Characterization of the immunostimulatory properties of Leishmania infantum HSP70 by fusion to the Escherichia coli maltose-binding protein in normal and nu/nu BALB/c mice

Abstract

Leishmania infantum HSP70 has been described as an immunodominant antigen in both humans and dogs suffering from visceral leishmaniasis. In this study, we used L. infantum HSP70 fused to Escherichia coli maltose-binding protein (MBP), as the reporter protein, to analyze the influence of HSP70 on the immunogenicity of MBP in BALB/c mice. Plasmids were constructed to produce the three recombinant proteins used in this study, namely, MBP, L. infantum HSP70, and MBP-HSP70, which consists of MBP fused to the L. infantum HSP70 amino terminus. Immunization of BALB/c mice with the MBP-HSP70 fusion protein elicited humoral and cellular responses against MBP that were higher by an order of magnitude than those elicited by immunization with MBP alone or with a mixture of MBP and HSP70. Covalent linkage of MBP to HSP70 was essential for eliciting a strong anti-MBP immune response. Cytokine secretion and immunoglobulin G isotype analyses indicated that immunization with the MBP-HSP70 fusion protein preferentially induces a Th1 immune response. Immunization of athymic nu/nu mice with the MBP-HSP70 fusion protein unexpectedly gave rise to an anti-MBP humoral response showing features of a T-cell-dependent response. Thus, we present evidence that L. infantum HSP70 demonstrates an adjuvant effect in the immune response against a covalently linked reporter protein.

FIG. 1

Analysis of recombinants MBP, HSP70, and MBP-HSP70 fusion protein. (A) Schematic representation of recombinants MBP, HSP70, and MBP-HSP70 fusion protein and of their encoding plasmids. (B) Recombinant MBP (lane 1), HSP70 (lane 2), and the MBP-HSP70 fusion protein (lane 3) after purification, migration (SDS-PAGE), and staining with Coomassie blue. The sizes (in kilodaltons) of molecular size markers (lane M) are indicated on the left.

FIG. 2

Humoral immune response of BALB/c mice to different recombinant proteins. (A) Groups of four mice were immunized i.p. with MBP, an MBP-HSP70 mixture, or the MBP-HSP70 fusion protein as indicated in the text. The anti-MBP and anti-HSP70 IgG antibody titers were determined with the FAST-ELISA. The titer is expressed as the highest serum dilution factor giving an absorbance value four times greater than the value obtained with preimmune sera (optical density, 0.05). (B) Serum samples from mice immunized with MBP, the MBP-HSP70 mixture, or the MBP-HSP70 fusion protein were analyzed for anti-MBP IgG1 and IgG2a antibodies. (C) The time course of the anti-MBP antibody response is shown. Sera were obtained at the times indicated (in weeks) after the second immunization of the mice with MBP, the MBP-HSP70 mixture, or the MBP-HSP70 fusion protein. MBP + HSP70, mixture; MBP-HSP70, fusion protein.

FIG. 3

Western blot analysis of the specificity of the anti-HSP70 antibodies in immunized mice. (A) Total proteins of 10⁷ human primary fibroblasts (lane 1), 10⁷ T. cruzi epimastigotes (lane 2), and 10⁷ L. infantum promastigotes (lane 3) were separated on SDS–10% PAGE gels and stained with Coomassie blue. The sizes (in kilodaltons) of the molecular size markers (lane M) are indicated on the right. (B to D) Equivalent protein gels were transferred to nitrocellulose membranes and incubated with anti-HSP70 monoclonal antibody 7.10 (B), serum from a mouse immunized with the MBP-HSP70 mixture (C), or serum from a mouse immunized with the MBP-HSP70 fusion protein (D). Sera were assayed at a 1:200 dilution.

FIG. 4

Analysis of the humoral immune response of athymic nu/nu mice. Groups of four BALB/c nu/nu were immunized i.p. with MBP, HSP70, or the MBP-HSP70 fusion protein on days 0 (first immunization) and 21 (second immunization). Details of the immunization protocol can be found in the text. Blood samples were taken a week after the first (A and C) and second (B and D) immunizations, and anti-MBP (empty bars) and anti-HSP70 (shaded bars) antibody titers were determined with the FAST-ELISA. Both IgG (A and B) and IgM (C and D) antibody classes were determined. This is a representative example of two independent experiments.