Failure to induce enhanced protection against tuberculosis by increasing T-cell-dependent interferon-gamma generation

Abstract

We evaluated the use of recombinant human interleukin-6 (rhIL-6) and a monoclonal antibody specific for interferon-gamma (IFN-gamma) as co-adjuvants in a subunit vaccine against tuberculosis consisting of the culture filtrate proteins of Mycobacterium tuberculosis (ST-CF) emulsified in the adjuvant dimethyl-dioctadecylammonium bromide (DDA). Both the addition of rhIL-6 and the neutralization of IFN-gamma resulted in an increased T helper type 1 (Th1) response characterized by enhanced IFN-gamma production and cell proliferation. Nevertheless, this did not result in the enhancement of protection against either an intravenous or an aerosol M. tuberculosis challenge. Our data stress the need to identify further correlates of protection in addition to IFN-gamma production to screen vaccines against tuberculosis infection.

Figure 1

Effects of the early addition of rhIL-6 in the priming of T cells during the immunization of C57BL/6 with ST-CF in DDA. C57BL/6 mice were immunized twice with the ST-CF/DDA vaccine (Vac) or injected with PBS/DDA (Non-Vac) at 2-week interval. Recombinant human IL-6 (30 µg per dose) was administered with the first immunization and in the two subsequent days by subcutaneous injection. Mouse spleens (three per group) were collected 3 weeks after the last immunization and the cells were pooled and cultured in triplicate wells, in vitro, with (shaded bars) or without (white bars) ST-CF (4 µg/ml). The amount of IFN-γ secreted as well as lymphoproliferation were evaluated (at 96 or 48 hr, respectively) by enzyme-linked immunosorbent assay and radioactive thymidine incorporation, respectively. (ND, not detectable). Statistical analysis was performed using Student's t-test (**P < 0·01, ***P < 0·001). The statistical analysis between the vaccinated group and the vaccinated group treated with IL-6 are shown.

Figure 2

Effects of the neutralization of IFN-γ in the priming of T cells during the immunization of C57BL/6 with ST-CF in DDA. C57BL/6 mice were immunized subcutaneously, three times at weekly intervals with ST-CF/DDA (50 µg and 250 µg, respectively, in 0·2 ml) (Vac) or with PBS/DDA (Non-Vac) and a monoclonal antibody specific for IFN-γ (anti-IFN-γ) or an irrelevant immunoglobulin preparation was administered 2 hr before the first and third immunizations, by i.p. injection (2 mg/animal). Mouse spleens (three per group) were collected 3 weeks after the last immunization and the cells were pooled and cultured in triplicate wells, in vitro, with (shaded bars) or without (white bars) ST-CF (4 µg/ml). The amount of IFN-γ secreted as well as lymphoproliferation were evaluated (at 96 or 48 hr, respectively) by enzyme-linked immunosorbent assay and radioactive thymidine incorporation, respectively. (ND, not detectable). Statistically significant effects of the antibody treatment are indicated by * (for P < 0·05), according to Student's t-test. The statistical analysis between the vaccinated group and the vaccinated group treated with anti-IFN-γ are shown.

Figure 3

Effect of the early addition of rhIL-6 or IFN-γ neutralization on the protection conferred by the ST-CF/DDA vaccine to an aerosol or an i.v. challenge with M. tuberculosis. Groups of five C57Bl/6 mice were immunized s.c. with ST-CF/DDA (Vac) three times at weekly intervals or injected with PBS/DDA. Recombinant human IL-6 (30 µg/animal) or phosphate-buffered saline alone were administered with the first immunization and on the two subsequent days by s.c. injection. A monoclonal antibody specific for IFN-γ (anti-IFN-γ) or an irrelevant immunoglobulin preparation were administered 2 hr before the first and third immunizations, and 2 weeks after the last immunization, by i.p. injection (2 mg/animal). Another group was immunized with 5 × 10⁴ CFU of BCG per animal while its control group was left untreated. Thirty-five days after the last immunization mice were challenged with M. tuberculosis Erdman either through an aerosol infection (15–20 CFU per lung) (a) or intravenously (i.v.) (5 × 10⁴ CFU per animal) (b) Bacterial organ count was determined 14 and 42 days after the i.v. and aerosol challenge, respectively. Results are represented as log₁₀ of resistance, which was calculated by subtracting the mean of log₁₀CFU in the immune groups from the mean of log₁₀CFU in the control, non-immune group. Statistical analysis was done by comparing the immune groups with the immune groups treated as indicated using Student's t-test (NS, P > 0·05).