{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,1]],"date-time":"2026-07-01T19:47:18Z","timestamp":1782935238362,"version":"3.54.5"},"reference-count":53,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2014,3,5]],"date-time":"2014-03-05T00:00:00Z","timestamp":1393977600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/3.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>The attenuated live M. bovis Bacille-Calmette-Gu\u00e9rin (BCG) is still the sole vaccine used against tuberculosis, but confers only variable efficacy against adult pulmonary tuberculosis (TB). Though no clear explanation for this limited efficacy has been given, different hypotheses have been advanced, such as the waning of memory T-cell responses, a reduced antigenic repertoire and the inability to induce effective CD8+ T-cell responses, which are known to be essential for latent tuberculosis control. In this study, a new  BCG-based vaccination protocol was studied, in which BCG was formulated in combination with a plasmid DNA vaccine. As BCG is routinely administered to neonates, we have evaluated a more realistic approach of a simultaneous intradermal coadministration of BCG with pDNA encoding the prototype antigen, PPE44. Strongly increased T- and B-cell responses were observed with this protocol in C57BL\/6 mice when compared to the administration of only BCG or in combination with an empty pDNA vector, as measured by Th1-type spleen cell cytokine secretion, specific IgG antibodies, as well as specific IFN-\u03b3 producing\/cytolytic-CD8+ T-cells. Moreover, we observed a bystander activation induced by the coding plasmid, resulting in increased immune responses against other  non-plasmid encoded, but BCG-expressed, antigens. In all, these results provide a proof of concept for a new TB vaccine, based on a BCG-plasmid DNA combination.<\/jats:p>","DOI":"10.3390\/vaccines2010181","type":"journal-article","created":{"date-parts":[[2014,3,5]],"date-time":"2014-03-05T11:48:39Z","timestamp":1394020119000},"page":"181-195","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Increasing the Vaccine Potential of Live M. bovis BCG by Coadministration with Plasmid DNA Encoding a Tuberculosis Prototype Antigen"],"prefix":"10.3390","volume":"2","author":[{"given":"Nicolas","family":"Bruffaerts","sequence":"first","affiliation":[{"name":"Scientific Institute of Public Health, Communicable and Infectious Diseases, Immunology, Brussels 1180, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marta","family":"Romano","sequence":"additional","affiliation":[{"name":"Scientific Institute of Public Health, Communicable and Infectious Diseases, Immunology, Brussels 1180, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Olivier","family":"Denis","sequence":"additional","affiliation":[{"name":"Scientific Institute of Public Health, Communicable and Infectious Diseases, Immunology, Brussels 1180, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fabienne","family":"Jurion","sequence":"additional","affiliation":[{"name":"Scientific Institute of Public Health, Communicable and Infectious Diseases, Immunology, Brussels 1180, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kris","family":"Huygen","sequence":"additional","affiliation":[{"name":"Scientific Institute of Public Health, Communicable and Infectious Diseases, Immunology, Brussels 1180, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2014,3,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1173","DOI":"10.1016\/S0140-6736(06)68507-3","article-title":"Effect of BCG vaccination on childhood tuberculous meningitis and miliary tuberculosis worldwide: A meta-analysis and assessment of cost-effectiveness","volume":"367","author":"Trunz","year":"2006","journal-title":"Lancet"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1339","DOI":"10.1016\/S0140-6736(95)92348-9","article-title":"Variation in protection by BCG: Implications of and for heterologous immunity","volume":"346","author":"Fine","year":"1995","journal-title":"Lancet"},{"key":"ref_3","unstructured":"World Health Organization (2013). Global Tuberculosis Report."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Weir, R.E., Gorak-Stolinska, P., Floyd, S., Lalor, M.K., Stenson, S., Branson, K., Blitz, R., Ben-Smith, A., Fine, P.E., and Dockrell, H.M. (2008). Persistence of the immune response induced by BCG vaccination. BMC Infect. Dis., 8.","DOI":"10.1186\/1471-2334-8-9"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"821","DOI":"10.1111\/j.1574-6976.2008.00118.x","article-title":"Influence of BCG vaccine strain on the immune response and protection against tuberculosis","volume":"32","author":"Ritz","year":"2008","journal-title":"FEMS Microbiol. Rev."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"3523","DOI":"10.1128\/IAI.01999-06","article-title":"Lack of immune responses to Mycobacterium tuberculosis DosR regulon proteins following Mycobacterium bovis BCG vaccination","volume":"75","author":"Lin","year":"2007","journal-title":"Infect. Immun."