The zebrafish breathes new life into the study of tuberculosis

Frontiers in Immunology

Volumn 7, Issue MAY, 2016, Pages

  Myllymäki, Henna ^a   Bäuerlein, Carina A ^a   Rämet, Mika ^a,b,c,d  

^a TAMPERE UNIVERSITY OF TECHNOLOGY   (Finland)

^b TAMPERE UNIVERSITY HOSPITAL   (Finland)

^c OULU UNIVERSITY HOSPITAL   (Finland)

^d UNIVERSITY OF OULU   (Finland)

Author keywords

Granuloma; Latency; Mycobacterium infections; Mycobacterium marinum; Mycobacterium tuberculosis; Tuberculosis; Vaccination; Zebrafish model system

Indexed keywords

DNA VACCINE; VASCULOTROPIN RECEPTOR;

ADAPTIVE IMMUNITY; BACTERIAL VIRULENCE; DEFENSE MECHANISM; DORMANCY; GRANULOMA; IMMUNE DEFICIENCY; INNATE IMMUNITY; LATENT PERIOD; MYCOBACTERIUM MARINUM; NONHUMAN; POSTPRIMARY TUBERCULOSIS; SHORT SURVEY; TUBERCULOSIS; ZEBRA FISH;

EID: 84973579918     PISSN: None     EISSN: 16643224     Source Type: Journal    
DOI: 10.3389/fimmu.2016.00196     Document Type: Short Survey

References (92)

