Clin Vaccine Immunol. 2013 Aug;20(8):1162-9.

A single dose of a DNA vaccine encoding Apa co-encapsulated with 6,6′-trehalose dimycolate in microspheres conferred long-term protection against tuberculosis in BCG-primed mice.

Carlétti D, Morais da Fonseca D, Gembre AF, Masson AP, Weijenborg Campos L, Leite LC, Rodrigues Pires A, Lannes-Vieira J, Lopes Silva C, Bonato VL, Horn C.

Laboratory of Immunology and Immunogenetics, Evandro Chagas Clinic Research Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.

 

ABSTRACT:

BCG-prime DNA (Mycobacterium tuberculosis genes)-booster vaccinations have been shown to induce greater protection against tuberculosis (TB) than BCG alone. This heterologous prime-boost strategy is perhaps the most realistic vaccination for the future of TB infection control, especially in endemic countries. Moreover, prime-boost regimen by using biodegradable microspheres seems to be a promising immunization to stimulate long lasting immune response. The alanine proline antigen (Apa) is a highly immunogenic glycoprotein secreted by M. tuberculosis. This study investigated the immune protection of Apa-DNA vaccine against intratracheal M. tuberculosis challenge in mice on the basis of a heterologous prime-boost regimen. BALB/c mice were subcutaneously primed with BCG and intramuscularly boosted with a single dose of plasmid encoding apa and 6,6′-trehalose dimycolate (TDM) adjuvant, co-encapsulated in microspheres (BCG-APA), and were evaluated 30 and 70 days after challenge. This prime-boost strategy (BCG-APA) resulted in a significant reduction in the bacterial load in the lungs, thus leading to better reservation of the lung parenchyma, 70 days post-infection compared to BCG vaccinated mice. The profound effect of this heterologous prime-boost regimen in experimental model supports its development as a feasible strategy for prevention of TB.

PMID: 23740922

 

SUPPLEMENT:

Tuberculosis (TB) causes more than 1,5 million deaths per year worldwide, affecting mostly young adults in their most productive years. 95% of TB deaths are in developing world. The high incidence of disease and the controversy effectiveness of BCG vaccine in adults indicate the need for a new vaccine to control TB. BCG vaccination confers protection in children and newborns for developing the most severe forms of disease and for these reasons its use is recommended and should not be discontinued in TB endemic countries.

Heterologous prime-boost vaccines are proposed to enhance specific immunity primed by childhood BCG vaccination and are one of the best strategies for introducing new vaccines into human populations. Specifically, DNA-based vaccines could be a good choice for improving BCG efficacy because they can stimulate both humoral and cell-mediated immunity.

Secreted proteins are considered the main targets for TB vaccines. Apa is one of the major immunodominant antigen secreted by the Mycobacterium tuberculosis complex. This glycoprotein is a fibronectin attachment protein (FAP) and can mediate mycobacterial binding to host cells as a potential adhesin. Moreover, an Apa-encoding DNA vaccine (Apa DNA) induces strong Th1 and Th2 responses and is capable of activating significant protective responses in experimental TB.

Adjuvants and delivery systems represent essential components of successful vaccines, and often a combination is required to elicit an optimal immune response. 6,6’-Trehalose dimycolate (TDM) is a glycolipid that is present in M. tuberculosis cell wall and acts as a potent immunomodulator, preferentially inducing Th1 responses, thus making it an interesting adjuvant for TB vaccines.

Delivery systems such as antigen-encapsulated microspheres enhance the binding, uptake, and half-life of antigens leading to a reduction in the number of doses in the immunization schedule. The slow degradation of these microspheres allows sustained delivery of the antigen. Moreover, biodegradable microspheres have an extensive record of safe use in medicine. To further improve the effectiveness of Apa vaccine, TDM was added in the vaccine formulation and the complex was coencapsulated in biodegradable microspheres.