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"390","DOI":"10.1111\/j.1365-2249.2006.03222.x","article-title":"The influence of previous exposure to environmental mycobacteria on the interferon-gamma response to bacille Calmette-Guerin vaccination in southern England and northern Malawi","volume":"146","author":"Weir","year":"2006","journal-title":"Clin. Exp. Immunol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"7172","DOI":"10.4049\/jimmunol.0801694","article-title":"Antigen load governs the differential priming of CD8 T cells in response to the bacille Calmette Guerin vaccine or Mycobacterium tuberculosis infection","volume":"182","author":"Ryan","year":"2009","journal-title":"J. Immunol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"3689","DOI":"10.1002\/1521-4141(200012)30:12<3689::AID-IMMU3689>3.0.CO;2-4","article-title":"Control of latent Mycobacterium tuberculosis infection is dependent on CD8 T cells","volume":"30","author":"Cassidy","year":"2000","journal-title":"Eur. J. Immunol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"830","DOI":"10.1128\/IAI.66.2.830-834.1998","article-title":"Protection against Mycobacterium tuberculosis infection by CD8+ T cells requires the production of gamma interferon","volume":"66","author":"Tascon","year":"1998","journal-title":"Infect. Immun."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1167","DOI":"10.1172\/JCI38482","article-title":"Anti-TNF immunotherapy reduces CD8+ T cell-mediated antimicrobial activity against Mycobacterium tuberculosis in humans","volume":"119","author":"Bruns","year":"2009","journal-title":"J. Clin. Investig."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1601","DOI":"10.1517\/14712598.2012.721768","article-title":"An update on vaccines for tuberculosis-there is more to it than just waning of BCG efficacy with time","volume":"12","author":"Romano","year":"2012","journal-title":"Exp. Opin. Biol. Ther."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1111\/j.1600-065X.2010.00980.x","article-title":"DNA vaccines: An historical perspective and view to the future","volume":"239","author":"Liu","year":"2011","journal-title":"Immunol. Rev."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"780","DOI":"10.1016\/j.vaccine.2004.07.036","article-title":"A polyvalent DNA vaccine expressing an ESAT6-Ag85B fusion protein protects mice against a primary infection with Mycobacterium tuberculosis and boosts BCG-induced protective immunity","volume":"23","author":"Derrick","year":"2004","journal-title":"Vaccine"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Lu, J., Wang, C., Zhou, Z., Zhang, Y., Cao, T., Shi, C., Chen, Z., Chen, L., Cai, C., and Fan, X. (2011). Immunogenicity and protective efficacy against murine tuberculosis of a prime-boost regimen with BCG and a DNA vaccine expressing ESAT-6 and Ag85A fusion protein. Clin. Dev. Immunol., 2011.","DOI":"10.1155\/2011\/617892"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1016\/j.vaccine.2012.11.042","article-title":"Prime-boost BCG vaccination with DNA vaccines based in beta-defensin-2 and mycobacterial antigens ESAT6 or Ag85B improve protection in a tuberculosis experimental model","volume":"31","author":"Biragyn","year":"2013","journal-title":"Vaccine"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Tan, K., Liang, J., Teng, X., Wang, X., Zhang, J., Yuan, X., and Fan, X. (2013). Comparison of BCG prime-DNA booster and rBCG regimens for protection against tuberculosis. Hum. Vaccin Immunother.","DOI":"10.4161\/hv.26969"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"4174","DOI":"10.1128\/IAI.69.6.4174-4176.2001","article-title":"Priming by DNA immunization augments protective efficacy of Mycobacterium bovis Bacille Calmette-Guerin against tuberculosis","volume":"69","author":"Feng","year":"2001","journal-title":"Infect. Immun."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"6945","DOI":"10.1128\/IAI.72.12.6945-6950.2004","article-title":"A heterologous DNA priming-Mycobacterium bovis BCG boosting immunization strategy using mycobacterial Hsp70, Hsp65, and Apa antigens improves protection against tuberculosis in mice","volume":"72","author":"Ferraz","year":"2004","journal-title":"Infect. Immun."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1111\/j.1744-313X.2011.01066.x","article-title":"Protection against Mycobacterium tuberculosis challenge in mice by DNA vaccine Ag85A-ESAT-6-IL-21 priming and BCG boosting","volume":"39","author":"Dou","year":"2012","journal-title":"Int. J. Immunogenet."