• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of cviPermissionsJournals.ASM.orgJournalAEM ArticleJournal InfoAuthorsReviewers
Clin Vaccine Immunol. Feb 2007; 14(2): 198–200.
Published online Dec 20, 2006. doi:  10.1128/CVI.00309-06
PMCID: PMC1797790

Dose-Dependent Immune Response to Mycobacterium bovis BCG Vaccination in Neonates[down-pointing small open triangle]

Abstract

In 10-week-old infants vaccinated at birth with Japanese Mycobacterium bovis BCG, the number of dermal needle penetrations correlated positively with frequency of proliferating CD4+ T cells in whole blood following BCG stimulation for 6 days but did not correlate with secreted cytokine levels after 7 h or interferon CD4+ T-cell frequency after 12 h of BCG stimulation.

The Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine is the only vaccine against tuberculosis (TB). Various BCG isolates, with well-characterized genetic differences, are currently used as commercial vaccines (1, 3, 9). In addition, different routes of BCG administration are used for vaccine delivery: i.e., intradermal injection or percutaneous puncture of the BCG-exposed skin by a multiprong device. The relative efficacies of the different strains of BCG and the different routes of vaccination are not clearly defined.

Protection against TB is dependent on T-cell-mediated immunity (4, 6-8, 12, 21, 24). The helper type 1 (Th1) CD4+ T cell producing interferon-gamma (IFN-γ) appears to be important for protection during primary infection with Mycobacterium tuberculosis and other mycobacteria (2, 5, 11, 18, 22). Similarly, following newborn and adult BCG vaccination, isolated peripheral blood mononuclear cells respond to ex vivo mycobacterial antigen stimulation by CD4+ T-cell proliferation and gamma interferon (IFN-γ) production. Therefore, these host responses have been widely used to measure antigen-specific memory T-cell immunity induced by BCG vaccination (16, 17, 20, 23).

Using two recently described short-term whole-blood assays and one 6-day T-cell proliferation assay to investigate BCG-induced T-cell immune responses in vaccinated neonates (10, 13, 14), we examined whether coincidental variation in the relative amounts of antigen introduced into the epidermis upon percutaneous administration of the Japanese BCG (JBCG) vaccine at birth correlates with any of the immune responses measured 10 weeks postvaccination.

Enumeration of the number of epidermal needle punctures following vaccination of neonates.

Thirty-one infants vaccinated at birth with JBCG (Tokyo-172 BCG substrain, from Japan BCG Laboratory) administered percutaneously were recruited into the study at their routine 10-week visit to the primary health care clinic in the Western Cape Province of South Africa. The study protocol was approved by the Institutional Review Boards of the University of Cape Town and the Public Health Research Institute/University of Medicine and Dentistry of New Jersey, and their respective human experimentation guidelines were adhered to. Written informed consent was obtained from the infant's parent or legal guardian. Human immunodeficiency virus-positive (by enzyme-linked immunosorbent assay) newborns and neonates exposed to a TB-positive index case during the first 10 weeks of life were excluded. The percutaneous immunization involved evenly spreading a drop containing 80 mg/ml JBCG (3 × 109 CFU/ml) over the skin followed by two superficial perforations of the epidermis with a nine-prong device (Japan BCG Laboratory) (Fig. (Fig.1A).1A). The numbers of needle puncture scars visible on each infant's forearm (Fig. (Fig.1B)1B) 10 weeks after neonatal vaccination were graphically recorded (Fig. (Fig.1C,1C, insert) and counted. Sixty-seven percent of infants (22 individuals) showed 18 clearly demarcated scars (the maximal number) (Fig. (Fig.1C).1C). Thirty-three percent of infants (9 individuals) had fewer than 18 scars visible (but no less than 15). Thus, it may be presumed that in 33% of infants the dose of JBCG inoculated into the skin was less than optimal.

