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.

Immune response induced by three Mycobacterium bovis BCG substrains with diverse regions of deletion in a C57BL/6 mouse model.

This study was performed to examine the adaptive immune response generated by three Mycobacterium bovis bacillus Calmette-Guérin (BCG) substrains to determine if the number of genomic regions of deletion played a significant role in determining the magnitude of the immune response or affected their ability to reduce the bacterial burden following low-dose aerosol challenge with a virulent M. tuberculosis strain. BCG Connaught, Pasteur, and Sweden were chosen as representative substrains, as they possessed many, intermediate, and few regions of deletion, respectively, as a result of changes in the genome in various regions. Mice were vaccinated subcutaneously and were then examined at 14, 21, and 42 days postvaccination. BCG was observed in the spleen, lung, and lymph nodes. BCG Connaught induced a greater pulmonary T-cell response than the other two substrains at day 14 postvaccination, although by 42 days postvaccination activated T-cell levels dropped to the levels observed in control mice for all three substrains. Among the three substrains, BCG Connaught induced significantly greater levels of interleukin-12 in bone marrow-derived macrophage cultures. Mice challenged at days 14, 21, and 42 postvaccination displayed an equal capacity to reduce the bacterial burden in the lungs and spleen. The data provide evidence that although the BCG substrains generated qualitatively and quantitatively different immune responses, they induced similar reductions in the bacterial burden against challenge with a virulent M. tuberculosis strain in the mouse model of tuberculosis. The data raise questions about the assessment of vaccine immune responses and the relationship to a vaccine's ability to reduce the bacterial burden.