Mycobacterium bovis BCG induces similar immune responses and protection by rectal and parenteral immunization routes

Abstract

We compared cellular immune responses to rectal, subcutaneous, and intradermal administration of Mycobacterium bovis BCG for 5 to 20 weeks in mice, guinea pigs, and macaques. Strong lymphoproliferative responses were induced in spleen cells after in vitro stimulation with purified protein derivative in guinea pigs and macaques, whatever the route of immunization. Comparable high numbers of gamma interferon- and tumor necrosis factor alpha-producing cells were found in the spleen after rectal, subcutaneous, and intradermal immunization of mice and macaques. Similar levels of precursors of cytotoxic T lymphocytes specific for mycobacterial antigens were observed in mice for all immunization routes. In macaques, cytotoxic activity, determined only at the end of the experiment (20 weeks), was similar after rectal and intradermal immunization. Six months after immunization, rectal and subcutaneous routes induced in mice similar levels of protective immunity against challenge with a virulent Mycobacterium tuberculosis strain (H37Rv). Rectal immunization gave immune responses and protective capacity similar to those for parenteral immunization and seemed to be a promising new route of vaccination against tuberculosis; in our study, immunization via the rectal route never induced side effects associated with parenteral routes (axillary adenitis) and could also effectively reduce the risks of viral transmission associated with unsafe injections in the developing world.

FIG. 1

Lymphoproliferative responses of guinea pig or macaque spleen cells stimulated with PPD, at various times after rectal and intradermal administration of BCG. Spleen cells collected from guinea pigs (A) or macaques (B) nonimmunized (empty bars) or immunized with BCG via rectal (fine hatches) or intradermal (coarse hatches) routes were stimulated by incubation for 4 days in vitro with PPD (10 μg/ml). The SI were calculated as (counts per minute with PPD)/(counts per minute without PPD). Results in guinea pigs (n = 4) and macaques (n = 3) (geometric means of triplicate determinations [error bars, standard deviations]) are representative of two different experiments.

FIG. 2

IFN-γ- and TNF-α-producing cells in the spleens of mice and macaques at various times after rectal, intradermal, or subcutaneous administration of BCG. Spleen cells collected from mice (A and C) or macaques (B and D) nonimmunized (empty bars) or immunized with BCG via rectal (fine hatches) or parenteral (coarse hatches) routes were stimulated in vitro with PPD (10 μg/ml) in the wells of nitrocellulose plates that were coated with an anti-mouse IFN-γ, an anti-mouse TNF-α, an anti-human IFN-γ, or an anti-human TNF-α antibody. The SFC were developed as indicated in Materials and Methods. Results in mice (n = 6) and macaques (n = 3) are representative of two different experiments (error bars, standard deviations).

FIG. 3

Precursors of cytotoxic T lymphocytes in the spleen after rectal or subcutaneous administration of BCG to BALB/c mice are mediated by CD8 T cells and are MHC class I restricted. (A) Spleens (n = 6) were collected at 5, 8, 12, or 20 weeks from nonimmunized (○) rectally (▴) or subcutaneously (▾) immunized mice. The cells were stimulated by incubating in vitro in the presence of PPD for 5 days. Their cytotoxic activity was tested against P815 cells presenting mycobacterial antigens after an osmotic shock in the presence of crude BCG culture filtrate. The results are representative of two different experiments. (B) Spleens (n = 6) were collected 12 weeks after immunization of BALB/c mice (H-2^d) via the rectal or subcutaneous (Sc) routes. Cells were incubated in vitro in the presence of PPD (10 μg/ml) for 5 days and their cytotoxic activity was tested against P815 (H-2^d) target cells in the presence of medium only (▴, ▾) or of anti-CD8 (▵, ▿) or anti-CD4 (●) MAbs. The cytotoxic activity of spleen cells was also tested against EL4 (H-2^b) target cells (□).

FIG. 4

Precursors of cytotoxic T lymphocytes in the spleen 20 weeks after rectal or intradermal administration of BCG to macaques. Cells were collected from spleen biopsies 20 weeks after immunization of macaques (n = 3) via the rectal (▴) or intradermal (▾) route. The cells were stimulated by incubating in vitro in the presence of PPD (10 μg/ml) for 5 days. Their cytotoxic activity was tested against syngenic macrophages obtained from bronchoalveolar lavages, which had taken up BCG by phagocytosis 24 h before the assay. The results are representative of two different experiments. Error bars, standard deviations.

FIG. 5

Assay of immune protection induced in mice 6 months after immunization by rectal or subcutaneous administration of BCG. Results are shown for BALB/c mice (n = 8-10) nonimmunized (empty bars) or immunized with BCG by the rectal (fine hatches) or by subcutaneous injection (coarse hatches) routes. Six months later, the mice were challenged intravenously with 10⁵ viable units of the virulent M. tuberculosis strain H37Rv. The spleen and lungs were collected 4 weeks later and dissociated. Their bacterial loads were evaluated after appropriate dilutions and growth on Middlebrook 7H11 agar medium. Results are expressed as geometric mean + standard deviation (error bar) ∗∗∗, P < 0.001.