Induction of a specific strong polyantigenic cellular immune response after short-term chemotherapy controls bacillary reactivation in murine and guinea pig experimental models of tuberculosis

Abstract

RUTI is a therapeutic vaccine that is generated from detoxified and liposomed Mycobacterium tuberculosis cell fragments that has demonstrated its efficacy in the control of bacillus reactivation after short-term chemotherapy. The aim of this study was to characterize the cellular immune response generated after the therapeutic administration of RUTI and to corroborate the lack of toxicity of the vaccine. Mouse and guinea pig experimental models were infected with a low-dose M. tuberculosis aerosol. RUTI-treated animals showed the lowest bacillary load in both experimental models. RUTI also decreased the percentage of pulmonary granulomatous infiltration in the mouse and guinea pig models. This was not the case after Mycobacterium bovis BCG treatment. Cellular immunity was studied through the characterization of the intracellular gamma interferon (IFN-gamma)-producing cells after the splenocytes' stimulation with M. tuberculosis-specific structural and growth-related antigens. Our data show that the difference between the therapeutic administration of BCG and RUTI resides mainly in the stronger activation of IFN-gamma(+) CD4(+) cells and CD8(+) cells against tuberculin purified protein derivative, ESAT-6, and Ag85B that RUTI generates. Both vaccines also triggered a specific immune response against the M. tuberculosis structural antigens Ag16kDa and Ag38kDa and a marked mRNA expression of IFN-gamma, tumor necrosis factor, interleukin-12, inducible nitric oxide synthase, and RANTES in the lung. The results show that RUTI's therapeutic effect is linked not only to the induction of a Th1 response but also to the stimulation of a quicker and stronger specific immunity against structural and growth-related antigens that reduces both the bacillary load and the pulmonary pathology.

Figures

FIG. 1.

Experiment designs. In both experimental animal models, animals were infected with a low-dose H37Rv aerosol. Treatment with immunotherapy (IT) with RUTI or BCG was administered after a short-term chemotherapy with INH plus RPN in mice or INH plus RIF in guinea pigs. The experiments were repeated twice in mice. The first guinea pig experiment was stopped at week 16 for the determination of the number of CFU. In this case, only two experimental groups were included (control and RUTI treated). A BCG treatment group was included in the experiment that was devoted to the pathology study, which ended at week 24.

FIG. 2.

Bacillary load in lungs of aerosol-infected mice and guinea pigs. (A) After infection, mice were treated with INH/RPN from weeks 6 to 14 (in white) and with two subcutaneous inoculations of RUTI at weeks 14 and 17 (in black) or BCG at week 14 (in gray). (B) After infection, guinea pigs were treated with INH/RIF from weeks 4 to 8 (in white) and with two subcutaneous inoculations of RUTI at weeks 8 and 11 (in black). The results are given as mean values with SD obtained from 4 (weeks 6 and 14) to 12 mice or three (weeks 4 and 8) to six guinea pigs for each time point. An asterisk indicates significant differences (P < 0.05 by t test). The color of the asterisk corresponds to the group with lower values to which the comparison was significant (i.e., black asterisks refer to the RUTI group).

FIG. 3.

Quantification of granulomatous infiltration in aerosol-infected mice's lung. After infection, mice were treated with INH/RPN from weeks 6 to 14 (in white) and with two subcutaneous inoculations of RUTI at weeks 14 and 17 (in black) or BCG at week 14 (in gray). Also shown are the percentages of pulmonary infiltration, which were obtained by dividing the area of granulomatous infiltration by the total area of the lobes, multiplied by 100. Values represent the means and SD. An asterisk indicates significant differences (P < 0.05 by t test). The color of the asterisk corresponds to the group with lower values to which the comparison was significant. Black and gray asterisks refer to RUTI and BCG groups, respectively.

FIG. 4.

Evolution of CD4+ and CD8+ IFN-γ+-specific cells from spleen in aerosol-infected mice. After infection, mice were treated with INH/RPN from weeks 6 to 14 (in white) and with two subcutaneous inoculations of RUTI at weeks 14 and 17 (in black) or BCG at week 14 (in gray). Data are expressed as the percentages of means and SD. An asterisk or ampersand indicates significant differences (P < 0.05 by t test). The color of the asterisk corresponds to the group with lower values to which the comparison was significant. Black and gray asterisks refer to RUTI and BCG groups, respectively; the ampersand refers to the control group.

FIG. 5.

Activated antigen-specific IFN-γ-secreting cells from the spleen of aerosol-infected mice. After infection, mice were treated with INH/RPN from weeks 6 to 14 (in white) and with two subcutaneous inoculations of RUTI at weeks 14 and 17 (w14 and w17, respectively; in black) or BCG at week 14 (in gray). Data represent the means and SD. An asterisk or ampersand indicates significant differences (P < 0.05 by t test). The color of the asterisk corresponds to the group with lower values to which the comparison was significant. Gray asterisks refer to the BCG group; the ampersand refers to the control group.

FIG. 6.

Evolution of mRNA expression of IFN-γ, TNF-α, IL-12, iNOS, and RANTES in lungs at different time points. After infection, mice were treated with INH/RPN from weeks 6 to 14 (in white) plus two subcutaneous inoculations of RUTI at weeks 14 and 17 (in black) or BCG at week 14 (in gray). Data are expressed as the log10 of the ratio obtained after dividing every value by the expression of HPRT in each sample and multiplying it by a factor (ranging from 101 to 106). Data represent the means and SD. An asterisk or ampersand indicates significant differences (P < 0.05 by t test). The color of the asterisk corresponds to the group with lower values to which the comparison was significant. Black and gray asterisks refer to RUTI and BCG groups, respectively; the ampersand refers to the control group.