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3353","DOI":"10.1016\/j.vaccine.2005.12.066","article-title":"Priming but not boosting with plasmid DNA encoding mycolyl-transferase Ag85A from Mycobacterium tuberculosis increases the survival time of Mycobacterium bovis BCG vaccinated mice against low dose intravenous challenge with M. tuberculosis H37Rv","volume":"24","author":"Romano","year":"2006","journal-title":"Vaccine"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"371","DOI":"10.1111\/j.1365-3083.2005.01674.x","article-title":"DNA vaccination using Bacillus Calmette-Guerin-DNA as an adjuvant to enhance immune response to three kinds of swine diseases","volume":"62","author":"Zhang","year":"2005","journal-title":"Scand. J. Immunol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"472","DOI":"10.1016\/j.vaccine.2012.11.024","article-title":"Enhancement of HIV-1 DNA vaccine immunogenicity by BCG-PSN, a novel adjuvant","volume":"31","author":"Sun","year":"2013","journal-title":"Vaccine"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"3310","DOI":"10.1128\/iai.56.12.3310-3312.1988","article-title":"Induction of nonspecific acquired resistance and delayed-type hypersensitivity, but not specific acquired resistance in mice inoculated with killed mycobacterial vaccines","volume":"56","author":"Orme","year":"1988","journal-title":"Infect. Immun."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"385","DOI":"10.1038\/nature10072","article-title":"Detection of prokaryotic mRNA signifies microbial viability and promotes immunity","volume":"474","author":"Sander","year":"2011","journal-title":"Nature"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"381","DOI":"10.1111\/j.1574-695X.2007.00315.x","article-title":"Variation of the expression of Mycobacterium tuberculosis ppe44 gene among clinical isolates","volume":"51","author":"Rindi","year":"2007","journal-title":"FEMS Immunol. Med. Microbiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"6053","DOI":"10.1016\/j.vaccine.2008.09.025","article-title":"Immunogenicity and protective efficacy of tuberculosis subunit vaccines expressing PPE44 (Rv2770c)","volume":"26","author":"Romano","year":"2008","journal-title":"Vaccine"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"893","DOI":"10.1038\/nm0896-893","article-title":"Immunogenicity and protective efficacy of a tuberculosis DNA vaccine","volume":"2","author":"Huygen","year":"1996","journal-title":"Nat. Med."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"886","DOI":"10.4049\/jimmunol.179.2.886","article-title":"Epidermal langerhans cells are dispensable for humoral and cell-mediated immunity elicited by gene gun immunization","volume":"179","author":"Stoecklinger","year":"2007","journal-title":"J. Immunol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1111\/j.1365-2567.2006.02373.x","article-title":"Immunogenicity and protective efficacy of tuberculosis DNA vaccines combining mycolyl-transferase Ag85A and phosphate transport receptor PstS-3","volume":"118","author":"Romano","year":"2006","journal-title":"Immunology"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"6913","DOI":"10.4049\/jimmunol.172.11.6913","article-title":"Induction of in vivo functional Db-restricted cytolytic T cell activity against a putative phosphate transport receptor of Mycobacterium tuberculosis","volume":"172","author":"Romano","year":"2004","journal-title":"J. Immunol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2900","DOI":"10.1128\/jb.179.9.2900-2906.1997","article-title":"Three different putative phosphate transport receptors are encoded by the Mycobacterium tuberculosis genome and are present at the surface of Mycobacterium bovis BCG","volume":"179","author":"Lefevre","year":"1997","journal-title":"J. Bacteriol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1113","DOI":"10.4049\/jimmunol.162.2.1113","article-title":"Immunogenicity and protective efficacy of tuberculosis DNA vaccines encoding putative phosphate transport receptors","volume":"162","author":"Tanghe","year":"1999","journal-title":"J. Immunol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"e1000392","DOI":"10.1371\/journal.ppat.1000392","article-title":"A critical role for CD8 T cells in a nonhuman primate model of tuberculosis","volume":"5","author":"Chen","year":"2009","journal-title":"PLoS Pathog."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"4297","DOI":"10.1128\/IAI.71.8.4297-4303.2003","article-title":"Investigation of the role of CD8+ T cells in bovine tuberculosis in vivo","volume":"71","author":"McAulay","year":"2003","journal-title":"Infect. Immun."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"475","DOI":"10.4161\/hv.8570","article-title":"The candidate tuberculosis vaccine Mtb72F\/AS02A: Tolerability and immunogenicity in humans","volume":"5","author":"Von","year":"2009","journal-title":"Hum. Vaccin."