FIG. 1.
(A) Multipuncture device used to administer the JBCG vaccine percutaneously. (B) Vaccination scars in a 10-week-old infant, vaccinated at birth with JBCG administered percutaneously. (C) Frequency histogram of the number of needle scars visible for each ...

BCG-specific T-cell intracellular IFN-γ and soluble cytokine production (short-term stimulation assays).

The frequency of mycobacterium-specific CD4+ T cells (as a percentage of total CD4+ cells) in peripheral blood of 10-week-old vaccinated infants was determined by quantifying IFN-γ+ CD4+ cells after short-term (12 h) ex vivo stimulation of whole blood with JBCG using flow cytometric detection of surface marker and intracellular cytokine (10, 14). Results were plotted against the number of vaccine scars visible on the arm for each infant (Fig. (Fig.1D).1D). Correlation analysis indicated that the relative dose of percutaneously administered JBCG, as assessed by the number of vaccine needle scars, did not predict the frequency of IFN-γ+ CD4+ T cells (Fig. (Fig.1D1D and Table Table1).1). A similar lack of correlation was observed when Danish BCG (DBCG) or purified protein derivative (PPD) was used for ex vivo stimulation of the whole blood to induce IFN-γ+ CD4+ T cells (Table (Table1).1). The frequency of IFN-γ+ CD8+ T cells induced by ex vivo exposure to JBCG, DBCG, or PPD also did not correlate with the number of scars. The levels of the six secreted cytokines and the IFN-γ/interleukin-4 (IL-4) cytokine ratio, measured by flow cytometric bead array kit (Th1/Th2 cytometric bead array; BD Biosciences) in the supernatants after 7 h of ex vivo stimulation of whole blood with JBCG or DBCG (10, 14), were also not significantly influenced by the dose of JBCG inoculated into the skin (Table (Table11).

TABLE 1.
Correlation between Japanese BCG vaccine scar number and ex vivo immune assay results

BCG-specific T-cell proliferation (6-day stimulation assay).

We next examined antigen-specific lymphoproliferation, using a novel whole-blood assay in which bromodeoxyuridine (BrdU) is incorporated into proliferating cells after 6 days of exposure to JBCG, DBCG, or PPD in culture. BrdU-positive cells were identified by flow cytometry (10). When this assay was used, the percentage of CD4+ T cells that proliferated in response to both JBCG and DBCG, as well as to PPD, correlated positively with the number of vaccine scars recorded (P < 0.05) (Fig. (Fig.1E1E and Table Table1).1). The frequency of CD8+ T cells proliferating in response to ex vivo stimulation with JBCG, DBCG, or PPD did not correlate significantly with vaccine dose (Table (Table1).1). These results are supported by a previous study by Lowry et al. looking at the effect of four different doses of percutaneous BCG on the cellular immune response in adults (19).

Twelve-hour (short term) stimulation of blood allows quantification of vaccination-induced effector/effector memory T cells that produce IFN-γ and other cytokines, without in vitro expansion of the T cells (14). When this short-term assay was compared with the 6-day whole-blood proliferation assay, the two assays did not correlate (10). This suggested that the “immediate” (12 h) effector/effector memory responses, observed prior to any ex vivo proliferation taking place, were different from the 6-day proliferating, central memory CD4+ T-cell response. Thus, T cells primed to produce IFN-γ (as part of the immediate effector/effector memory response) after only a few hours of ex vivo mycobacterial stimulation may belong to a different subset of T cells from those primed to proliferate following longer ex vivo stimulation. It seems, therefore, that the short- and long-term assays measure different aspects of BCG-induced immunity. Similarly, Hoft et al. have reported that different immunologic assays measure unique aspects of mycobacterial immunity (15). This suggests that only one of the T-cell populations evaluated (i.e., the proliferating central memory CD4+ T cells) is sensitive to small changes in the dose of antigen delivered into the skin during vaccination.

