Safety and Immunogenicity of the Recombinant Mycobacterium bovis BCG Vaccine VPM1002 in HIV-Unexposed Newborn Infants in South Africa

André G Loxton, Julia K Knaul, Leander Grode, Andrea Gutschmidt, Christiane Meller, Bernd Eisele, Hilary Johnstone, Gian van der Spuy, Jeroen Maertzdorf, Stefan H E Kaufmann, Anneke C Hesseling, Gerhard Walzl, Mark F Cotton, André G Loxton, Julia K Knaul, Leander Grode, Andrea Gutschmidt, Christiane Meller, Bernd Eisele, Hilary Johnstone, Gian van der Spuy, Jeroen Maertzdorf, Stefan H E Kaufmann, Anneke C Hesseling, Gerhard Walzl, Mark F Cotton

Abstract

Tuberculosis is a global threat to which infants are especially vulnerable. Effective vaccines are required to protect infants from this devastating disease. VPM1002, a novel recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine previously shown to be safe and immunogenic in adults, was evaluated for safety in its intended target population, namely, newborn infants in a region with high prevalence of tuberculosis. A total of 48 newborns were vaccinated intradermally with VPM1002 (n = 36) or BCG Danish strain (n = 12) in a phase II open-labeled, randomized trial with a 6-month follow-up period. Clinical and laboratory measures of safety were evaluated during this time. In addition, vaccine-induced immune responses to mycobacteria were analyzed in whole-blood stimulation and proliferation assays. The safety parameters and immunogenicity were comparable in the two groups. Both vaccines induced interleukin-17 (IL-17) responses; however, VPM1002 vaccination led to an increase of CD8+ IL-17+ T cells at the week 16 and month 6 time points. The incidence of abscess formation was lower for VPM1002 than for BCG. We conclude that VPM1002 is a safe, well-tolerated, and immunogenic vaccine in newborn infants, confirming results from previous trials in adults. These results strongly support further evaluation of the safety and efficacy of this vaccination in larger studies. (This study has been registered at ClinicalTrials.gov under registration no. NCT01479972.).

Keywords: HIV-unexposed; IL-17; VPM1002; immunogenicity; newborn; recombinant; recombinant BCG; safety; vaccines; whole blood assay.

Copyright © 2017 Loxton et al.

Figures

FIG 1
FIG 1
Flowchart representing consented, screened, and enrolled newborn infants of the trial. MDR-TB, multidrug-resistant TB.
FIG 2
FIG 2
Proportions of distinct subsets of specific CD4+ (A) and CD8+ (B) T cells showing single or combined expression of IFN-γ, TNF-α, and/or IL-2 in whole blood after restimulation with PPD for 12 h. Patterns are shown for different time points before and after vaccination with BCG (n = 12) and VPM1002 (n = 36). The median proportion of each cytokine-expressing cell subset is represented by the horizontal line, the interquartile range (IQR) by the box, and the range by the whiskers. Differences in subset proportions between the vaccination groups were analyzed for each time point using a Wilcoxon rank sum test and were not significant (P > 0.050).
FIG 3
FIG 3
Vaccine-induced IFN-γ responses as determined by ELISA in whole-blood samples after stimulation with PPD for 7 h (A) and 7 days (B). Medians (lines) and ranges (error bars) are shown. Both BCG (n = 12) and VPM1002 (n = 36) vaccination groups showed positive IFN-γ responses to vaccination, peaking at week 6. Within each group, changes from baseline were analyzed using a Wilcoxon signed-rank test and were significant in both assays at all time points excepting BCG at day 14. The proliferative IFN-γ response in the 7 day assay was significantly greater in response to BCG than VPM1002 (P = 0.0141), as analyzed by a Wilcoxon rank sum test.
FIG 4
FIG 4
Effect of vaccination on IL-17 production by CD8+ T cells. (A) Proportions of CD8+ T cells expressing IL-17 after restimulation with BCG for 7 days. Median proportions for BCG (n = 12) and VPM1002 (n = 36) vaccination groups at each time point are expressed by the horizontal line, the interquartile range (IQR) by the box, and the range by the whiskers. Corresponding individual responses are illustrated by dots adjacent to each box-and-whisker. Changes from the baseline to each time point postvaccination were assessed using a Wilcoxon signed-rank test. These were significant in only the VPM1002 group at day 14 (P = 0.0156) and month 6 (P = 0.0002). (B) Individual longitudinal expression of IL-17 among CD8+ T cells from baseline to month 6 is shown for individual infants in the BCG and VPM1002 vaccination groups. Positive responses were seen in 2 (16.7%) subjects from the BCG and 13 (36.1%) from the VPM1002 vaccination groups. Median responses were similar in the two vaccination groups (P = 0.0836, Wilcoxon rank sum test).
FIG 5
FIG 5
Gene set enrichment of differentially expressed genes. Enrichment was calculated for each time point relative to baseline. Red indicates the proportion of genes in a particular module which show significantly increased expression. Likewise, blue indicates significantly lower expression of genes. Modules which are gray are enriched but individual genes in that module are not significantly changed. The columns on the right indicated by “dif” show differences between the BCG and VPM1002 group at each time point.
FIG 6
FIG 6
IL-17A gene expression in whole-blood samples throughout the study period from VPM1002- or BCG-vaccinated infants. d, day; w, week; m, month.