1. World Health Organization. Global Tuberculosis Report 2015. (2015). Available from: http://www.who.int/tb/publications/global_report/en/
2. Frieden TR, Sterling TR, Munsiff SS, Watt CJ, Dye C. Tuberculosis. Lancet (2003) 362:887-99. doi:10.1016/S0140-6736(03)14333-4
3. Adams DO. The structure of mononuclear phagocytes differentiating in vivo. I. Sequential fine and histologic studies of the effect of Bacillus Calmette-Guerin (BCG). Am J Pathol (1974) 76:17-48
4. Ramakrishnan L. Revisiting the role of the granuloma in tuberculosis. Nat Rev Immunol (2012) 12:352-66. doi:10.1038/nri3211
5. Adams DO. The granulomatous inflammatory response. A review. Am J Pathol (1976) 84(1):164-92
6. Renshaw SA, Trede NS. A model 450 million years in the making: zebrafish and vertebrate immunity. Dis Model Mech (2012) 5:38-47. doi:10.1242/dmm.007138
7. Langenau DM, Ferrando AA, Traver D, Kutok JL, Hezel JP, Kanki JP, et al. In vivo tracking of T cell development, ablation, and engraftment in transgenic zebrafish. Proc Natl Acad Sci U S A (2004) 101:7369-74. doi:10.1073/pnas.0402248101
8. Meijer AH, Spaink HP. Host-pathogen interactions made transparent with the zebrafish model. Curr Drug Targets (2011) 12:1000-17. doi:10.2174/138945011795677809
9. Decostere A, Hermans K, Haesebrouck F. Piscine mycobacteriosis: a literature review covering the agent and the disease it causes in fish and humans. Vet Microbiol (2004) 99:159-66. doi:10.1016/j.vetmic.2003.07.011
10. O'Garra A, Redford PS, McNab FW, Bloom CI, Wilkinson RJ, Berry MP. The immune response in tuberculosis. Annu Rev Immunol (2013) 31:475-527. doi:10.1146/annurev-immunol-032712-095939
11. Linell F, Norden A. Mycobacterium balnei, a new acid-fast bacillus occurring in swimming pools and capable of producing skin lesions in humans. Acta Tuberc Scand Suppl (1954) 33:1-84
12. Harriff MJ, Bermudez LE, Kent ML. Experimental exposure of zebrafish, Danio rerio (Hamilton), to Mycobacterium marinum and Mycobacterium peregrinum reveals the gastrointestinal tract as the primary route of infection: a potential model for environmental mycobacterial infection. J Fish Dis (2007) 30:587-600. doi:10.1111/j.1365-2761.2007.00839.x
13. Parikka M, Hammaren MM, Harjula SK, Halfpenny NJ, Oksanen KE, Lahtinen MJ, et al. Mycobacterium marinum causes a latent infection that can be reactivated by gamma irradiation in adult zebrafish. PLoS Pathog (2012) 8:e1002944. doi:10.1371/journal.ppat.1002944
14. Swaim LE, Connolly LE, Volkman HE, Humbert O, Born DE, Ramakrishnan L. Mycobacterium marinum infection of adult zebrafish causes caseating granulomatous tuberculosis and is moderated by adaptive immunity. Infect Immun (2006) 74:6108-17. doi:10.1128/IAI.00887-06
15. Prouty MG, Correa NE, Barker LP, Jagadeeswaran P, Klose KE. Zebrafish-Mycobacterium marinum model for mycobacterial pathogenesis. FEMS Microbiol Lett (2003) 225:177-82. doi:10.1016/S0378-1097(03)00446-4
16. Davis JM, Ramakrishnan L. The role of the granuloma in expansion and dissemination of early tuberculous infection. Cell (2009) 136:37-49. doi:10.1016/j.cell.2008.11.014
17. Volkman HE, Pozos TC, Zheng J, Davis JM, Rawls JF, Ramakrishnan L. Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium. Science (2010) 327:466-9. doi:10.1126/science.1179663
18. Davis JM, Clay H, Lewis JL, Ghori N, Herbomel P, Ramakrishnan L. Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos. Immunity (2002) 17:693-702. doi:10.1016/S1074-7613(02)00475-2
19. Marais BJ, Gie RP, Schaaf HS, Beyers N, Donald PR, Starke JR. Childhood pulmonary tuberculosis: old wisdom and new challenges. Am J Respir Crit Care Med (2006) 173:1078-90. doi:10.1164/rccm.200511-1809SO
20. Roy A, Eisenhut M, Harris RJ, Rodrigues LC, Sridhar S, Habermann S, et al. Effect of BCG vaccination against Mycobacterium tuberculosis infection in children: systematic review and meta-analysis. BMJ (2014) 349:g4643. doi:10.1136/bmj.g4643
