Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system

Journal of Computer Science & Systems Biology- Open Access www.omicsonline.com Research Article In Silico Prediction of the Tertiary Structure of M. leprae Hsp65 Protein Shows an Unusual Structure in Carboxy-terminal Region

Copyright: © 2008 Rossetti RAM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. DNA vaccines have been used with great success in experimental and some clinical therapy. However, the mechanisms of activation of the immune system by these vaccines are not utterly understood yet. Hsp65 is a Mycobacterium leprae chaperone whose gene has been efficiently used as experimental DNA vaccine against tuberculosis and clinical trial against tumor. Since little is know about the three-dimensional (3D) structure of hsp65 and modeling of 3D protein structure can increase the information to improve the knowledge about the mechanism action as well as the design of new DNA vaccine formulation, here we used the bioinformatics to get the design in silico of hsp65 (heat shock protein) molecule. The determination of hsp65 3D structure was obtained by homology using the software Modeller (Eswar et al., 2001). It was used two proteins as models: 1SJP, a 60kDa chaperonin from Mycobacterium tuberculosis in the PDB, and the 1WE3, the crystal structure of the chaperoni

Immune regulatory effect of pHSP65 DNA therapy in pulmonary tuberculosis: activation of CD8(+) cells, interferon-gamma recovery and reduction of lung injury

A DNA vaccine based on the heat-shock protein 65 Mycobacterium leprae gene (pHSP65) presented a prophylactic and therapeutic effect in an experimental model of tuberculosis. In this paper, we addressed the question of which protective mechanisms are activated in Mycobacterium tuberculosis-infected mice after immune therapy with pHSP65. We evaluated activation of the cellular immune response in the lungs of infected mice 30 days after infection (initiation of immune therapy) and in those of uninfected mice. After 70 days (end of immune therapy), the immune responses of infected untreated mice, infected pHSP65-treated mice and infected pCDNA3-treated mice were also evaluated. Our results show that the most significant effect of pHSP65 was the stimulation of CD8(+) lung cell activation, interferon-gamma recovery and reduction of lung injury. There was also partial restoration of the production of tumour necrosis factor-alpha. Treatment with pcDNA3 vector also induced an immune stimulatory effect. However, only infected pHSP65-treated mice were able to produce significant levels of interferon-gamma and to restrict the growth of bacilli

Immunomodulation and protection induced by DNA-hsp65 vaccination in an animal model of arthritis

We described a prophylactic and therapeutic effect of a DNA vaccine encoding the Mycobacterium leprae 65- kDa heat shock protein (DNA-hsp65) in experimental murine tuberculosis. However, high homology of the vaccine to the corresponding mammalian hsp60, together with the CpG motifs in the plasmidial vector, could trigger or exacerbate an autoimmune disease. in the present study, we evaluate the potential of DNA- hsp65 vaccination to induce or modulate arthritis in mice genetically selected for acute inflammatory reaction (AIR), either maximal (AIRmax) or minimal (AIRmin). Mice immunized with DNA-hsp65 or injected with the corresponding DNA vector (DNAv) developed no arthritis, whereas pristane injection resulted in arthritis in 62% of AIRmax mice and 7.3% of AIRmin mice. Administered after pristane, DNA- hsp65 downregulated arthritis induction in AIRmax animals. Levels of interleukin (IL)- 12 were significantly lower in mice receiving pristane plus DNA- hsp65 or DNAv than in mice receiving pristane alone. However, when mice previously injected with pristane were inoculated with DNA- hsp65 or DNAv, the protective effect was significantly correlated with lower IL-6 and IL-12 levels and higher IL-10 levels. Our results strongly suggest that DNA-hsp65 has no arthritogenic potential and is actually protective against experimentally induced arthritis in mice.Universidade Federal de São Paulo, Dept Bioquim & Imunol, Ctr Pesquisa TB, Fac Med Ribeirao Preto, BR-14049900 Ribeirao Preto, SP, BrazilUniv Estadual Paulista Julio Mesquita Filho, Inst Biociencias, BR-18618000 Botucatu, SP, BrazilInst Butantan, Lab Imunogenet, BR-05503900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim & Imunol, Ctr Pesquisa TB, Fac Med Ribeirao Preto, BR-14049900 Ribeirao Preto, SP, BrazilWeb of Scienc