References

    1. World Health Organization. 2016. Global tuberculosis report—2015. World Health Organization, Geneva, Switzerland.
    1. Heyns L, Gie RP, Goussard P, Beyers N, Warren RM, Marais BJ. 2006. Nosocomial transmission of Mycobacterium tuberculosis in kangaroo mother care units: a risk in tuberculosis-endemic areas. Acta Paediatr 95:535–539. doi:10.1080/08035250600636560.
    1. Marais BJ, Gie RP, Schaaf HS, Hesseling AC, Obihara CC, Starke JJ, Enarson DA, Donald PR, Beyers N. 2004. The natural history of childhood intra-thoracic tuberculosis: a critical review of literature from the pre-chemotherapy era. Int J Tuberc Lung Dis 8:392–402.
    1. Vanden DK, Persson A, Marais BJ, Fink PJ, Urdahl KB. 2013. Immune vulnerability of infants to tuberculosis. Clin Dev Immunol 2013:781320. doi:10.1155/2013/781320.
    1. Hesseling AC, Johnson LF, Jaspan H, Cotton MF, Whitelaw A, Schaaf HS, Fine PE, Eley BS, Marais BJ, Nuttall J, Beyers N, Godfrey-Faussett P. 2009. Disseminated bacille Calmette-Guérin disease in HIV-infected South African infants. Bull World Health Organ 87:505–511. doi:10.2471/BLT.08.055657.
    1. Marais BJ. 2004. Childhood tuberculosis: reflections from the front line. Pediatr Ann 33:695–698. doi:10.3928/0090-4481-20041001-13.
    1. Swaminathan S, Ramachandran G. 2015. Challenges in childhood tuberculosis. Clin Pharmacol Ther 98:240–244. doi:10.1002/cpt.175.
    1. Wiseman CA, Gie RP, Starke JR, Schaaf HS, Donald PR, Cotton MF, Hesseling AC. 2012. A proposed comprehensive classification of tuberculosis disease severity in children. Pediatr Infect Dis J 31:347–352. doi:10.1097/INF.0b013e318243e27b.
    1. Dodd PJ, Gardiner E, Coghlan R, Seddon JA. 2014. Burden of childhood tuberculosis in 22 high-burden countries: a mathematical modeling study. Lancet Glob Health 2:e453–e459. doi:10.1016/S2214-109X(14)70245-1.
    1. Statens Serum Institut. 2007. SmPC of BCG 1331 SSI: summary of product characteristics. Statens Serum Institut, Copenhagen, Denmark.
    1. Serum Institute of India Pvt, Ltd. 2016. SII BCG vaccine. Serum Institute of India Pvt, Ltd, Pune, India.
    1. Trunz BB, Fine P, Dye C. 2006. Effect of BCG vaccination on childhood tuberculous meningitis and miliary tuberculosis worldwide: a meta-analysis and assessment of cost-effectiveness. Lancet 367:1173–1180. doi:10.1016/S0140-6736(06)68507-3.
    1. van Well GT, Paes BF, Terwee CB, Springer P, Roord JJ, Donald PR, van Furth AM, Schoeman JF. 2009. Twenty years of pediatric tuberculous meningitis: a retrospective cohort study in the western cape of South Africa. Pediatrics 123:e1–e8. doi:10.1542/peds.2008-1353.
    1. Mangtani P, Abubakar I, Ariti C, Beynon R, Pimpin L, Fine PE, Rodrigues LC, Smith PG, Lipman M, Whiting PF, Sterne JA. 2014. Protection by BCG vaccine against tuberculosis: a systematic review of randomized controlled trials. Clin Infect Dis 58:470–480. doi:10.1093/cid/cit790.