Acknowledgments

This work was supported by the AERAS Global Tuberculosis Vaccine Foundation. Partial funding was provided by grants from the National Institutes of Health NIAID (RO1 AI-22616 and RO1 AI-54361 to G.K. and RO1 AI-065653 to W.H.) and by NRF and DAAD fellowships (to V.D.). Additional funding was provided by the EDCTP and the Dana Foundation (to W.H) and by the Medical Research Council Tuberculosis Vaccine Initiative (to S.R.R.).

There are no potential conflicts of interest for the authors of this study.

Footnotes

[down-pointing small open triangle]Published ahead of print on 20 December 2006.

REFERENCES

1. Anonymous. 2004. BCG vaccine. WHO position paper. Wkly. Epidemiol. Rec. 79:27-38. [PubMed]
2. Barnes, P. F., S. Lu, J. S. Abrams, E. Wang, M. Yamamura, and R. L. Modlin. 1993. Cytokine production at the site of disease in human tuberculosis. Infect. Immun. 61:3482-3489. [PMC free article] [PubMed]
3. Behr, M. A., and P. M. Small. 1999. A historical and molecular phylogeny of BCG strains. Vaccine 17:915-922. [PubMed]
4. Caruso, A. M., N. Serbina, E. Klein, K. Triebold, B. R. Bloom, and J. L. Flynn. 1999. Mice deficient in CD4 T cells have only transiently diminished levels of IFN-gamma, yet succumb to tuberculosis. J. Immunol. 162:5407-5416. [PubMed]
5. Casanova, J. L., and L. Abel. 2002. Genetic dissection of immunity to mycobacteria: the human model. Annu. Rev. Immunol. 20:581-620. [PubMed]
6. Castillo-Rodal, A. I., M. Castañón-Arreola, R. Hernández-Pando, J. J. Calva, E. Sada-Diaz, and Y. López-Vidal. 2006. Mycobacterium bovis BCG substrains confer different levels of protection against Mycobacterium tuberculosis infection in a BALB/c model of progressive pulmonary tuberculosis. Infect. Immun. 74:1718-1724. [PMC free article] [PubMed]
7. Chackerian, A. A., T. V. Perera, and S. M. Behar. 2001. Gamma interferon-producing CD4+ T lymphocytes in the lung correlate with resistance to infection with Mycobacterium tuberculosis. Infect. Immun. 69:2666-2674. [PMC free article] [PubMed]
8. Chen, L., J. Wang, A. Zganiacz, and Z. Xing. 2004. Single intranasal mucosal Mycobacterium bovis BCG vaccination confers improved protection compared to subcutaneous vaccination against pulmonary tuberculosis. Infect. Immun. 72:238-246. [PMC free article] [PubMed]
9. Corbel, M. J., U. Fruth, E. Griffiths, and I. Knezevic. 2004. Report on a WHO consultation on the characterisation of BCG strains, Imperial College, London, 15-16 December 2003. Vaccine 22:2675-2680. [PubMed]
10. Davids, V., W. A. Hanekom, N. Mansoor, H. Gamieldien, S. J. Gelderbloem, A. Hawkridge, G. D. Hussey, E. J. Hughes, J. Soler, R. A. Murray, S. R. Ress, and G. Kaplan. 2006. The effect of bacille Calmette-Guerin vaccine strain and route of administration on induced immune responses in vaccinated infants. J. Infect. Dis. 193:531-536. [PubMed]
11. Doffinger, R., S. Dupuis, C. Picard, C. Fieschi, J. Feinberg, G. Barcenas-Morales, and J. L. Casanova. 2002. Inherited disorders of IL-12- and IFNgamma-mediated immunity: a molecular genetics update. Mol. Immunol. 38:903-909. [PubMed]
12. Goter-Robinson, C., S. C. Derrick, A. L. Yang, B. Y. Jeon, and S. L. Morris. 2006. Protection against an aerogenic Mycobacterium tuberculosis infection in BCG-immunized and DNA-vaccinated mice is associated with early type I cytokine responses. Vaccine 24:3522-3529. [PubMed]
