Improved Immunogenicity and Protective Efficacy of a Tuberculosis DNA Vaccine Encoding Ag85 by Protein Boosting

Abstract

C57BL/6 mice were vaccinated with plasmid DNA encoding Ag85 from Mycobacterium tuberculosis, with Ag85 protein in adjuvant, or with a combined DNA prime-protein boost regimen. While DNA immunization, as previously described, induced robust Th1-type cytokine responses, protein-in-adjuvant vaccination elicited very poor cytokine responses, which were 10-fold lower than those observed with DNA immunization alone. Injection of Ag85 DNA-primed mice with 30 to 100 g of purified Ag85 protein in adjuvant increased the interleukin-2 and gamma interferon (IFN-␥) response in spleen two-to fourfold. Further, intracellular cytokine analysis by flow cytometry also showed an increase in IFN-␥-producing CD4 ؉ T cells in DNA-primedprotein-boosted animals, compared to those that received only the DNA vaccination. Moreover, these responses appeared to be better sustained over time. Antibodies were readily produced by all three methods of immunization but were exclusively of the immunoglobulin G1 (IgG1) isotype following protein immunization in adjuvant and preferentially of the IgG2a isotype following DNA and DNA prime-protein boost vaccination. Finally, protein boosting increased the protective efficacy of the DNA vaccine against an intravenous M. tuberculosis H37Rv challenge infection, as measured by CFU or relative light unit counts in lungs 1 and 2 months after infection. The capacity of exogenously given protein to boost the DNA-primed vaccination effect underlines the dominant role of Th1-type CD4؉ helper T cells in mediating protection.Tuberculosis (TB) remains a major health problem affecting millions of people worldwide. The only TB vaccine presently available is an attenuated strain of Mycobacterium bovis termed M. bovis BCG. The efficacy of BCG remains controversial, particularly against pulmonary TB in young adults (5), and development of a better vaccine is urgently needed to counter the global threat of this disease (22).Secreted and surface-exposed cell wall proteins are major antigens recognized by the protective immune response against TB and immunization with whole-culture filtrate, a rich source of these extracellular proteins, can protect mice and guinea pigs to some extent against subsequent challenge with the tubercle bacillus (1, 14, 15). A major portion of the secreted proteins in Mycobacterium tuberculosis and BCG culture filtrate is formed by the Ag85 complex, a 30-to 32-kDa family of three proteins (Ag85A, Ag85B, and Ag85C) (38) which all possess a mycoloyltransferase enzyme activity required for the biogenesis of cord factor (4), a dominant structure necessary for maintaining cell wall integrity (19,29). Ag85 complex induces strong T-cell proliferation and gamma interferon (IFN-␥) production in most healthy individuals infected with M. tuberculosis and/or Mycobacterium leprae (24) and in BCGvaccinated mice (16), making it a promising candidate as a protective antigen. Vaccination with naked plasmid DNA encoding Ag85A and Ag85B can stimulate strong humoral and cell-mediated immune responses and confer si...

DNA Vaccines: Progress and Challenges

“…Therefore, various approaches were tested that sought to take advantage of combining the ability of DNA to prime Ab responses with the ability of recombinant proteins to boost them. DNA-protein primeboost regimens have been studied extensively in HIV (58), providing partial protection from simian-human immunodeficiency virus challenge (59) and also have been studied in anthrax (60), tuberculosis (61), and in transmission-blocking vaccines for both vivax and falciparium malaria (62).…”

Therapeutic effect of DNA vaccines combined with chemotherapy in a latent infection model after aerosol infection of mice with Mycobacterium tuberculosis

“…Prophylactic DNA vaccines expressing M. tuberculosis antigens or cytokines are of particular interest, because of their efficiency and long-lasting effect in animal TB models. For example, prophylactic DNA vaccines expressing various M. tuberculosis antigens, including Ag85A, [15][16][17] Ag85B, 17,18 65 kDa heat shock protein, 19,20 and PstS-3 21 , were found to be effective at limiting the growth of M. tuberculosis in mice. In contrast, there has been little evidence that DNA vaccines are useful as therapeutic vaccines against TB.…”

Protective effect of DNA vaccine during chemotherapy on reactivation and reinfection of Mycobacterium tuberculosis

“…In prophylactic settings, there are several reports that DNA vaccines expressing M. tuberculosis antigens are effective for limiting the bacterial growth in mice. [10][11][12][13] However, it is still controversial whether DNA vaccines work against TB reactivation in postexposure models. [14][15][16] For example, the vaccination of plasmid DNA expressing hsp65 after completion of chemotherapy was shown to be effective in preventing the reactivation of intravenously infected M. tuberculosis.…”

“…Firstly, it has been reported that the Ag85A epitopes are frequently expressed on the surface of the infected macrophages during early phase, but downregulated at the late phase, while PstS-3-specific epitopes are more abundantly expressed during late phase. 13 Thus, DNA immunization with combination of these two genes might induce both Ag85A-and PstS-3-specific immune responses required for suppressing bacterial growth during both early and late phases. Secondly, it has also been known that CD4 + T cells play an important role in the protection for the acute Figure 2 Preventive effect of DNA vaccines given during chemotherapy on reinfection of M. tuberculosis.…”