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The heat shock proteins (HSP) are produced by prokaryotic and eukaryotic cells in response to a variety of physiological insults. Those HSP belonging to the HSP90, HSP70, and HSP60 families are among the most highly conserved and abundant proteins found in living prokaryotic and eukaryotic organisms. Although they are classified as stress proteins, the HSP also have essential functions in the cell under normal growth conditions. These proteins are involved in the folding of newly synthesized proteins by preventing incorrect interactions within and between nonnative proteins (reviewed in reference 18). The HSP are also involved in several molecular processes of the immune system, such as immunoglobulin chain assembly (17), antigen processing and presentation (29), and the assembly of functional major histocompatibility complexes I and II (12). Although the abundance of the HSP may explain why they behave as dominant antigens in the immune response to a variety of pathogens (25, 26, 35), it is surprising that the immune system focuses its attention on a family of proteins with such a high degree of evolutionary conservation. It has been suggested that immune recognition of HSP of pathogens serves as a first line of defense as well as a source of autoimmune cascades that may develop through inappropriate cross-reactivity with self-HSP (8, 40). Of the HSP families, HSP70, in spite of being the most conserved protein, present in all organisms studied to date (16), is a major immunogen in infections caused by a number of pathogens, such as Schistosoma mansoni (19), Onchocerca volvulus (34), Plasmodium falciparum (2, 4, 5, 21), Histoplasma capsulatum (1), Trypanosoma cruzi (14, 23, 32), Trypanosoma congolense (6), and Leishmania (11, 24, 30, 36) and Mycobacterium (9, 15, 27, 33) spp. Epitope analysis of several of these HSP70 indicates the existence of multiple B- and T-cell epitopes throughout their primary structure (4, 13, 28, 31, 32, 41).

The immunogenic properties of the HSP have been demonstrated in particular for Mycobacterium tuberculosis HSP70, which has been used successfully as an adjuvant-free carrier molecular (3). Recently, Suzue and Young (38) have further investigated the immunogenic potential of M. tuberculosis HSP70 by immunizing mice with a recombinant human immunodeficiency virus type 1 p24-HSP70 fusion protein. They demonstrated that in the absence of adjuvants, covalent linking of HSP70 to p24 was essential to elicit humoral and cellular immune responses to p24. Here, we have addressed the question of whether the immunostimulatory property of M. tuberculosis HSP70 is possessed by other HSP70. Based on the powerful immunogenic characteristics of Leishmania infantum HSP70 (30, 31), we analyzed whether L. infantum HSP70, when fused to an accompanying protein, can induce humoral and cell-mediated responses to the fused protein.

Plasmid constructions, purification of proteins, mice, and immunizations. DNA coding for L. infantum HSP70 was amplified by PCR as described elsewhere (30). The amplification product was digested with EcoRI and HindIII restriction enzymes, purified on spin bind columns (FMC Bio Products, Rockland, Maine), and cloned in frame to the carboxyl terminus of the maltose-binding protein (MBP) encoded by the vector pMAL-cRI (New England Biolabs, Inc., Beverly, Mass.). The resulting clone was named pMAL-rLiHsp70. To obtain the recombinant protein MBP alone, the pMAL-cRI plasmid was digested with EcoRI, filled in with Klenow fragment, and religated with T4 DNA ligase. Consequently, the modified vector, pMAL-cRI*, includes an in-frame stop codon four triplets downstream of the EcoRI restriction site. The pMAL-cRI* vector thus expresses the MBP moiety alone rather than the MBP-lacZa expressed by the original pMAL-cRI plasmid. The pMAL-rLiHsp70 clone was digested with EcoRI and HindIII, the insert was cloned in the corresponding restriction sites of the vector pBluescript (Stratagene, La Jolla, Calif.), and the resulting clone was denominated pBlsc70Li. Clone pQE/hsp70 was created by subcloning the BamHI-HindIII insert of plasmid pBlsc70Li into the appropriate restriction sites of vector pQE9 (Qiagen, Hilden, Germany).

View larger version (35K): [in this window] [in a new window]   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.

L. infantum HSP70 potentiates a specific humoral response against the covalently linked antigen. By using the data of Suzue and Young (38), we studied whether L. infantum HSP70 can elicit an immunostimulatory effect when used as a carrier molecule in adjuvant-free immunizations of mice. Escherichia coli MBP was chosen as the reporter antigen. Plasmid constructions encoding MBP, L. infantum HSP70, or the MBP-HSP70 fusion protein were obtained (Fig. 1). Groups of four BALB/c mice were immunized with MBP, HSP70, an MBP-HSP70 mixture, or the MBP-HSP70 fusion protein. One week after the last immunization, the anti-MBP immunoglobulin G (IgG) level in serum was determined with the FAST-ELISA (Becton Dickinson, Lincoln Park, N.J.) by using the coating antigen at a concentration of 2 µg/ml. Horseradish peroxidase-conjugated anti-mouse IgG (Nordic Immunological Laboratories, Tilburg, The Netherlands) was used as the second antibody at a 1:2,000 dilution. The titer is expressed as the highest serum dilution giving an absorbance value four times higher than the preimmune serum reading (optical density, 0.050). Differences in antibody titers between groups of mice were compared with the two-tailed unpaired Student t test. Excel software (Microsoft Corporation) was used for statistical analysis.

View larger version (23K): [in this window] [in a new window]   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.

