Article Figures & Data
Figures
- FIG. 1.
Clinical outcomes following L. donovani challenge in BALB/c mice immunized via four different routes. Mice were immunized three times with 20 μg of LAg, alone or entrapped in liposomes, at 2-week intervals through the i.p., i.v., s.c., and i.m. routes. Control groups received nothing or empty liposomes (Lip) through the respective routes. Ten days after the last immunization, the mice were challenged intravenously with 2.5 × 107 promastigotes of L. donovani. The parasite loads in the livers and spleens of i.p. (A and B), i.v. (C and D), s.c. (E and F), and i.m. (G and H) immunized animals are expressed in Leishman Donovan units. Data represent mean values ± standard errors (SE) for five mice per group at the designated time points and are representative of two independent experiments with similar results. #, P < 0.05; *, P < 0.01; **, P < 0.001 for comparison to control groups (mice left unimmunized or immunized with empty liposomes by the respective routes).
- FIG. 2.
LAg-specific immune responses in mice immunized via four different routes. (A) Serum samples were collected from liposomal LAg-immunized mice and assayed for LAg-specific IgG, IgG1, and IgG2a levels by ELISA. Data are presented as the absorbance at 450 nm and are means ± SE for five mice per group in duplicate wells. Data are representative of two independent experiments with similar results. (B to E) Mice were immunized and infected with L. donovani. DTH responses were measured in i.p. (B), i.v. (C), s.c. (D), and i.m. (E) immunized animals. Results are shown as means ± SE for five mice per group at the designated time points and are representative of two independent experiments with similar results. (F) Four months after infection, splenocytes derived from different vaccinated groups were stimulated with LAg (10 μg/ml) for 72 h, and the level of nitrite in the culture supernatants was assessed. Results are means ± SE for five mice per group done in duplicate and are representative of two independent experiments with similar results. *, P < 0.01; **, P < 0.001 for comparison to control groups (mice left unimmunized or immunized with empty liposomes by the respective routes).
- FIG. 3.
Effects of splenocyte supernatants (SN) of immunized mice on intracellular parasite growth in peritoneal macrophages in the absence (A) and presence of anticytokine antibodies (Ab) (B) or TGF-β (C). Peritoneal macrophages harvested from naive mice were pretreated for 12 to 18 h with supernatants from s.c. immunized mice either alone or in the presence of 10 μg/ml of each of the anticytokine antibodies and appropriate isotype-matched control antibodies. IFN-γ at 10 ng/ml was used as a positive control. Again, macrophages were pretreated with supernatants from i.p. immunized mice alone or with various concentrations of TGF-β ranging from 0.5 to 100 ng/ml. After 3 h of infection, unphagocytosed parasites were removed by washing with PBS and cultured in the same medium with supernatants, IFN-γ, antibodies, or TGF-β, using similar concentrations. Seventy-two hours later, the number of intracellular amastigotes was determined. Results are means ± SE for five experiments done in triplicate. **, P < 0.001 for comparison to control groups.
- FIG. 4.
In vivo effects of anti-TGF-β antibody (Ab) and TGF-β during vaccination via nonprotective s.c. and protective i.p. routes. Mice were vaccinated three times with liposomal LAg with 100 μg of anti-TGF-β or control antibody at 2-week intervals through the s.c. route. Control groups received nothing or anti-TGF-β antibody. Ten days after the last immunization, the mice were challenged intravenously with 2.5 × 107 promastigotes of L. donovani. After 4 months, parasite loads in the liver (A) and spleen (B) were measured and expressed in Leishman Donovan units. Mice were vaccinated three times with liposomal LAg, alone or in combination with 5 μg TGF-β, at 2-week intervals through the i.p. route and infected with L. donovani. After 4 months of challenge infection, parasite loads in the liver (C) and spleen (D) were measured and expressed in Leishman Donovan units. Data represent mean values ± SE for five mice per group. **, P < 0.001 for comparison to control groups.
Tables
- TABLE 1.
Cytokine levels in spleen cell supernatants from mice immunized via different routes
Vaccination group Cytokine concn (pg/ml)a IFN-γ IL-12 IL-4 IL-10 TNF-α TGF-β Groups after immunization Control 21 ± 3 22 ± 3 15 ± 2 16 ± 4 11 ± 1 93 ± 16 Liposomes i.p. 22 ± 3 24 ± 3 14 ± 2 20 ± 1 13 ± 5 105 ± 6 Liposomes s.c. 23 ± 4 22 ± 2 13 ± 3 18 ± 4 12 ± 2 102 ± 8 Liposomal LAg i.p. 120 ± 7b 95 ± 8b 48 ± 5b 22 ± 4 19 ± 5 114 ± 6 Liposomal LAg s.c. 28 ± 3 25 ± 3 21 ± 3 31 ± 3 14 ± 4 924 ± 87b Groups 4 mo after infection Control 31 ± 2 27 ± 4 93 ± 8 230 ± 21 24 ± 4 1,652 ± 81 Liposomes i.p. 37 ± 2 28 ± 3 87 ± 8 228 ± 14 24 ± 3 1,624 ± 113 Liposomes s.c. 32 ± 4 30 ± 4 86 ± 6 220 ± 11 29 ± 4 1,540 ± 125 Liposomal LAg i.p. 274 ± 18b 168 ± 10b 37 ± 4b 33 ± 4b 126 ± 9b 113 ± 9b Liposomal LAg s.c. 38 ± 3 32 ± 3 97 ± 7 228 ± 8 28 ± 4 1,680 ± 77