13. Hanekom, W. A. 2005. The immune response to BCG vaccination of newborns. Ann. N. Y. Acad. Sci. 1062:69-78. [PubMed]
14. Hanekom, W. A., J. Hughes, M. Mavinkurve, M. Mendillo, M. Watkins, H. Gamieldien, S. J. Gelderbloem, M. Sidibana, N. Mansoor, V. Davids, R. A. Murray, A. Hawkridge, P. A. Haslett, S. Ress, G. D. Hussey, and G. Kaplan. 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. [PubMed]
15. Hoft, D. F., S. Worku, B. Kampmann, C. C. Whalen, J. J. Ellner, C. S. Hirsch, R. B. Brown, R. Larkin, Q. Li, H. Yun, and R. F. Silver. 2002. Investigation of the relationships between immune-mediated inhibition of mycobacterial growth and other potential surrogate markers of protective Mycobacterium tuberculosis immunity. J. Infect. Dis. 186:1448-1457. [PubMed]
16. Hussey, G. D., M. L. Watkins, E. A. Goddard, S. Gottschalk, E. J. Hughes, K. Iloni, M. A. Kibel, and S. R. Ress. 2002. Neonatal mycobacterial specific cytotoxic T-lymphocyte and cytokine profiles in response to distinct BCG vaccination strategies. Immunology 105:314-324. [PMC free article] [PubMed]
17. Kemp, E. B., R. B. Belshe, and D. F. Hoft. 1996. Immune responses stimulated by percutaneous and intradermal bacille Calmette-Guerin. J. Infect. Dis. 174:113-119. [PubMed]
18. Koscielniak, E., T. de Boer, S. Dupuis, L. Naumann, J. L. Casanova, and T. H. Ottenhoff. 2003. Disseminated Mycobacterium peregrinum infection in a child with complete interferon-gamma receptor-1 deficiency. Pediatr. Infect. Dis. J. 22:378-380. [PubMed]
19. Lowry, P. W., T. S. Ludwig, J. A. Adams, M. L. Fitzpatrick, S. M. Grant, G. A. Andrle, M. R. Offerdahl, S. N. Cho, and D. R. Jacobs, Jr. 1998. Cellular immune responses to four doses of percutaneous bacille Calmette-Guerin in healthy adults. J. Infect. Dis. 178:138-146. [PubMed]
20. Marchant, A., T. Goetghebuer, M. O. Ota, I. Wolfe, S. J. Ceesay, D. De Groote, T. Corrah, S. Bennett, J. Wheeler, K. Huygen, P. Aaby, K. P. McAdam, and M. J. Newport. 1999. Newborns develop a Th1-type immune response to Mycobacterium bovis bacillus Calmette-Guerin vaccination. J. Immunol. 163:2249-2255. [PubMed]
21. Ordway, D. J., L. Costa, M. Martins, H. Silveira, L. Amaral, M. J. Arroz, F. A. Ventura, and H. M. Dockrell. 2004. Increased interleukin-4 production by CD8 and gammadelta T cells in health-care workers is associated with the subsequent development of active tuberculosis. J. Infect. Dis. 190:756-766. [PubMed]
22. Ottenhoff, T. H., T. De Boer, J. T. van Dissel, and F. A. Verreck. 2003. Human deficiencies in type-1 cytokine receptors reveal the essential role of type-1 cytokines in immunity to intracellular bacteria. Adv. Exp. Med. Biol. 531:279-294. [PubMed]
23. Ravn, P., H. Boesen, B. K. Pedersen, and P. Andersen. 1997. Human T cell responses induced by vaccination with Mycobacterium bovis bacillus Calmette-Guerin. J. Immunol. 158:1949-1955. [PubMed]
24. Turner, J., and I. M. Orme. 2004. The expression of early resistance to an infection with Mycobacterium tuberculosis by old mice is dependent on IFN type II (IFN-gamma) but not IFN type I. Mech. Ageing Dev. 125:1-9. [PubMed]

Articles from Clinical and Vaccine Immunology : CVI are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...