Comparison of BCG, MPL and cationic liposome adjuvant systems in leishmanial antigen vaccine formulations against murine visceral leishmaniasis

Abstract

Background: The development of an effective vaccine against visceral leishmaniasis (VL) caused by Leishmania donovani is an essential aim for controlling the disease. Use of the right adjuvant is of fundamental importance in vaccine formulations for generation of effective cell-mediated immune response. Earlier we reported the protective efficacy of cationic liposome-associated L. donovani promastigote antigens (LAg) against experimental VL. The aim of the present study was to compare the effectiveness of two very promising adjuvants, Bacille Calmette-Guerin (BCG) and Monophosphoryl lipid A (MPL) plus trehalose dicorynomycolate (TDM) with cationic liposomes, in combination with LAg, to confer protection against murine VL.

Results: All the three formulations afforded significant protection against L. donovani in both the visceral organs, liver and spleen. Although comparable level of protection was observed in BCG+LAg and MPL-TDM+LAg immunized mice, highest level of protection was exhibited by the liposomal LAg immunized group. Significant increase in anti-LAg IgG levels were detected in both MPL-TDM+LAg and liposomal LAg immunized animals with higher levels of IgG2a than IgG1. But BCG+LAg failed to induce any antibody response. As an index of cell-mediated immunity DTH responses were measured and significant response was observed in mice vaccinated with all the three different formulations. However, highest responses were observed with liposomal vaccine immunization. Comparative evaluation of IFN-gamma and IL-4 responses in immunized mice revealed that MPL-TDM+LAg group produced the highest level of IFN-gamma but lowest IL-4 level, while BCG+LAg demonstrated generation of suboptimum levels of both IFN-gamma and IL-4 response. Elicitation of moderate levels of prechallenge IFN-gamma along with optimum IL-4 corresponds with successful vaccination with liposomal LAg.

Conclusion: This comparative study reveals greater effectiveness of the liposomal vaccine for protection against progressive VL in BALB/c. Again, evaluation of the immune responses by vaccination emphasizes the need of stimulation of potent cellular immunity based on both Th1 and Th2 cell responses to confer protection against VL.

Figure 1

Evaluation of protection against L. donovani in differently adjuvanted LAg vaccinated mice . Kinetics of liver (A) and spleen (B) parasite burden of mice immunized intraperitoneally three times at 2-week intervals with BCG-LAg, MPL-TDM+LAg and LAg entrapped in cationic liposomes. Control animals received PBS or adjuvant only. At 10 days after the last immunization, mice were challenged intravenously with 2 × 10⁷ promastigotes of L. donovani. At the designated times mice were sacrificed and LDU were calculated from the weight and microscopic examination of impression smears of liver and spleen tissues. Each bar represents the mean ± SE for five individual mice per group. The results are those from one experiment representative of two performed. Asterisks over each bar indicate significant differences in comparison to control groups. Asterisks over line indicate significant differences between groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Figure 2

Specific antibody responses in differently adjuvanted LAg vaccinated mice . Mice were immunized three times at 2-week intervals. Ten days after immunization mice were challenged with L. donovani. Serum samples were collected after the last booster (A) and 2 (B) and 4 months (C) after infection and assayed for LAg specific IgG and its isotypes IgG1 and IgG2a antibodies by ELISA. Each sample was examined in duplicate. Each bar represents the mean absorbance values at 450 nm ± SE of five individual mice per group at designated time points. The results are those from one experiment representative of two performed. Asterisks over each bar indicate significant differences in comparison to control groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3

DTH responses in differently adjuvanted LAg vaccinated mice . Mice were immunized three times at 2-week intervals. Ten days after immunization mice were challenged with L. donovani. After the last immunization and 2 and 4 months after infection LAg-specific DTH responses were measured. The response is expressed as the difference (in mm) between the thickness of the test (LAg-injected) and control (PBS-injected) footpads at 24 h. Each bar represents the mean ± SE for five individual mice per group at designated time points. The results are those from one experiment representative of two performed. Asterisks over each bar indicate significant differences in comparison to control groups. Asterisks over line indicate significant differences between groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Figure 4

IFN-γ and IL-4 responses in differently adjuvanted LAg vaccinated mice . Mice were immunized three times at 2-week intervals. Ten days after last immunization spleens were collected from mice and restimulated in vitro with LAg (10 μg/ml). After 72 h supernatants were collected and concentrations of released IFN-γ (A) and IL-4 (B) levels were determined by ELISA. Each sample was examined in duplicate. Each bar represents the mean ± SE for five individual mice per group. The results are those from one experiment representative of two performed. Asterisks over each bar indicate significant differences in comparison to control groups. Asterisks over line indicate significant differences between groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001.