21. Mak TK, Hesseling AC, Hussey GD, Cotton MF. Making BCG vaccination programmes safer in the HIV era. Lancet (2008) 372:786-7. doi:10.1016/S0140-6736(08)61318-5
22. Myllymaki H, Niskanen M, Oksanen KE, Ramet M. Animal models in tuberculosis research-where is the beef? Expert Opin Drug Discov (2015) 10:871-83. doi:10.1517/17460441.2015.1049529
23. Tobin DM, Ramakrishnan L. Comparative pathogenesis of Mycobacterium marinum and Mycobacterium tuberculosis. Cell Microbiol (2008) 10:1027-39. doi:10.1111/j.1462-5822.2008.01133.x
24. Stinear TP, Seemann T, Harrison PF, Jenkin GA, Davies JK, Johnson PD, et al. Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis. Genome Res (2008) 18:729-41. doi:10.1101/gr.075069.107
25. Barker LP, George KM, Falkow S, Small PL. Differential trafficking of live and dead Mycobacterium marinum organisms in macrophages. Infect Immun (1997) 65:1497-504
26. Benard EL, van der Sar AM, Ellett F, Lieschke GJ, Spaink HP, Meijer AH. Infection of zebrafish embryos with intracellular bacterial pathogens. J Vis Exp (2012) 61:e3781. doi:10.3791/3781
27. Meijer AH. Protection and pathology in TB: learning from the zebrafish model. Semin Immunopathol (2016) 38(2):261-73. doi:10.1007/s00281-015-0522-4
28. van Leeuwen LM, van der Sar AM, Bitter W. Animal models of tuberculosis: zebrafish. Cold Spring Harb Perspect Med (2014) 5:a018580. doi:10.1101/cshperspect.a018580
29. Cronan MR, Tobin DM. Fit for consumption: zebrafish as a model for tuberculosis. Dis Model Mech (2014) 7:777-84. doi:10.1242/dmm.016089
30. Hall C, Flores MV, Crosier K, Crosier P. Live cell imaging of zebrafish leukocytes. Methods Mol Biol (2009) 546:255-71. doi:10.1007/978-1-60327-977-2_16
31. Renshaw SA, Loynes CA, Trushell DM, Elworthy S, Ingham PW, Whyte MK. A transgenic zebrafish model of neutrophilic inflammation. Blood (2006) 108:3976-8. doi:10.1182/blood-2006-05-024075
32. Wittamer V, Bertrand JY, Gutschow PW, Traver D. Characterization of the mononuclear phagocyte system in zebrafish. Blood (2011) 117:7126-35. doi:10.1182/blood-2010-11-321448
33. Lohi O, Parikka M, Rämet M. The zebrafish as a model for paediatric diseases. Acta Paediatr (2013) 102:104-10. doi:10.1111/j.1651-2227.2012.02835.x
34. Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol (2013) 31:227-9. doi:10.1038/nbt.2501
35. Varshney GK, Sood R, Burgess SM. Understanding and editing the zebrafish genome. Adv Genet (2015) 92:1-52. doi:10.1016/bs.adgen.2015.09.002
36. Traver D, Herbomel P, Patton EE, Murphey RD, Yoder JA, Litman GW, et al. The zebrafish as a model organism to study development of the immune system. Adv Immunol (2003) 81:253-330
37. Traver D, Paw BH, Poss KD, Penberthy WT, Lin S, Zon LI. Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants. Nat Immunol (2003) 4:1238-46. doi:10.1038/ni1007
38. Kissa K, Murayama E, Zapata A, Cortes A, Perret E, Machu C, et al. Live imaging of emerging hematopoietic stem cells and early thymus colonization. Blood (2008) 111:1147-56. doi:10.1182/blood-2007-07-099499
39. Lugo-Villarino G, Balla KM, Stachura DL, Banuelos K, Werneck MB, Traver D. Identification of dendritic antigen-presenting cells in the zebrafish. Proc Natl Acad Sci U S A (2010) 107:15850-5. doi:10.1073/pnas.1000494107
40. Koul A, Herget T, Klebl B, Ullrich A. Interplay between mycobacteria and host signalling pathways. Nat Rev Microbiol (2004) 2:189-202. doi:10.1038/nrmicro840
41. Benard EL, Roobol SJ, Spaink HP, Meijer AH. Phagocytosis of mycobacteria by zebrafish macrophages is dependent on the scavenger receptor Marco, a key control factor of pro-inflammatory signalling. Dev Comp Immunol (2014) 47:223-33. doi:10.1016/j.dci.2014.07.022
42. Fink IR, Benard EL, Hermsen T, Meijer AH, Forlenza M, Wiegertjes GF. Molecular and functional characterization of the scavenger receptor CD36 in zebrafish and common carp. Mol Immunol (2015) 63:381-93. doi:10.1016/j.molimm.2014.09.010
