Evaluation of the immunogenicity of different fragments of the pCDNA3-hsp65 vaccine

Author(s)

Claudia Andréa Alves de Araújo

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2002-12-19

Examining board members

Celio Lopes Silva; Marcelo Brocchi; Luciana Cezar de Cerqueira Leite

• Advisor: Celio Lopes Silva

Abstract

Recently, it had been demonstrated that pCDNA3-hsp65 DNA vaccine has prophylactic and therapeutic activities in experimental tuberculosis. This vaccine contains hsp65 gene from Mycobacterium leprae and 1324 bp downstream fragment. However, an investigation more refined of immune response triggered by different fragments from vaccine insert was not performed. Also, the influence of 1324 bp downstream fragment to induce immune response was not studied. Thus, DNA constructs were engineered from pCDNA3-hsp65 vaccine. These new DNA constructs were called pCDNA3-hsp65G (G = hsp65 gene, to differ of pCDNA3-hsp65, that contain hsp65 gene and 1324 bp downstream fragment), pCDNA3-N (N = Hsp65 N-terminal), pCDNA3-N59 (N59 = 59 bp codifying Hsp65 N-terminal) e pCDNA3-DS (DS = downstream). BALB/c mice were immunized by intradermal (Gene Gun) and intramuscular route, with 10 µg/dose and 100 µg/dose, respectively. Three immunizations were performed with 15 days between each one; 30 days after the last immunization, the mice were sacrificed and the immune response was evaluated. The humoral immune response was evaluated by anti-Hsp65 IgG, lgG1 and lgG2a antibody titles by ELISA. The cellular immune response was evaluated using three parameters: proliferation of spleen and lymph nodes cells by thimide tritiated incorporation, production of IL-4, IL-10, IFN-γ, IL-12 Hsp65- specific by ELISA and determination of B, T, T CD4+ and T CD8+ lymphocytes, macrophages and dendritic cells influx to spleen of the immunized mice. By intradermal (Gene Gun) route, the immunizations with pCDNA3-hsp65 and pCDNA3-hsp65G induced high anti-Hsp65 IgG, lgG1 and lgG2a antibody titles. These DNA constructs also triggered specific cellular immune response. Spleen and lymph node cells from mice immunized with leveis pCDNA3-hsp65 and pCDNA3-hsp65G proliferated significantly with recombinant Hsp65. The leveis of IL-4, IL-10, IFN-γ and IL-12 antigen-specific was high, characterizing Th1/Th2 profile, but tending to Th2 response. These data correlated to high lgG1 and lgG2a antibody titles. Also, it was found that there is a influx of macrophages, dendritic cells and lymphocytes B, T, T CD4+ and T CD8+ into mice spleens in relation to control groups (pCDNA3 and non immunized). However, the humoral and cellular immune response were not evoked by pCDNA3-hsp65 was higher than that one triggered by pCDNA3-hsp65G. The pCDNA3-N, pCDNA3-N\\DS and pCDNA3-N59 did not induce specific immune response. By intramuscular route, pCDNA3-hsp65, pCDNA3-hsp65G and pCDNA3-N induced high anti-Hsp65 IgG, lgG1 and lgG2a, cellular proliferation, significant cytokine leveis and increase of macrophages, dendritic cells, lymphocytes influx comparing to contrais (pCDNA3 and non immunized). Th1 response was evoked by these DNA constructs because it was detected high IFN-y and IL-12 leveis and lgG2a titles. It not found differences in humoral and cellular immune response evoked by pCDNA3-hsp65 and pCDNA3-hsp65G. The pCDNA3-DS and pCDNA3-N59 were not immunogenic. These results indicated that the absence of downstream fragment interfere significantly in immune response activation, when mice were immunized by intradermal route with pCDNA3-hsp65, construct that not contain downstream fragment. This suggest that there is a putative participation of CpG motifs, contained into downstream fragment, to induce immune response, that was decreased when this fragment was excised. By the other hand, by intramuscular route, the downstream fragment interfere in immune response triggering. It is believed that these differences in role of downstream fragment is related with DNA quantity injected into mice and administration route. (AU)