Blo t 5-specific T cell receptor (TCR) transgenic mice upon intranasal Blo t 5 sensitization. Intra-tracheal administration of pVAX-IL-35 was also found to effectively reduce total cellular infiltration as well as neutrophil, lymphocyte infiltration, pro-inflammatory cytokines and chemotactic chemokines in BALF from Blo t 5-specfic TCR transgenic mice upon intranasal Blo t 5 instillation.