Influence of advanced age on Mycobacterium bovis BCG vaccination in guinea pigs aerogenically infected with Mycobacterium tuberculosis

Abstract

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only tuberculosis (TB) vaccine currently available, but its efficacy against adult pulmonary TB remains controversial. BCG induces specific immune responses to mycobacterial antigens and may elicit protective immunity against TB. TB remains a major public health problem, especially among the elderly, yet the efficacy of BCG in the elderly is unknown. We investigated the ability of BCG vaccination to prevent TB in young (6-week-old), middle-aged (18-month-old), and old (60-month-old) guinea pigs. BCG-Tokyo vaccination reduced the growth of Mycobacterium tuberculosis H37Rv in all three groups. By use of an enzyme-linked immunospot (ELISPOT) assay, antigen-specific gamma interferon (IFN-γ)-producing cells were detected in the 60-month-old guinea pigs after a booster vaccination with BCG-Tokyo. Our findings suggest that BCG-Tokyo has a protective effect against tuberculosis infection regardless of age.

FIG. 1.

Experimental design. Guinea pigs in the old group were vaccinated with BCG at the age of 6 weeks and were then maintained for 60 months before revaccination with BCG 6 weeks before M. tuberculosis infection (group 1). Guinea pigs in the middle-aged groups either were vaccinated with BCG at the age of 6 weeks and were then maintained for 18 months before infection (group 2) or were not vaccinated with BCG until 6 weeks before infection (group 3). Guinea pigs in the young groups either were vaccinated 6 weeks before infection (group 4) or were not vaccinated (group 5).

FIG. 2.

PPD skin reactions of the guinea pigs. DTH was assessed on the basis of the skin reactions of guinea pigs 6 or 11 weeks (6w or 11w) after BCG inoculation both before and after challenge with M. tuberculosis. Error bars represent standard deviations. No significant difference among the groups vaccinated with BCG was observed.

FIG. 3.

BCG-induced PPD-specific T-cell responses. To investigate T-cell functions specific for PPD in groups 1, 4, and 5, an IFN-γ ELISPOT assay was performed at weeks zero, 6, and 11 after BCG vaccination. Error bars represent standard deviations. Asterisks indicate that the mean numbers of IFN-γ SFCs were significantly different. **, P < 0.01, as determined by analysis of variance (ANOVA) followed by a posthoc Tukey-Kramer test. There was no difference in IFN-γ SFCs between the old and young guinea pigs vaccinated with BCG.

FIG. 4.

Effects of BCG vaccination on bacterial growth in young, middle-aged, and old guinea pigs challenged with M. tuberculosis H37Rv. To determine the impact of BCG vaccination on bacterial growth, bacterial replication in lung (A), tracheal lymph node (B), and spleen (C) specimens from each guinea pig was examined. The minimum detectable level of bacilli in the tissue homogenate was 1.52 log₁₀ CFU. Error bars represent standard deviations. Asterisks indicate that the mean numbers of M. tuberculosis CFU in an organ were significantly different. *, P < 0.05, and **, P < 0.01, as determined by analysis of variance (ANOVA) followed by a posthoc Tukey-Kramer test.

FIG. 5.

Correlation between IFN-γ production and bacterial growth in the lungs. There was a statistically significant negative correlation between the number of IFN-γ SFCs detected 5 weeks after M. tuberculosis challenge and bacterial growth in the lungs (r, −0.6696; P, 0.04852 as determined by Pearson's correlation coefficient test). Circles, group 4 (young, vaccinated); squares, group 5 (young, unvaccinated); triangles: group 1 (old, revaccinated).

FIG. 6.

Histopathology of lungs from guinea pigs infected with M. tuberculosis H37Rv. Shown are histopathological observations in the lungs of young unvaccinated (a and b), young BCG-vaccinated (c), and old BCG-revaccinated (d) guinea pigs 5 weeks after M. tuberculosis challenge. Bars, 1 mm (a, c, and d) and 50 μm (b).