DNA immunization with Trypanosoma cruzi HSP70 fused to the KMP11 protein elicits a cytotoxic and humoral immune response against the antigen and leads to protection

Abstract

Murine immunization with Trypanosoma cruzi KMP11-HSP70 fused genes but not the KMP11 gene alone elicited both an immunoglobulin G2a long-lasting humoral immune response against KMP11 protein and activation of CD8+ cytotoxic T lymphocytes specific for two KMP11 peptides containing A2 motifs. Moreover, protection against the parasite challenge was observed after immunization with the chimeric gene.

FIG. 1

(A) Construction of the DNA vaccines. T. cruzi KMP11 and KMP11-HSP70 genes were cloned separately between the cytomegalovirus promoter sequence and the bovine growth hormone polyadenylation sequence in the pCMV4 expression vector, whose characteristics are summarized in this figure, generating pCMV4.11 and pCMV4.11.70 clones. To construct the vector pCMV4.11.70 containing the fused genes, the KMP11 coding sequence with the stop codon deleted was cloned upstream and in frame with the HSP70 gene previously cloned in the pCMV4 vector. (B) Expression of KMP11 and KMP11-HSP70 proteins in COS-7 cells. Protein expression was checked in vitro by plasmid transient transfection with lipofectin (Gibco) into COS-7 cells, followed by Western blotting of the cell extracts (29). Antisera produced in rabbits and directed against the GMPG repeated motif located at the C termini of the T. cruzi HSP70 protein (15) and the KMP11 protein (24) were used (panels 1 and 2, respectively). Lanes p4, cells transfected with the control vector; lane p4.11.70, cells transfected with the vector bearing the coding sequence for the KMP11-HSP70 fusion protein; lane p4.11, cells transfected with the DNA plasmid containing the gene coding for the KMP11 protein. Double and single asterisks indicate the locations of the KMP11-HSP70 fusion protein and the KMP11 protein, respectively. MW, molecular weights of standard proteins in thousands.

FIG. 2

Detection of anti-KMP11 IgG antibody levels in the sera of mice immunized with DNA plasmids or saline solution. BALB/c (top panel) and C57BL/6-A2.1/K^b (bottom panel) mice were immunized intramuscularly four times with saline solution (◊) or 100 μg of each the DNA vectors pCMV4 (□), pCMV4.11 (▵), and pCMV4.11.70 (●). Production of IgG antibodies to KMP11 was evaluated by ELISA (29) on days 0, 21, 42, 56, 63, 77, 91, 105, 119, and 126 using 1 μg of recombinant KMP11 protein/well. Data are optical density (OD) values of pooled sera from six mice per group. These and all subsequent data show representative results of at least three independent experiments. Asterisks indicate immunization days.

FIG. 3

IgG isotype level generated against KMP11 protein in mice immunized with pCMV4.11.70. The antibody level was determined by ELISA with sera from BALB/c (top panel) and C57BL/6-A2.1/K^b (bottom panel) mice, intramuscularly immunized with pCMV4.11.70 DNA, using 1 μg of recombinant KMP11 protein/well. IgG1 (○) and IgG2a (●) antibodies produced against KMP11 were evaluated in serum samples obtained on days 0, 21, 42, 56, 63, 77, 91, 105, 119, and 126. Data are optical density (OD) values of pooled sera from 6 mice. Asterisks indicate immunization days.

FIG. 4

KMP11 peptide-specific CTL response. (A) KMP11 deduced amino acid sequence and composition of synthesized peptides containing theoretically estimated A2 union motifs (17). The asterisk indicates the protein stop codon. The positions of the designed A2 peptides K1, K2, K3, and K4 are marked. (B) KMP11 peptide-specific CTLs elicited after immunization with the DNA vector carrying the KMP11-HSP70 fusion. Spleens from C57BL/6-A2.1/K^b mice immunized with saline solution (□) or the pCMV4 (∗), pCMV4.11 (▵), and pCMV4.11.70 (⧫) vectors were removed 2 weeks after the last immunization. Splenocytes were used as effector cells after being incubated for 6 days with EL4-A2.1/K^b cells treated with 50 μg of mitomycin/ml and loaded separately with each of the four KMP11 A2 peptides. CTL activity was measured against EL4-A2.1/K^b cells pulsed with or without each one of the respective peptides by a standard ⁵¹Cr release assay (19). Each panel corresponds to results with one A2 peptide. The level of lysis of EL4-A2.1/K^b cells in the absence of peptide was <5% for all groups (data not shown). Specific lysis was calculated using the following formula: percent specific lysis = (experimental release [cpm] − spontaneous release [cpm]/(total release [cpm] − spontaneous release [cpm] × 100), where cpm is counts per minute. Spontaneous release represents the counts obtained when the target cells were incubated in culture medium without effectors, and total release was obtained after treatment of target cells with 2.5% Triton X-100. Experiments with more than 20% spontaneous lysis were discarded. Data are representative of results with three mice per group.

FIG. 5

Parasitemia and survival percentages for immunized mice after challenge with T. cruzi. Mice were immunized with saline solution (□) or pCMV4 (▵), pCMV4.11 (○), and pCMV4.11.70 (⧫). Six mice per group were challenged with 10³ T. cruzi blood trypomastigotes (Y Brazil strain) 9 weeks after the fourth immunization (A). The levels of parasites in the bloodstream were determined individually for three mice per group using a Neubauer chamber. Values are means ± standard deviations (SD) of the means of results for three mice. (B) Survival percentages of immunized mice challenged 2 weeks after the last immunization were regularly recorded.

FIG. 6

Anti-KMP11 IgG2a antibodies in immunized mice after challenge with T. cruzi. Nine weeks after the last immunization, six BALB/c mice from each group were challenged with 10³ T. cruzi blood trypomastigotes (Y Brazil strain). On days 10, 14, 17, and 22 after challenge, anti-KMP11 IgG2a levels in all the mice were individually tested by ELISA using 1 μg of recombinant KMP11 protein per well. The bars represent the means of optical density (OD) values ± SD of the results for six mice immunized with saline solution (□), pCMV4 (▤), or pCMV4.11 (▨). ■ and  represent the means of optical density (OD) values ± SD of the sera of three mice immunized with pCMV4.11.70 that survived or died, respectively, after the T. cruzi challenge.