    1. Nguipdop-Djomo P, Heldal E, Rodrigues LC, Abubakar I, Mangtani P. 2016. Duration of BCG protection against tuberculosis and change in effectiveness with time since vaccination in Norway: a retrospective population-based cohort study. Lancet Infect Dis 16:219–226. doi:10.1016/S1473-3099(15)00400-4.
    1. Bonifachich E, Chort M, Astigarraga A, Diaz N, Brunet B, Pezzotto SM, Bottasso O. 2006. Protective effect of bacillus Calmette-Guerin (BCG) vaccination in children with extra-pulmonary tuberculosis, but not the pulmonary disease: a case-control study in Rosario, Argentina. Vaccine 24:2894–2899. doi:10.1016/j.vaccine.2005.12.044.
    1. Zodpey SP, Shrikhande SN, Maldhure BR, Vasudeo ND, Kulkarni SW. 1998. Effectiveness of bacillus Calmette-Guerin (BCG) vaccination in the prevention of childhood pulmonary tuberculosis: a case control study in Nagpur, India. Southeast Asian J Trop Med Public Health 29:285–288.
    1. Marais BJ, Seddon JA, Detjen AK, van der Werf MJ, Grzemska M, Hesseling AC, Curtis N, Graham SM, WHO Child TB Subgroup. 2016. Interrupted BCG vaccination is a major threat to global child health. Lancet Respir Med 4:251–253. doi:10.1016/S2213-2600(16)00099-0.
    1. Jasenosky LD, Scriba TJ, Hanekom WA, Goldfeld AE. 2015. T cells and adaptive immunity to Mycobacterium tuberculosis in humans. Immunol Rev 264:74–87. doi:10.1111/imr.12274.
    1. Lin PL, Flynn JL. 2015. CD8 T cells and Mycobacterium tuberculosis infection. Semin Immunopathol 37:239–249. doi:10.1007/s00281-015-0490-8.
    1. da Costa AC, Nogueira SV, Kipnis A, Junqueira-Kipnis AP. 2014. Recombinant BCG: innovations on an old vaccine. Scope of BCG strains and strategies to improve long-lasting memory. Front Immunol 5:152. doi:10.3389/fimmu.2014.00152.
    1. Lyadova IV, Panteleev AV. 2015. Th1 and Th17 cells in tuberculosis: protection, pathology, and biomarkers. Mediators Inflamm 2015:854507. doi:10.1155/2015/854507.
    1. Muller I, Cobbold SP, Waldmann H, Kaufmann SH. 1987. Impaired resistance to Mycobacterium tuberculosis infection after selective in vivo depletion of L3T4+ and Lyt-2+ T cells. Infect Immun 55:2037–2041.
    1. Murray RA, Mansoor N, Harbacheuski R, Soler J, Davids V, Soares A, Hawkridge A, Hussey GD, Maecker H, Kaplan G, Hanekom WA. 2006. Bacillus Calmette-Guerin vaccination of human newborns induces a specific, functional CD8+ T cell response. J Immunol 177:5647–5651. doi:10.4049/jimmunol.177.8.5647.
    1. Roy A, Eisenhut M, Harris RJ, Rodrigues LC, Sridhar S, Habermann S, Snell L, Mangtani P, Adetifa I, Lalvani A, Abubakar I. 2014. Effect of BCG vaccination against Mycobacterium tuberculosis infection in children: systematic review and meta-analysis. BMJ 349:g4643. doi:10.1136/bmj.g4643.
    1. Jason J, Archibald LK, Nwanyanwu OC, Kazembe PN, Chatt JA, Norton E, Dobbie H, Jarvis WR. 2002. Clinical and immune impact of Mycobacterium bovis BCG vaccination scarring. Infect Immun 70:6188–6195. doi:10.1128/IAI.70.11.6188-6195.2002.
