An in vivo comparison of bacillus Calmette-Guérin (BCG) and cytokine-secreting BCG vaccines

Abstract

A recombinant bacillus Calmette-Guérin (BCG) vaccine has been developed, which constitutively secretes interleukin (IL)-2. Groups of deer were immunized with either normal BCG (Pasteur 1173 P2 strain) or recombinant BCG (rBCG/IL-2) and their immune responses were monitored over 3 months. Animals gained weight over this period and showed no signs of adverse reactions to either vaccine. Lymphocyte transformation responses did not differ significantly between the two groups. No antibody that was specific for BCG was detected in any animal. Intradermal skin-test responses to BCG antigens showed that the rBCG/IL-2 induced a smaller delayed-type hypersensitivity response than the normal BCG. Cytokine transcription was determined by reverse transcription-polymerase chain reaction (RT-PCR). While IL-2 and interferon-gamma (IFN-gamma) levels did not differ significantly between the two groups, the level of IL-4 was found to be lower in the group given rBCG/IL-2. This resulted in a strong interferon-gamma:IL-4 ratio, suggesting a skewing of the immune response towards a Type 1 response. The rate at which the vaccine was eliminated from the host was the same regardless of whether BCG or rBCG was used. At autopsy (3 months after vaccination) 99.99% of the organisms had been eliminated. The small number of organisms isolated from the draining lymph node of animals given rBCG/IL-2 were grown in antibiotic-containing media. They were shown to still contain the shuttle plasmid and to secrete biologically active IL-2, indicating that the plasmid was stably maintained despite the host's immune response and in the absence of antibiotic selection.

Figure 1

The immunopotentiating effect of recombinant interleukin 2 (rIL-2) on in vitro antigen-specific lymphocyte transformation responses. Peripheral blood lymphocytes from tuberculous (diseased) or non-diseased, skin-test positive, in-contact (resistant) animals were cultured with antigen from Mycobacterium bovis. An optimal concentration (10 μg/ml) of rIL-2 was given to one set of cultures, while media alone was given to the control cultures.

Figure 2

The delayed-type hypersensitivity (DTH) responses to specific and non-specific antigens were measured in animals 3 months after vaccination with either bacillus Calmette–Guérin (BCG) or recombinant BCG secreting interleukin-2(rBCG). DTH responses to intradermal injection of antigens from either Mycobacterium bovis (bovine purified-protein derivative) or M. avium (avian purified-protein derivative) were measured as skin thickening (mm). The data represents the mean±SE of the group (n = 6).

Figure 3

The peripheral blood lymphocyte responses to Mycobacterium bovis (PPD) antigens were measured in animals vaccinated with either bacillus Calmette–Guérin (BCG) or recombinant BCG (rBCG). Blood samples were taken at 2, 4, 8, 12 and 14 weeks. The lymphocytes were assayed for antigen-specific lymphocyte transformation. The data represents the mean±SE of the group (n = 6).

Figure 4

The transcription of interleukin-2 (IL-2), interleukin-4 (IL-4) and interferon-γ (IFN-γ) mRNA was measured after antigen restimulation of lymphocytes from animals vaccinated 8 weeks (a) and 14weeks (b) previously with either bacillus Calmette–Guérin (BCG) or recombinant BCG (rBCG). Units of mRNA were calculated from autoradiographs of probed blots of electrophoresed PCR product. The optical density of the product was then standardized to a control mRNA (β-actin). The data represents the mean±SE of the results from each group (n = 6).

Figure 5

Expression of interleukin-2 (IL-2) was measured from recombinant bacillus Calmette–Guérin (BCG) (rBCG+IL-2) that had been isolated from the lymph nodes of animals vaccinated 14 weeks previously. Levels of IL-2 activity were measured in the culture supernatants of colonies of rBCG grown to OD₆₀₀ = 1. Supernatants were tested for their ability to induce proliferation in peripheral blood lymphocytes suboptimally stimulated with concanavalin A (Con A blast assay). Doubling dilutions of supernatants from rBCG plus IL-2 were compared to recombinant BCG with the IL-2 gene deleted (BCG minus IL-2) and purified recombinant IL-2 protein (rIL-2).