43. Velez DR, Wejse C, Stryjewski ME, Abbate E, Hulme WF, Myers JL, et al. Variants in Toll-like receptors 2 and 9 influence susceptibility to pulmonary tuberculosis in Caucasians, African-Americans, and West Africans. Hum Genet (2010) 127:65-73. doi:10.1007/s00439-009-0741-7
44. Kleinnijenhuis J, Oosting M, Joosten LA, Netea MG, Van Crevel R. Innate immune recognition of Mycobacterium tuberculosis. Clin Dev Immunol (2011) 2011:405310. doi:10.1155/2011/405310
45. Benard EL, Racz PI, Rougeot J, Nezhinsky AE, Verbeek FJ, Spaink HP, et al. Macrophage-expressed perforins mpeg1 and mpeg1.2 have an anti-bacterial function in zebrafish. J Innate Immun (2015) 7:136-52. doi:10.1159/000366103
46. van der Vaart M, van Soest JJ, Spaink HP, Meijer AH. Functional analysis of a zebrafish myd88 mutant identifies key transcriptional components of the innate immune system. Dis Model Mech (2013) 6:841-54. doi:10.1242/dmm.010843
47. van der Sar AM, Stockhammer OW, van der Laan C, Spaink HP, Bitter W, Meijer AH. MyD88 innate immune function in a zebrafish embryo infection model. Infect Immun (2006) 74:2436-41. doi:10.1128/IAI.74.4.2436-2441.2006
48. Elks PM, Brizee S, van der Vaart M, Walmsley SR, van Eeden FJ, Renshaw SA, et al. Hypoxia inducible factor signaling modulates susceptibility to mycobacterial infection via a nitric oxide dependent mechanism. PLoS Pathog (2013) 9:e1003789. doi:10.1371/journal.ppat.1003789
49. Elks PM, van der Vaart M, van Hensbergen V, Schutz E, Redd MJ, Murayama E, et al. Mycobacteria counteract a TLR-mediated nitrosative defense mechanism in a zebrafish infection model. PLoS One (2014) 9:e100928. doi:10.1371/journal.pone.0100928
50. Deretic V, Saitoh T, Akira S. Autophagy in infection, inflammation and immunity. Nat Rev Immunol (2013) 13:722-37. doi:10.1038/nri3532
51. van der Vaart M, Korbee CJ, Lamers GE, Tengeler AC, Hosseini R, Haks MC, et al. The DNA damage-regulated autophagy modulator DRAM1 links mycobacterial recognition via TLR-MYD88 to authophagic defense. Cell Host Microbe (2014) 15:753-67. doi:10.1016/j.chom.2014.05.005
52. Hosseini R, Lamers GE, Hodzic Z, Meijer AH, Schaaf MJ, Spaink HP. Correlative light and electron microscopy imaging of autophagy in a zebrafish infection model. Autophagy (2014) 10:1844-57. doi:10.4161/auto.29992
53. Yang CT, Cambier CJ, Davis JM, Hall CJ, Crosier PS, Ramakrishnan L. Neutrophils exert protection in the early tuberculous granuloma by oxidative killing of mycobacteria phagocytosed from infected macrophages. Cell Host Microbe (2012) 12:301-12. doi:10.1016/j.chom.2012.07.009
54. Ramakrishnan L. Looking within the zebrafish to understand the tuberculous granuloma. Adv Exp Med Biol (2013) 783:251-66. doi:10.1007/978-1-4614-6111-1_13
55. Oehlers SH, Cronan MR, Scott NR, Thomas MI, Okuda KS, Walton EM, et al. Interception of host angiogenic signalling limits mycobacterial growth. Nature (2015) 517:612-5. doi:10.1038/nature13967
56. Houben D, Demangel C, van Ingen J, Perez J, Baldeon L, Abdallah AM, et al. ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria. Cell Microbiol (2012) 14:1287-98. doi:10.1111/j.1462-5822.2012.01799.x
57. Cambier CJ, Takaki KK, Larson RP, Hernandez RE, Tobin DM, Urdahl KB, et al. Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids. Nature (2014) 505:218-22. doi:10.1038/nature12799
58. Kaufman CK, White RM, Zon L. Chemical genetic screening in the zebrafish embryo. Nat Protoc (2009) 4:1422-32. doi:10.1038/nprot.2009.144
59. Takaki K, Davis JM, Winglee K, Ramakrishnan L. Evaluation of the pathogenesis and treatment of Mycobacterium marinum infection in zebrafish. Nat Protoc (2013) 8:1114-24. doi:10.1038/nprot.2013.068