    1. Schaible UE, Winau F, Sieling PA, Fischer K, Collins HL, Hagens K, Modlin RL, Brinkmann V, Kaufmann SH. 2003. Apoptosis facilitates antigen presentation to T lymphocytes through MHC-I and CD1 in tuberculosis. Nat Med 9:1039–1046. doi:10.1038/nm906.
    1. Grode L, Seiler P, Baumann S, Hess J, Brinkmann V, Nasser EA, Mann P, Goosmann C, Bandermann S, Smith D, Bancroft GJ, Reyrat JM, van Soolingen D, Raupach B, Kaufmann SH. 2005. Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guérin mutants that secrete listeriolysin. J Clin Invest 115:2472–2479. doi:10.1172/JCI24617.
    1. Hess J, Miko D, Catic A, Lehmensiek V, Russell DG, Kaufmann SH. 1998. Mycobacterium bovis bacille Calmette-Guerin strains secreting listeriolysin of Listeria monocytogenes. Proc Natl Acad Sci U S A 95:5299–5304. doi:10.1073/pnas.95.9.5299.
    1. Farinacci M, Weber S, Kaufmann SH. 2012. The recombinant tuberculosis vaccine rBCG ΔureC::hly+ induces apoptotic vesicles for improved priming of CD4+ and CD8+ T cells. Vaccine 30:7608–7614. doi:10.1016/j.vaccine.2012.10.031.
    1. Saiga H, Nieuwenhuizen N, Gengenbacher M, Koehler AB, Schuerer S, Moura-Alves P, Wagner I, Mollenkopf HJ, Dorhoi A, Kaufmann SH. 2015. The recombinant BCG ΔureC::hly vaccine targets the AIM2 inflammasome to induce autophagy and inflammation. J Infect Dis 211:1831–1841. doi:10.1093/infdis/jiu675.
    1. Kaufmann SH, Cotton MF, Eisele B, Gengenbacher M, Grode L, Hesseling AC, Walzl G. 2014. The BCG replacement vaccine VPM1002: from drawing board to clinical trial. Expert Rev Vaccines 13:619–630. doi:10.1586/14760584.2014.905746.
    1. Winau F, Weber S, Sad S, de Diego J, Hoops SL, Breiden B, Sandhoff K, Brinkmann V, Kaufmann SH, Schaible UE. 2006. Apoptotic vesicles crossprime CD8 T cells and protect against tuberculosis. Immunity 24:105–117. doi:10.1016/j.immuni.2005.12.001.
    1. Vogelzang A, Perdomo C, Zedler U, Kuhlmann S, Hurwitz R, Gengenbacher M, Kaufmann SH. 2014. Central memory CD4+ T cells are responsible for the recombinant bacillus Calmette-Guerin ΔureC::hly vaccine's superior protection against tuberculosis. J Infect Dis 210:1928–1937. doi:10.1093/infdis/jiu347.
    1. Desel C, Dorhoi A, Bandermann S, Grode L, Eisele B, Kaufmann SH. 2011. Recombinant BCG ΔureC hly+ induces superior protection over parental BCG by stimulating a balanced combination of type 1 and type 17 cytokine responses. J Infect Dis 204:1573–1584. doi:10.1093/infdis/jir592.
    1. Casanova JL, Blanche S, Emile JF, Jouanguy E, Lamhamedi S, Altare F, Stéphan JL, Bernaudin F, Bordigoni P, Turck D, Lachaux A, Albertini M, Bourrillon A, Dommergues JP, Pocidalo MA, Le Deist F, Gaillard JL, Griscelli C, Fischer A. 1996. Idiopathic disseminated bacillus Calmette-Guerin infection: a French national retrospective study. Pediatrics 98:774–778.