Thus, our goal was test heterologous vaccination by priming with BCG and boosting with a single dose of Apa DNA vaccine, containing TDM adjuvant, coencapsulated in biodegradable microspheres. Great reduction of bacterial load and preservation of lung parenchyma was achieved by using heterologous immunization with Apa-DNA and conferred long term protection against experimental TB. The advantageous properties of the microsphere-based delivery system likely account for the superior protection obtained in this study, and most importantly, this protection was accomplished by a boosting immunization with only one dose of pVAXapa-TDM-Me. These findings strongly support further evaluation of Apa-based vaccine to prevent TB.

 

References

  1. Horn C, Namane A, Pescher P, Riviere M, Romain F, Puzo G, Barzu O, Marchal G. 1999. Decreased capacity of recombinant 45/47-kDa molecules (Apa) of Mycobacterium tuberculosis to stimulate T lymphocyte responses related to changes in their mannosylation pattern. J. Biol. Chem. 32030.
  2. Romain F, Horn C, Romain F, Horn C, Pescher P, Namane A, Riviere M, Puzo G, Barzu O, Marchal G. 1999. Deglycosylation of the 45/47-kilodalton antigen complex of Mycobacterium tuberculosis decreases its capacity to elicit in vivo or in vitro cellular immune responses. Infect Immun 67(11):5567-72.
  3. Garapin A, Ma L, Pescher P, Lagranderie M, Marchal G. 2001. Mixed immune response induced in rodents by two naked DNA genes coding for mycobacterial glycosylated proteins. Vaccine 19:2830–2841.
  4. Sable SB, Cheruvu M, Nandakumar S, Sharma S, Bandyopadhyay K, Kellar KL, Posey JE, Plikaytis BB, Amara RR, Shinnick TM. 2011. Cellular immune responses to nine Mycobacterium tuberculosis vaccine candidates following intranasal vaccination. PLoS One 6:(7)e22718.

 

Acknowledgements

We thank Eliane N. Miyage, Butantan Institute, São Paulo, Brazil, for technical assistance in the gene expression studies. We also thank Elizabeth Albuquerque, Fiocruz, for helpful discussions on statistical analysis. We are grateful to Marcos de Almeida and Francisco Carvalho from the Pathology Service, IPEC-Fiocruz, for their technical assistance in histological sections. This study was supported by grants from Fiocruz, Rio de Janeiro, Brazil, and USP, Ribeirão Preto, São Paulo, Brazil.

Contact

Cynthia Horn – PhD
Titular Researcher
FIOCRUZ, INI/Ipec, Laboratory of Immunology and Immunogenetics
cynthia.horn@ipec.fiocruz.br

fig1

Figure 1. Design of protection against experimental TB conferred by heterologous prime-boost regimen. The time points for immunizations, infection, and sample collection are shown. BALB/c mice were immunized subcutaneously (s.c.) with a single dose of BCG (5 x 105 CFU; BCG group) or with one dose of BCG administered s.c. followed by one dose of pVAX-TDM-Me (BCG-PVAX) or pVAXapa-TDM-Me (BCG-APA) administered intramuscularly (i.m.) after 30 days. Thirty days after vaccination, mice were challenged intratracheally with a virulent strain of M. tuberculosis (1 x 105 CFU). At 30 or 70 days after infection, the lungs were processed to determine bacterial loads by counting the CFU and hematoxylin-eosin (HE) staining.

 

fig2Figure 2. Evaluation of lungs from immunized mice at 30 and 70 days after M. tuberculosis infection. At 30 (A, above) or 70 (B, below) days after intratracheal infection. BALB/c’s lungs were processed to determine bacterial loads (CFU assay) or fixed and stained with Haematoxilin-Eosin. Overview of lung tissues is shown below the images with higher magnification (x100). Bacterial load is expressed as log10 CFU/g of lung, derived from the means ± SD of serial dilutions individually counted for each group. P values indicating statistically significant differences among the groups are displayed in the representative graphs. *, P < 0.05 versus nonimmunized infected mice (TB group) at 30 days postinfection. The following symbols indicate significance at 70 days postinfection: *, P < 0.001 versus the TB group; X, P < 0.05 versus the BCG-PVAX group; and +, P < 0.005 versus the BGG-vaccinated group. Results of one representative experiment out of three independent experiments are shown.

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