Incorporation of immunostimulatory motifs in the transcribed region of a plasmid DNA vaccine enhances Th1 immune responses and therapeutic effect against Mycobacterium tuberculosis in mice.

T-helper type 1 (Th1) immune response is involved in the development of protective immunity against Mycobacterium tuberculosis. Thus, an increase in Th1 and cellular immune responses should lead to enhanced anti-mycobacterial activity. In this study, we aimed to improve Th1 immune responses to a DNA vaccine by adding potentially immunostimulatory nucleotide sequences into the transcribed region downstream of the antigen. The Mycobacterium leprae gene for hsp65, codon-optimized for expression in mammalian cells, was inserted into pVAX1 with and without 3'-sequences containing CpG and dsRNA motifs. When the plasmid contained both motifs, transfected murine macrophage-like RAW264.7 cells showed markedly increased levels of mRNA for immune molecules of Th1 (IFN-α, IL-12) and Th17 (IL-17, IL-23 and IL-6) responses and for T cell co-stimulatory molecules (CD80 and CD86) but not for a Th2 response (IL-4 and IL-10). Immunized mice showed substantially increased serum anti-Hsp65 IgG2a antibody levels and IFN-γ production by spleen cells, confirming enhancement of the Th1 response in vivo. Furthermore, when non-vaccinated mice were infected with H37Rv by low-dose aerosol challenge, and then 4 weeks later were treated with plasmids by intramuscular injection, the mice that had been treated with plasmids containing immunostimulatory motifs showed an enhanced reduction in mycobacterial loads in lung and spleen. We conclude that DNA vaccines may be made more highly immunogenic and more effective for treatment by including transcribed stimulatory sequences.

A subunit vaccine based on biodegradable microspheres carrying rHsp65 protein and KLK protects BALB/c mice against tuberculosis infection.

Of the hundreds of new tuberculosis (TB) vaccine candidates, some have therapeutic value in addition to their prophylactic properties. This is the case for the DNA vaccine encoding heat-shock protein 65 (DNAhsp65) from Mycobacterium leprae. However, there are concerns about the use of DNA vaccines in certain populations such as newborns and pregnant women. Thus, the optimization of vaccination strategies that circumvent this limitation is a priority. This study evaluated the efficacy of a single dose subunit vaccine based on recombinant Hsp65 protein against infection with M. tuberculosis H37Rv. The Hsp65 protein in this study was either associated or not with immunostimulants, and was encapsulated in biodegradable PLGA microspheres. Our results demonstrate that the protein was entrapped in microspheres of adequate diameter to be engulfed by phagocytes. Mice vaccinated with a single dose of Hsp65-microspheres or Hsp65+CpG-microspheres developed both humoral and cellular-specific immune responses. However, they did not protect mice against challenge with M. tuberculosis. By contrast, Hsp65+KLK-microspheres induced specific immune responses that reduced bacilli loads and minimized lung parenchyma damage. These data suggest that a subunit vaccine based on recombinant protein Hsp65 is feasible.

Vaccination with a Sindbis virus-based DNA vaccine expressing antigen 85B induces protective immunity against Mycobacterium tuberculosis.

To improve DNA vaccination against Mycobacterium tuberculosis, we evaluated the effectiveness of a Sindbis virus-based DNA construct expressing the tuberculosis antigen 85B (Sin85B). The protective efficacy of Sin85B was initially assessed by aerogenically challenging immunized C57BL/6 mice with virulent Mycobacterium tuberculosis. At 1 and 7 months postinfection, the lung bacterial burdens were considerably reduced and the lung pathology was improved in vaccinated mice compared to naive controls. Furthermore, the mean survival period for Sin85B-immunized mice (305 +/- 9 days) after the tuberculous challenge was extended 102 days relative to the naive mice (203 +/- 13 days) and was essentially equivalent to the survival time of Mycobacterium bovis BCG-vaccinated mice (294 +/- 15 days). The essential role of gamma interferon (IFN-gamma) in Sin85B-mediated protection was established by showing that significantly increased levels of IFN-gamma mRNA were present postinfection in lung cells from vaccinated mice relative to control mice and by demonstrating that IFN-gamma depletion prior to challenge abolished the vaccine-induced protection. The substantial antituberculosis protective responses induced by Sin85B immunization of CD4-/- mice strongly suggested that CD8 cells partially mediate Sin85B-induced protective immunity. Interestingly, Sin85B vaccination did not protect RNase L-/- (a key enzyme in the innate antiviral response) mice while significant protection was detected in RNase L-/- mice immunized with either BCG or a conventional DNA plasmid expressing antigen 85B. These data show that immunization with Sin85B offers protection similar to BCG in a murine model of pulmonary tuberculosis and suggest that Sin85B-induced protection is dependent upon both innate and acquired immune mechanisms.