    1. Hesseling AC, Cotton MF, Jennings T, Whitelaw A, Johnson LF, Eley B, Roux P, Godfrey-Faussett P, Schaaf HS. 2009. High incidence of tuberculosis among HIV-infected infants: evidence from a South African population-based study highlights the need for improved tuberculosis control strategies. Clin Infect Dis 48:108–114. doi:10.1086/595012.
    1. Talbot EA, Perkins MD, Silva SF, Frothingham R. 1997. Disseminated bacille Calmette-Guerin disease after vaccination: case report and review. Clin Infect Dis 24:1139–1146. doi:10.1086/513642.
    1. Gengenbacher M, Nieuwenhuizen N, Vogelzang A, Liu H, Kaiser P, Schuerer S, Lazar D, Wagner I, Mollenkopf HJ, Kaufmann SH. 2016. Deletion of nuoG from the vaccine candidate Mycobacterium bovis BCG ΔureC::hly improves protection against tuberculosis. mBio 7:e00679-16. doi:10.1128/mBio.00679-16.
    1. Velmurugan K, Grode L, Chang R, Fitzpatrick ML, Laddy D, Hokey D, Derrick S, Morris S, McCown D, Kidd R, Gengenbacher M, Eisele B, Kaufmann SH, Fulkerson J, Brennan MJ. 2013. Nonclinical development of BCG replacement vaccine candidates. Vaccines (Basel) 1:120–138. doi:10.3390/vaccines1020120.
    1. Grode L, Ganoza CA, Brohm C, Weiner J III, Eisele B, Kaufmann SH. 2013. Safety and immunogenicity of the recombinant BCG vaccine VPM1002 in a phase 1 open-label randomized clinical trial. Vaccine 31:1340–1348. doi:10.1016/j.vaccine.2012.12.053.
    1. Srenathan U, Steel K, Taams LS. 2016. IL-17+ CD8+ T cells: differentiation, phenotype and role in inflammatory disease. Immunol Lett 178:20–26. doi:10.1016/j.imlet.2016.05.001.
    1. da Silva MV, Tiburcio MG, Machado JR, Silva DA, Rodrigues DB, Rodrigues V, Oliveira CJ. 2015. Complexity and controversies over the cytokine profiles of T helper cell subpopulations in tuberculosis. J Immunol Res 2015:639107. doi:10.1155/2015/639107.
    1. Hamada H, Garcia-Hernandez ML, Reome JB, Misra SK, Strutt TM, McKinstry KK, Cooper AM, Swain SL, Dutton RW. 2009. Tc17, a unique subset of CD8 T cells that can protect against lethal influenza challenge. J Immunol 182:3469–3481. doi:10.4049/jimmunol.0801814.
    1. Hamada H, Bassity E, Flies A, Strutt TM, Garcia-Hernandez ML, McKinstry KK, Zou T, Swain SL, Dutton RW. 2013. Multiple redundant effector mechanisms of CD8+ T cells protect against influenza infection. J Immunol 190:296–306. doi:10.4049/jimmunol.1200571.
    1. Clapp B, Yang X, Thornburg T, Walters N, Pascual DW. 2016. Nasal vaccination stimulates CD8+ T cells for potent protection against mucosal Brucella melitensis challenge. Immunol Cell Biol 94:496–508. doi:10.1038/icb.2016.5.
    1. Ravichandran J, Jackson RJ, Trivedi S, Ranasinghe C. 2015. IL-17A expression in HIV-specific CD8 T cells is regulated by IL-4/IL-13 following HIV-1 prime-boost immunization. J Interferon Cytokine Res 35:176–185. doi:10.1089/jir.2014.0078.
    1. . 1994. Practice parameter: management of hyperbilirubinemia in the healthy term newborn. Pediatrics 94:558–565.