60. Adams KN, Takaki K, Connolly LE, Wiedenhoft H, Winglee K, Humbert O, et al. Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism. Cell (2011) 145:39-53. doi:10.1016/j.cell.2011.02.022
61. Adams KN, Szumowski JD, Ramakrishnan L. Verapamil, and its metabolite norverapamil, inhibit macrophage-induced, bacterial efflux pump-mediated tolerance to multiple anti-tubercular drugs. J Infect Dis (2014) 210:456-66. doi:10.1093/infdis/jiu095
62. Torraca V, Cui C, Boland R, Bebelman JP, van der Sar AM, Smit MJ, et al. The CXCR3-CXCL11 signaling axis mediates macrophage recruitment and dissemination of mycobacterial infection. Dis Model Mech (2015) 8:253-69. doi:10.1242/dmm.017756
63. Tobin DM, Roca FJ, Oh SF, McFarland R, Vickery TW, Ray JP, et al. Host genotype-specific therapies can optimize the inflammatory response to mycobacterial infections. Cell (2012) 148:434-46. doi:10.1016/j.cell.2011.12.023
64. Roca FJ, Ramakrishnan L. TNF dually mediates resistance and susceptibility to mycobacteria via mitochondrial reactive oxygen species. Cell (2013) 153:521-34. doi:10.1016/j.cell.2013.03.022
65. Kaufmann SH, Lange C, Rao M, Balaji KN, Lotze M, Schito M, et al. Progress in tuberculosis vaccine development and host-directed therapies-a state of the art review. Lancet Respir Med (2014) 2:301-20. doi:10.1016/S2213-2600(14)70033-5
66. Deng W, Tang X, Hou M, Li C, Xie J. New insights into the pathogenesis of tuberculosis revealed by Mycobacterium marinum: the zebrafish model from the systems biology perspective. Crit Rev Eukaryot Gene Expr (2011) 21:337-45. doi:10.1615/CritRevEukarGeneExpr.v21.i4.40
67. Case ED, Samuel JE. Contrasting lifestyles within the host cell. Microbiol Spectr (2016) 4. doi:10.1128/microbiolspec.VMBF-0014-2015
68. Oksanen KE, Halfpenny NJ, Sherwood E, Harjula SK, Hammaren MM, Ahava MJ, et al. An adult zebrafish model for preclinical tuberculosis vaccine development. Vaccine (2013) 31:5202-9. doi:10.1016/j.vaccine.2013.08.093
69. Ojanen MJ, Turpeinen H, Cordova ZM, Hammaren MM, Harjula SK, Parikka M, et al. The proprotein convertase subtilisin/kexin furinA regulates zebrafish host response against Mycobacterium marinum. Infect Immun (2015) 83:1431-42. doi:10.1128/IAI.03135-14
70. Hammaren MM, Oksanen KE, Nisula HM, Luukinen BV, Pesu M, Ramet M, et al. Adequate Th2-type response associates with restricted bacterial growth in latent mycobacterial infection of zebrafish. PLoS Pathog (2014) 10:e1004190. doi:10.1371/journal.ppat.1004190
71. van Meijgaarden KE, Haks MC, Caccamo N, Dieli F, Ottenhoff TH, Joosten SA. Human CD8+ T-cells recognizing peptides from Mycobacterium tuberculosis (Mtb) presented by HLA-E have an unorthodox Th2-like, multifunctional, Mtb inhibitory phenotype and represent a novel human T-cell subset. PLoS Pathog (2015) 11:e1004671. doi:10.1371/journal.ppat.1004671
72. van der Sar AM, Abdallah AM, Sparrius M, Reinders E, Vandenbroucke-Grauls CM, Bitter W. Mycobacterium marinum strains can be divided into two distinct types based on genetic diversity and virulence. Infect Immun (2004) 72:6306-12. doi:10.1128/IAI.72.11.6306-6312.2004
73. Oksanen KE, Myllymäki H, Ahava MJ, Mäkinen L, Parikka M, Rämet M. DNA vaccination boosts Bacillus Calmette-Guérin protection against mycobacterial infection in zebrafish. Dev Comp Immunol (2016) 54:89-96. doi:10.1016/j.dci.2015.09.001
74. Cui Z, Samuel-Shaker D, Watral V, Kent ML. Attenuated Mycobacterium marinum protects zebrafish against mycobacteriosis. J Fish Dis (2010) 33:371-5. doi:10.1111/j.1365-2761.2009.01115.x
75. Stoop EJ, Schipper T, Rosendahl Huber SK, Nezhinsky AE, Verbeek FJ, Gurcha SS, et al. Zebrafish embryo screen for mycobacterial genes involved in the initiation of granuloma formation reveals a newly identified ESX-1 component. Dis Model Mech (2011) 4:526-36. doi:10.1242/dmm.006676
