Abstract
Vaccines with synthetic peptides induce the immune response to epitopes that bind to several HLA alleles. By using a TEPITOPE algorithm, we selected and analyzed the T-cell responses of peripheral blood mononuclear cells from 29 paracoccidioidomycosis (PCM) patients to peptides of the immunodominant gp43 antigen of Paracoccidioides brasiliensis, the causative agent of PCM.
Paracoccidioidomycosis (PCM) is caused by the fungus Paracoccidioides brasiliensis and is the prevalent human systemic mycosis in Latin America (4, 14, 19, 20). Current chemotherapy for the treatment of PCM is usually successful, although the treatment regimens are very long and relapses are frequent. Patients with chronic PCM would greatly benefit from a specific and active immunotherapy that could shorten the treatment period, increase the efficiency of chemotherapy, and protect against the relapse of disease.
The immunodominant 43-kDa glycoprotein 43 (gp43) is the major diagnostic antigen for P. brasiliensis infection (17, 18; reviewed in reference 23) because it is recognized in virtually all sera from infected patients with active PCM, using different serological methods (6, 21). Although gp43 may be a virulence factor under some experimental conditions involving cell adhesion (9, 24) and PCM patients have blood circulating gp43 (12, 16), this antigen elicits a protective cellular immune response in mice of different haplotypes. Epitope mapping of the entire gp43 protein in the murine model identified the 15-mer peptide at positions 181 to 195, named P10, as the carrier of the immunodominant epitope in lymphocyte proliferation assays. The immunization of mice with either purified gp43 or P10 peptide was protective against subsequent intratracheal challenge with virulent P. brasiliensis agents (22), and use of P10 as an adjuvant to chemotherapy shortened the period of treatment and protected against relapsing disease (13). Additionally, previous data from our group identified, with the aid of the TEPITOPE algorithm that predicts binding to 25 different HLA-DR molecules (2, 3), the P10-analogous peptide gp43 at sequence positions 180 to 194 [gp43(180-194)] as the immunodominant peptide recognized by up to 53% of the peripheral blood mononuclear cells (PBMC) from 19 treated PCM patients (10). In this work, we extended the data previously reported by testing the previously selected peptides with PBMC from 10 additional PCM patients and 13 healthy donor individuals to evaluate specificity. All PCM patients were typed for HLA class II (Fig. 1, HLA distribution), and the observation that a great diversity of HLA-DR molecules is associated with the recognition of each peptide (Table 1) confirms the absence of a correlation of the disease with the HLA molecule (7), suggesting that peptides predicted to bind promiscuously were able to be presented by multiple HLA-DR molecules. Additionally, the frequency of allelic variation in the distribution of the HLA-DRs of the tested patients is similar to that of the Brazilian population spectra, so that the lack of some haplotypes (Fig. 1) reflects the low frequency of these haplotypes in the Brazilian population (8). The proliferative response of PBMC from 29 different PCM patients (Table 1) showed that 76% (22/29) responded to at least one peptide and that the ideal peptide concentration for recognition varied among individuals and peptides tested. Peptide gp43(181-195) was the most promiscuous peptide, recognized by 48% (14/29) of the PCM patients tested, followed by gp43(180-194), gp4(179-199), gp43(95-109), gp43(183-197), gp43(45-59), gp43(106-120), and gp43(283-298), which were recognized by 45%, 45%, 41%, 40%, 39%, 33%, and 27% of patients, respectively (Table 1). The pooling effect of peptides gp43(45-59), gp43(94-108), gp43(106-120), gp43(283-298), and gp43(181-195) showed that six out of seven (86%) individuals tested recognized the pool of the five selected peptides, increasing the frequency of