    1. DuPreez K, Hesseling AC, Mandalakas AM, Marais BJ, Schaaf HS. 2011. Opportunities for chemoprophylaxis in children with culture-confirmed tuberculosis. Ann Trop Paediatr 31:301–310. doi:10.1179/1465328111Y.0000000035.
    1. Fan L, Xiao H, Mai G, Su B, Ernst J, Hu Z. 2015. Impaired Mycobacterium tuberculosis antigen-specific IFN-gamma response without IL-17 enhancement in patients with severe cavitary pulmonary tuberculosis. PLoS One 10:e0127087. doi:10.1371/journal.pone.0127087.
    1. Nunnari G, Pinzone MR, Vancheri C, Palermo F, Cacopardo B. 2013. Interferon-gamma and interleukin-17 production from PPD-stimulated PBMCs of patients with pulmonary tuberculosis. Clin Invest Med 36:E64–E71.
    1. Kozakiewicz L, Chen Y, Xu J, Wang Y, Dunussi-Joannopoulos K, Ou Q, Flynn JL, Porcelli SA, Jacobs WR Jr, Chan J. 2013. B cells regulate neutrophilia during Mycobacterium tuberculosis infection and BCG vaccination by modulating the interleukin-17 response. PLoS Pathog 9:e1003472. doi:10.1371/journal.ppat.1003472.
    1. Perreau M, Rozot V, Welles HC, Belluti-Enders F, Vigano S, Maillard M, Dorta G, Mazza-Stalder J, Bart PA, Roger T, Calandra T, Nicod L, Harari A. 2013. Lack of Mycobacterium tuberculosis-specific interleukin-17A-producing CD4+ T cells in active disease. Eur J Immunol 43:939–948. doi:10.1002/eji.201243090.
    1. Caccamo N, Guggino G, Meraviglia S, Gelsomino G, Di CP, Titone L, Bocchino M, Galati D, Matarese A, Nouta J, Klein MR, Salerno A, Sanduzzi A, Dieli F, Ottenhoff TH. 2009. Analysis of Mycobacterium tuberculosis-specific CD8 T cells in patients with active tuberculosis and in individuals with latent infection. PLoS One 4:e5528. doi:10.1371/journal.pone.0005528.
    1. Nunes-Alves C, Booty MG, Carpenter SM, Rothchild AC, Martin CJ, Desjardins D, Steblenko K, Kløverpris HN, Madansein R, Ramsuran D, Leslie A, Correia-Neves M, Behar SM. 2015. Human and murine clonal CD8+ T cell expansions arise during tuberculosis because of TCR selection. PLoS Pathog 11:e1004849. doi:10.1371/journal.ppat.1004849.
    1. Kagina BM, Abel B, Scriba TJ, Hughes EJ, Keyser A, Soares A, Gamieldien H, Sidibana M, Hatherill M, Gelderbloem S, Mahomed H, Hawkridge A, Hussey G, Kaplan G, Hanekom WA, other members of the South African Tuberculosis Vaccine Initiative. 2010. Specific T cell frequency and cytokine expression profile do not correlate with protection against tuberculosis, following BCG vaccination of newborns. Am J Respir Crit Care Med 182:1073–1079. doi:10.1164/rccm.201003-0334OC.
    1. Qiu Z, Zhang M, Zhu Y, Zheng F, Lu P, Liu H, Graner MW, Zhou B, Chen X. 2012. Multifunctional CD4 T cell responses in patients with active tuberculosis. Sci Rep 2:216. doi:10.1038/srep00216.