76. Volkman HE, Clay H, Beery D, Chang JC, Sherman DR, Ramakrishnan L. Tuberculous granuloma formation is enhanced by a mycobacterium virulence determinant. PLoS Biol (2004) 2:e367. doi:10.1371/journal.pbio.0020367
77. Knudsen NP, Norskov-Lauritsen S, Dolganov GM, Schoolnik GK, Lindenstrom T, Andersen P, et al. Tuberculosis vaccine with high predicted population coverage and compatibility with modern diagnostics. Proc Natl Acad Sci U S A (2014) 111:1096-101. doi:10.1073/pnas.1314973111
78. Hoang T, Aagaard C, Dietrich J, Cassidy JP, Dolganov G, Schoolnik GK, et al. ESAT-6 (EsxA) and TB10.4 (EsxH) based vaccines for pre-and post-exposure tuberculosis vaccination. PLoS One (2013) 8:e80579. doi:10.1371/journal.pone.0080579
79. Bottai D, Frigui W, Clark S, Rayner E, Zelmer A, Andreu N, et al. Increased protective efficacy of recombinant BCG strains expressing virulence-neutral proteins of the ESX-1 secretion system. Vaccine (2015) 33:2710-8. doi:10.1016/j.vaccine.2015.03.083
80. Lin PL, Dietrich J, Tan E, Abalos RM, Burgos J, Bigbee C, et al. The multistage vaccine H56 boosts the effects of BCG to protect cynomolgus macaques against active tuberculosis and reactivation of latent Mycobacterium tuberculosis infection. J Clin Invest (2012) 122:303-14. doi:10.1172/JCI46252
81. Dietrich J, Andersen C, Rappuoli R, Doherty TM, Jensen CG, Andersen P. Mucosal administration of Ag85B-ESAT-6 protects against infection with Mycobacterium tuberculosis and boosts prior bacillus Calmette-Guerin immunity. J Immunol (2006) 177:6353-60. doi:10.4049/jimmunol.177.9.6353
82. Yuan W, Dong N, Zhang L, Liu J, Lin S, Xiang Z, et al. Immunogenicity and protective efficacy of a tuberculosis DNA vaccine expressing a fusion protein of Ag85B-Esat6-HspX in mice. Vaccine (2012) 30:2490-7. doi:10.1016/j.vaccine.2011.06.029
83. Capuano SVI, Croix DA, Pawar S, Zinovik A, Myers A, Lin PL, et al. Experimental Mycobacterium tuberculosis infection of cynomolgus macaques closely resembles the various manifestations of human M. tuberculosis infection. Infect Immun (2003) 71:5831-44. doi:10.1128/IAI.71.10.5831-5844.2003
84. Murray KN, Bauer J, Tallen A, Matthews JL, Westerfield M, Varga ZM. Characterization and management of asymptomatic mycobacterium infections at the Zebrafish International Resource Center. J Am Assoc Lab Anim Sci (2011) 50:675-9
85. Pasnik DJ, Smith SA. Immunogenic and protective effects of a DNA vaccine for Mycobacterium marinum in fish. Vet Immunol Immunopathol (2005) 103:195-206. doi:10.1016/j.vetimm.2004.08.017
86. Kato G, Kondo H, Aoki T, Hirono I. BCG vaccine confers adaptive immunity against Mycobacterium sp. infection in fish. Dev Comp Immunol (2010) 34:133-40. doi:10.1016/j.dci.2009.08.013
87. Ernst JD. The immunological life cycle of tuberculosis. Nat Rev Immunol (2012) 12:581-91. doi:10.1038/nri3259
88. Berry MP, Graham CM, McNab FW, Xu Z, Bloch SA, Oni T, et al. An interferon-inducible neutrophil-driven blood transcriptional signature in human tuberculosis. Nature (2010) 466:973-7. doi:10.1038/nature09247
89. Kaufmann SH, Dorhoi A. Inflammation in tuberculosis: interactions, imbalances and interventions. Curr Opin Immunol (2013) 25:441-9. doi:10.1016/j.coi.2013.05.005
90. Fletcher HA. Profiling the host immune response to tuberculosis vaccines. Vaccine (2015) 33:5313-5. doi:10.1016/j.vaccine.2015.07.090
91. Tameris MD, Hatherill M, Landry BS, Scriba TJ, Snowden MA, Lockhart S, et al. Safety and efficacy of MVA85A, a new tuberculosis vaccine, in infants previously vaccinated with BCG: a randomised, placebo-controlled phase 2b trial. Lancet (2013) 381:1021-8. doi:10.1016/S0140-6736(13)60177-4
92. Kagina BM, Abel B, Scriba TJ, Hughes EJ, Keyser A, Soares A, et al. Specific T cell frequency and cytokine expression profile do not correlate with protection against tuberculosis after Bacillus Calmette-Guérin vaccination of newborns. Am J Respir Crit Care Med (2010) 182:1073. doi:10.1164/rccm.201003-0334OC