Immunization with the MBP-HSP70 fusion protein induces a powerful cellular response against MBP. To examine the ability of the MBP-HSP70 fusion protein to induce anti-MBP cellular responses, groups of four BALB/c mice were immunized subcutaneously at the tail base with 5 µg of the MBP-HSP70 fusion protein, MBP, or bovine serum albumin (BSA; Sigma, St. Louis, Mo.). One week after immunization, draining inguinal and periaortic lymph nodes were removed, and single-cell suspensions of lymph node cells (LNC) were prepared and suspended in complete medium (RPMI 1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 10 µM 2-mercaptoethanol). LNC (10⁶/well) were dispensed into 96-well microtiter plates. Cultures were challenged in triplicate with 2 µg of MBP or concanavalin A (ConA; Sigma) per ml and incubated for 3 days at 37°C in 5% CO₂; after this, 1 µCi of [³H]thymidine (5 Ci/mmol; Amersham Corp., Aylesbury, United Kingdom) was added to each well. After 16 h, the cells were harvested and [³H]thymidine incorporation into DNA was measured by liquid scintillation counting. The specific incorporation was determined by subtracting the mean incorporation of triplicate control wells incubated with medium alone from the mean incorporation of the triplicate wells incubated with antigen. Table 1 shows the MBP-specific immune response of each group following in vitro T-cell stimulation with MBP. LNC from animals immunized with the MBP-HSP70 fusion protein proliferated in response to the antigen, whereas those from animals immunized with MBP alone did not. The proliferation levels of LNC from MBP-immunized animals were similar to those of LNC from control animals to which BSA had been administered (Table 1). The ability of the fusion protein to elicit cellular responses was also examined by determination of gamma interferon (IFN-), interleukin 2 (IL-2), and IL-4 cytokines from LNC supernatants (Table 1). For cytokine determinations, LNC (5 × 10⁶/well) from the same mice were seeded in 24-well plates and the cultures were challenged with 2 µg of MBP or ConA per ml for 72 h at 37°C in 5% CO₂. IFN-, IL-4, and IL-2 production was measured in supernatants with an enzyme-linked immunosorbent assay kit (Genzyme Corp., Cambridge, Mass.). Results of the cytokine determinations were consistent with the proliferation assays in that only LNC from fusion protein-immunized mice secreted detectable amounts of IFN- and IL-2 when stimulated with MBP. IL-4 levels were low for all groups (<20 pg/ml), in accord with the relatively low IgG1 (Th2-mediated) antibody response observed upon in vivo immunization.

Analysis of the specificity of anti-HSP70 antibodies elicited by the MBP-HSP70 fusion protein. Since HSP70 is considered to be the most conserved protein present in all organisms (16), we analyzed the humoral response against L. infantum HSP70 and the specificity of the response elicited by immunization with the MBP-HSP70 fusion protein. This is of particular importance if this molecule is to be considered a practical alternative in the development of adjuvant-free vaccines. An autoimmune cascade caused by an inappropriate cross-reaction with self-HSP would obviously be undesirable. Immunization of mice with the MBP-HSP70 fusion protein induced high anti-HSP70 antibody titers, although they were lower (P < 0.001) than those induced against the MBP moiety (Fig. 2A). The mean anti-HSP70 IgG titer was 3.4 × 10⁵ (SD = 2.4 × 10⁵), while, as indicated above, the mean anti-MBP IgG titer in the same mice was 2 × 10⁶. The difference was even greater when anti-HSP70 IgG titers from MBP-HSP70 fusion protein-immunized mice were compared with those of mice immunized with the MBP-HSP70 mixture. In this latter group, the mean anti-HSP70 IgG titer was relatively low (1.1 × 10⁴; SD = 9.4 × 10³). The specificity of the humoral response was assayed by analysis of the recognition pattern of the antibodies to HSP70 from human fibroblasts, T. cruzi epimastigotes, and L. infantum promastigotes (Fig. 3A). The proteins were analyzed by standard SDS-PAGE (22) on 10% polyacrylamide gels. Samples of total protein were prepared from L. infantum promastigotes (WHO code MHOM/FR/78/LEM75), T. cruzi epimastigotes (strain G) (20), or human primary fibroblasts. After electrophoresis, the proteins were transferred to nitrocellulose membranes (Amersham). The filters were blocked with 5% nonfat dried milk powder in phosphate-buffered saline-0.5% Tween 20 and probed sequentially with primary and secondary antisera in blocking solution. A peroxidase immunoconjugate (Nordic Immunological Laboratories) was used as a secondary antibody, and specific binding was developed with 0.5 mg of 4-chloronaphthol (Sigma) per ml and 0.025% hydrogen peroxide.

View larger version (45K): [in this window] [in a new window]   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.

Immunization with the MBP-HSP70 fusion protein induces a humoral response in athymic BALB/c nu/nu mice. To study T-cell involvement in the MBP-HSP70 fusion protein-induced humoral response, athymic BALB/c nu/nu mice were immunized twice with MBP, HSP70, or the MBP-HSP70 fusion protein. A positive anti-MBP antibody response was detected in nu/nu mice only after immunization with the MBP-HSP70 fusion protein (Fig. 4). The humoral response elicited in these mice was a typical, T-cell-dependent response. Following the first immunization, an IgM isotype was detected, whereas IgG predominated after challenge (Fig. 4B). The anti-MBP antibodies elicited in the fusion protein-immunized mice were essentially IgG2a (antibody titers, >1,200). Immunization of nu/nu mice with MBP or HSP70 elicited no humoral response (Fig. 4).

View larger version (20K): [in this window] [in a new window]   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.