    1. Anderson ST, Kaforou M, Brent AJ, Wright VJ, Banwell CM, Chagaluka G, Crampin AC, Dockrell HM, French N, Hamilton MS, Hibberd ML, Kern F, Langford PR, Ling L, Mlotha R, Ottenhoff TH, Pienaar S, Pillay V, Scott JA, Twahir H, Wilkinson RJ, Coin LJ, Heyderman RS, Levin M, Eley B, ILULU Consortium, KIDS TB Study Group. 2014. Diagnosis of childhood tuberculosis and host RNA expression in Africa. N Engl J Med 370:1712–1723. doi:10.1056/NEJMoa1303657.
    1. Cliff JM, Lee JS, Constantinou N, Cho JE, Clark TG, Ronacher K, King EC, Lukey PT, Duncan K, Van Helden PD, Walzl G, Dockrell HM. 2013. Distinct phases of blood gene expression pattern through tuberculosis treatment reflect modulation of the humoral immune response. J Infect Dis 207:18–29. doi:10.1093/infdis/jis499.
    1. Kaufmann SH. 2013. Tuberculosis vaccines: time to think about the next generation. Semin Immunol 25:172–181. doi:10.1016/j.smim.2013.04.006.
    1. Ottenhoff TH, Kaufmann SH. 2012. Vaccines against tuberculosis: where are we and where do we need to go? PLoS Pathog 8:e1002607. doi:10.1371/journal.ppat.1002607.
    1. Hawn TR, Day TA, Scriba TJ, Hatherill M, Hanekom WA, Evans TG, Churchyard GJ, Kublin JG, Bekker LG, Self SG. 2014. Tuberculosis vaccines and prevention of infection. Microbiol Mol Biol Rev 78:650–671. doi:10.1128/MMBR.00021-14.
    1. Groschel MI, Prabowo SA, Cardona PJ, Stanford JL, van der Werf TS. 2014. Therapeutic vaccines for tuberculosis: a systematic review. Vaccine 32:3162–3168. doi:10.1016/j.vaccine.2014.03.047.
    1. World Health Organization. 2013. WHO Expert Committee on Biological Standardization—Annex 3: recommendations to assure the quality, safety and efficacy of BCG vaccines. Technical report series 979 2013. World Health Organization, Geneva, Switzerland.
    1. U.S. Department of Health and Human Services. 2009. Division of AIDS table for grading the severity of adult and pediatric adverse events, version 1.0. U.S. Department of Health and Human Services, Washington, DC: .
    1. Hanekom WA, Hughes J, Mavinkurve M, Mendillo M, Watkins M, Gamieldien H, Gelderbloem SJ, Sidibana M, Mansoor N, Davids V, Murray RA, Hawkridge A, Haslett PA, Ress S, Hussey GD, Kaplan G. 2004. Novel application of a whole blood intracellular cytokine detection assay to quantitate specific T-cell frequency in field studies. J Immunol Methods 291:185–195. doi:10.1016/j.jim.2004.06.010.
    1. Smyth GK. 2005. limma: linear models for microarray data, p 397–420. In Gentleman R, Carey VJ, Huber W, Irizarry RA, Dudoit S (ed), Bioinformatics and computational biology solutions using R bioconductor. Springer, New York, NY.
    1. Weiner J III, Domaszewska T. 2016. tmod: an R package for general and multivariate enrichment analysis. PeerJ Preprints 4:e2420v1.
    1. World Medical Association. 2013. Declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA 310:2191–2194. doi:10.1001/jama.2013.281053.

Source: PubMed

Sponsorer og samarbejdspartnere

Medicinske tilstande

Narkotikainterventioner

3
Abonner