SUMMARYA 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-c recovery and reduction of lung injury. There was also partial restoration of the production of tumour necrosis factor-a. Treatment with pcDNA3 vector also induced an immune stimulatory effect. However, only infected pHSP65-treated mice were able to produce significant levels of interferon-c and to restrict the growth of bacilli.

Tuberculosis (TB) remains a threat for public health, killing around 3 million people a year. Despite the fact that most cases can be cured with antibiotics, the treatment is long and patients relapse if chemotherapy is not continued for at least 6 months. Thus, a better characterization of the working principles of the immune system in TB and identification of new immunotherapeutic products for the development of shorter regimens of treatment are essential to achieve an effective management of this disease. In the present work, we demonstrate that immunotherapy with a plasmid DNA encoding the Mycobacterium leprae 65 kDa heat-shock protein (hsp65 ) in order to boost the efficiency of the immune system, is a valuable adjunct to antibacterial chemotherapy to shorten the duration of treatment, improve the treatment of latent TB infection and be effective against multidrug-resistant bacilli (MDR-TB). We also showed that the use of DNA-hsp65 alone or in combination with other drugs influence the pathway of the immune response or other types of inflammatory responses and should augment our ability to alter the course of immune response/inflammation as needed, evidencing an important target for immunization or drug intervention. Gene Therapy (2005) 12, 281-287.

Background: The greatest challenges in vaccine development include optimization of DNA vaccines for use in humans, creation of effective single-dose vaccines, development of delivery systems that do not involve live viruses, and the identification of effective new adjuvants. Herein, we describe a novel, simple technique for efficiently vaccinating mice against tuberculosis (TB). Our technique consists of a single-dose, genetic vaccine formulation of DNA-hsp65 complexed with cationic liposomes and administered intranasally.

The high incidence of tuberculosis around the world and the inability of BCG to protect certain populations clearly indicate that an improved vaccine against tuberculosis is needed. A single antigen, the mycobacterial heat shock protein hsp65, is sufficient to protect BALB/c mice against challenge infection when administered as DNA vaccine in a threedose-based schedule. In order to simplify the vaccination schedule, we coencapsulated hsp65-DNA and trehalose dimicolate (TDM) into biodegradable poly(DL-lactide-co-glycolide) (PLGA) microspheres. BALB/c mice immunized with a single dose of DNA-hsp65/TDM-loaded microspheres produced high levels of IgG2a subtype antibody and high amounts of IFN-g in the supernatant of spleen cell cultures. DNA-hsp65/TDM-loaded microspheres were also able to induce high IFN-g production in bulk lung cells from challenged mice and confer protection as effective as that attained after three doses of naked DNA administration. This new formulation also allowed a ten-fold reduction in the DNA dose when compared to naked DNA. Thus, this combination of DNA vaccine and adjuvants with immunomodulatory and carrier properties holds the potential for an improved vaccine against tuberculosis. Gene Therapy (2003) 10, 678-685.

Host cell invasion by Toxoplasma gondii is a multistep process with one of the first steps being the apical release of micronemal proteins that interact with host receptors. We demonstrate here that micronemal protein 1 (MIC1) is a lactose-binding lectin. MIC1 and MIC4 were recovered in the lactose-eluted (Lac(+)) fraction on affinity chromatography on immobilized lactose of the soluble antigen fraction from tachyzoites of the virulent RH strain. MIC1 and MIC4 were both identified by N-terminal microsequencing. MIC4 was also identified by sequencing cDNA clones isolated from an expression library following screening with mouse polyclonal anti-60/70 kDa (Lac(+) proteins) serum. This antiserum localized the Lac(+) proteins on the apical region of T. gondii tachyzoites by confocal microscopy. The Lac(+) fraction induced hemagglutination (mainly type A human erythrocytes), which was inhibited by beta-galactosides (3 mM lactose and 12 mM galactose) but not by up to 100 mM melibiose (alpha-galactoside), fucose, mannose, or glucose or 0.2 mg/ml heparin. The lectin activity of the Lac(+) preparation was attributed to MIC1, because blotted MIC1, but not native MIC4, bound human erythrocyte type A and fetuin. The copurification of MIC1 and MIC4 may have been due to their association, as reported by others. These data suggest that MIC1 may act through its lectin activity during T. gondii infection.