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1016\/j.tube.2012.10.011","article-title":"The candidate tuberculosis vaccine Mtb72F\/AS02 in PPD positive adults: A randomized controlled phase I\/II study","volume":"93","author":"Spertini","year":"2013","journal-title":"Tuberculosis"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"3937","DOI":"10.1016\/j.vaccine.2005.03.003","article-title":"Protection of mice and guinea pigs against tuberculosis induced by immunization with a single Mycobacterium tuberculosis recombinant antigen, MTB41","volume":"23","author":"Skeiky","year":"2005","journal-title":"Vaccine"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1099\/jmm.0.45960-0","article-title":"Immunogenicity of mycobacterial PPE44 (Rv2770c) in Mycobacterium bovis BCG-infected mice","volume":"54","author":"Bonanni","year":"2005","journal-title":"J. Med. Microbiol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"211","DOI":"10.4049\/jimmunol.179.1.211","article-title":"A reduced antigen load in vivo, rather than weak inflammation, causes a substantial delay in CD8+ T cell priming against Mycobacterium bovis (Bacillus Calmette-Guerin)","volume":"179","author":"Russell","year":"2007","journal-title":"J. Immunol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"496","DOI":"10.1016\/j.immuni.2013.08.034","article-title":"Antigen availability determines CD8+ T cell-dendritic cell interaction kinetics and memory fate decisions","volume":"39","author":"Henrickson","year":"2013","journal-title":"Immunity"},{"key":"ref_42","unstructured":"Bruffaerts, N., Vandermeulen, G., Romano, M., Pr\u00e9at, V., Stockhofe-Zurwieden, N., and Huygen, K. (2014). Unpublished data."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1947","DOI":"10.1126\/science.272.5270.1947","article-title":"Induction of bystander T cell promiferation by viruses and type I interferon in vivo","volume":"272","author":"Tough","year":"1996","journal-title":"Science"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1016\/S1074-7613(00)80564-6","article-title":"Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15","volume":"8","author":"Zhang","year":"1998","journal-title":"Immunity"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"976","DOI":"10.1002\/eji.200940017","article-title":"Bystander stimulation of activated CD4+ T cells of unrelated specificity following a booster vaccination with tetanus toxoid","volume":"40","author":"Savelyeva","year":"2010","journal-title":"Eur. J. Immunol."},{"key":"ref_46","unstructured":"Bruffaerts, N., Romano, M., Denis, O., Jurion, F., and Huygen, K. (2014). Unpublished data."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"557","DOI":"10.1016\/j.imbio.2006.05.004","article-title":"Toll-like receptor 9 contributes to recognition of Mycobacterium bovis Bacillus Calmette-Guerin by Flt3-ligand generated dendritic cells","volume":"211","author":"Schaefer","year":"2006","journal-title":"Immunobiology"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"479","DOI":"10.1038\/nri3247","article-title":"Nucleic acid sensing at the interface between innate and adaptive immunity in vaccination","volume":"12","author":"Desmet","year":"2012","journal-title":"Nat. Rev. Immunol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1715","DOI":"10.1084\/jem.20051782","article-title":"TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis","volume":"202","author":"Bafica","year":"2005","journal-title":"J. Exp. Med."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"1503","DOI":"10.4049\/jimmunol.1001022","article-title":"CpG promotes cross-presentation of dead cell-associated antigens by pre-CD8\u03b1+ dendritic cells","volume":"186","author":"Tomkowiak","year":"2011","journal-title":"J. Immunol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"6341","DOI":"10.1016\/j.vaccine.2007.06.016","article-title":"Priming of CD8+ T-cell responses after DNA immunization is impaired in TLR9- and MyD88-deficient mice","volume":"25","author":"Pavlenko","year":"2007","journal-title":"Vaccine"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1169","DOI":"10.1586\/erv.11.66","article-title":"Intradermal naked plasmid DNA immunization: Mechanisms of action","volume":"10","author":"Elnekave","year":"2011","journal-title":"Exp. Rev. Vaccines"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"978","DOI":"10.1038\/ni.2680","article-title":"The skin-resident and migratory immune system in steady state and memory: Innate lymphocytes, dendritic cells and T cells","volume":"14","author":"Heath","year":"2013","journal-title":"Nat. Immunol."}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/2\/1\/181\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T21:08:56Z","timestamp":1760216936000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/2\/1\/181"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,3,5]]},"references-count":53,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2014,3]]}},"alternative-id":["vaccines2010181"],"URL":"https:\/\/doi.org\/10.3390\/vaccines2010181","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2014,3,5]]}}}