Recombinant adenovirus delivery of calreticulin-ESAT-6 produces an antigen-specific immune response but no protection against a Mycobacterium tuberculosis challenge.

Bacillus Calmette-Guerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT-6 and CFP10 provides protection against M. tuberculosis in a murine infection model. Further, previous studies have shown that calreticulin increases the cell-mediated immune responses to antigens. Therefore, to test whether calreticulin enhances the immune response against M. tuberculosis antigens, we fused ESAT-6 to calreticulin and constructed a recombinant replication-deficient adenovirus to express the resulting fusion protein (AdCRT-ESAT-6). The adjuvant effect of calreticulin was assayed by measuring cytokine responses specific to ESAT-6. Recombinant adenovirus expressing the fusion protein produced higher levels of interferon-γ and tumour necrosis factor-α in response to ESAT-6. This immune response was not improved by the addition of CFP-10 to the CRT-ESAT-6 fusion protein (AdCRT-ESAT-6-CFP10). Mice immunized with these recombinant adenoviruses did not decrease the mycobacterial burden after low-dose aerosol infection with M. tuberculosis. We conclude that calreticulin can be used as an adjuvant to enhance the immune response against mycobacterial antigens, but it is not enough to protect against tuberculosis.

Safety, pharmacokinetics, and tissue distribution of liposomal P-ethoxy antisense oligonucleotides targeted to Bcl-2.

Antisense oligonucleotides (oligos) have the ability to selectively block disease-causing genes, thereby inhibiting production of disease-associated proteins. However, their effectiveness has been limited by their low intracellular delivery. We had previously demonstrated that liposomes could increase the intracellular uptake of P-ethoxy oligos, hydrophobic analogs of phosphodiesters, and that liposomal Bcl-2 P-ethoxy antisense oligos (L-Bcl-2) could selectively inhibit Bcl-2 protein production, thereby inducing growth inhibition in Follicular Lymphoma cell lines. To understand the in vivo behavior of L-Bcl-2, we conducted a series of studies to evaluate the safety, pharmacokinetics, and tissue distribution of i.v. injections of L-Bcl-2 in normal rodents. Daily administration of 20 mg of L-Bcl-2/kg of body weight in 5 consecutive days had no adverse effects on renal or hepatic functions, nor on hematological parameters. Histopathology also did not reveal any significant changes in the morphology of the organs studied. In rats, the area under the curve of L-Bcl-2 reflects a two-compartment model of distribution with a biphasic plasma clearance. The T(1/2alpha) and T(1/2beta) were approximately 8 min and 4.2 h, respectively, and the V(d) was 79 ml, indicating a broad body distribution. The highest concentrations of L-Bcl-2 were found in spleen > liver > kidneys. These studies showed that in the schedules studied no significant toxicity associated with L-Bcl-2 was observed over 6 weeks, and that L-Bcl-2 could be widely distributed in the body.