Neonatal BCG immunization followed by DNAhsp65 boosters: highly immunogenic but not protective against tuberculosis - a paradoxical effect of the vector?

A new tuberculosis vaccine is urgently needed. Prime-boost strategies are considered very promising and the inclusion of BCG is highly desirable. In this investigation, we tested the protective efficacy of BCG delivered in the neonatal period followed by boosters in the adult phase with a DNA vaccine containing the hsp65 gene from Mycobacterium leprae (pVAXhsp65). Immune responses were characterized by serum anti-hsp65 antibody levels and IFN-gamma and IL-5 production by the spleen. Amounts of these cytokines were also determined in lung homogenates. Protective efficacy was established by the number of colony-forming units (CFU) and histopathological analysis of the lungs after challenge with Mycobacterium tuberculosis. Immunization with BCG alone triggered a significant reduction of CFU in the lungs and also clearly preserved the pulmonary parenchyma. BCG priming also increased the immunogenicity of pVAXhsp65. However, boosters with pVAXhsp65 or the empty vector abolished the protective efficacy of BCG. Also, higher IL-5 levels were produced by spleen and lungs after DNA boosters. These results demonstrated that neonatal BCG immunization followed by DNAhsp65 boosters is highly immunogenic but is not protective against tuberculosis.

Anal cytology in patients with AIDS.

The objective of the present study was to study the prevalence of abnormal anal cytology in patients with AIDS. Anal smears, obtained with a cytobrush, of 102 HIV-positive patients of the Emilio Ribas Institute (Sao Paulo, Brazil) were collected, and only after that, the patients were submitted to anoscopy. Thirty-two patients had LSIL and 14 others had HSIL. Squamous intra-epithelial lesions were also observed in 38% of the patients without condyloma (18/47): in 9 of the 33 patients without history of condyloma (27%) and in 9 of the 14 patients who had previously treated condyloma (64%). An invasive squamous cell carcinoma was observed in one patient without history of condyloma. In all 13 patients with HSIL, biopsies guided by high resolution anoscopy confirmed high grade dysplasia. Our findings suggest that anal cytology is mandatory in AIDS even in patients without macroscopic anal lesions or without previous history of anal condyloma.

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.