recognition compared to that of peptides tested individually (Table 1). The proliferative responses of 13 healthy individuals against the eight peptides selected by TEPITOPE analysis showed that 7 individuals recognized the gp43 protein but with lower stimulation index (SI) values (2.0 to 4.4) than those of the PCM patients. Moreover, two individuals recognized three different peptides from the P. brasiliensis gp43 protein with similarly low SI (2.0 to 2.4) responses (Table 2). The fact that PBMC from some healthy individuals with typical urban lives, and therefore not exposed to P. brasiliensis, recognized gp43 and some of its derived peptides suggests that these individuals may have been fortuitously exposed to P. brasiliensis on a trip to reserve areas of the fungus or that they have developed a cross-recognition response to other related fungal antigens. Previous data have shown that upon cloning and sequencing of the whole gp43 sequence, gp43 presents 54 to 60% sequence homology with the N- and C-terminal domains of exo-β-1,3-d-glucanases from Saccharomyces cerevisiae and Candida albicans (5). Using the Internet-available FASTS tool (11) to search for the exo-β-1,3-d-glucanase region from several different microorganisms presenting sequence homology to these recognized peptides, we identified sequence positions 181 to 201 of the exo-β-1,3-d-glucanase region from Blumeria graminis {GenBank accession number Q96V64; VPNTLRAIQALAERYAPQTDV [named herein bg(181-201)]} and sequence positions 251 to 265 from the glucanase-like protein of Aspergillus nidulans {a hypothetical protein within the endoglucanase region; GenBank accession number XP661656; EQTILAFETLAQRYL [named herein as(251-265)]}, which present sequence homology with gp43(179-199) and also contain the gp43(181-195) peptide. In the same way, we also identified that the position sequence 122 to 137 of the exo-β-1,3-d-glucanase region from Saccharomyces castelli {GenBank accession number Q875Z0; IDDHVKVACSWGTGVL [named herein sc(122-137)]} has sequence homology with the P. brasiliensis gp43(283-298) peptide (data not shown). Moreover, TEPITOPE analysis showed that these peptides from different fungi could be presented by several HLA-DR molecules (data not shown), among which were those HLA-DR molecules carried by healthy individuals whose T lymphocytes recognized the peptides, strongly supporting the results obtained. These data suggest that these individuals may have had previous contact with fungi carrying the related exo-β-1,3-d-glucanases as important enzymes responsible for the synthesis of the fungal cell wall. The antigen may have been released by cell lysis and peptide bg(181-201), as(350-370), or sc(122-137) or functionally similar peptides from other fungi with similar antigenic β-glucanases, presented by antigen-presenting cells to T lymphocytes.
FIG. 1.
HLA-DR distribution of the 29 PCM patients examined.
TABLE 1.
Proliferation responses of PBMC from 29 PCM patients to the gp43 and eight gp43 peptides selected by a TEPITOPE algorithma
| Patient ID no.b | SI value for indicated concn (μM) of peptide:
|
SI for control
|
|||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| gp43 (45-59)
|
gp43 (94-108)
|
gp43 (106-120)
|
gp43 (283-295)
|
gp43 (180-194)
|
gp43 (183-197)
|
gp43 (179-199)
|
gp43 (181-195)
|
Peptide pool
|
gp43 (1 μg) | PHA | |||||||||||||||||||
| 0.1 | 1 | 10 | 0.1 | 1 | 10 | 0.1 | 1 | 10 | 0.1 | 1 | 10 | 0.1 | 1 | 10 | 0.1 | 1 | 10 | 0.1 | 1 | 10 | 0.1 | 1 | 10 | 0.1 | 1 | 10 | |||
| 02* | 3.2 | 17.5 | 14.9 | - | 2.6 | 2.7 | 4.5 | 3.9 | 4.5 | 3.0 | 2.5 | 8.4 | 5.0 | 12.0 | 8.1 | 18.5 | 19.5 | 4.4 | 32.6 | 8.3 | 7.8 | 22.2 | 7.3 | 5.5 | 210.6 | 2,368.6 | |||
| 03* | - | - | - | 2.8 | 5.2 | 3.9 | 2.0 | 2.1 | - | 2.8 | 8.3 | 2.9 | 3.2 | 5.3 | 4.3 | 5.1 | 4.1 | 15.7 | 2.4 | - | - | 5.2 | 7.8 | 5.5 | 26.9 | 16.9 | |||
| 18* | 5.7 | 3.8 | - | 3.4 | 4.6 | - | 2.9 | 2.3 | - | 3.3 | 2.9 | - | 3.1 | 9.2 | - | 7.1 | 4.5 | 2.3 | 2.1 | - | - | 8.8 | 3.4 | - | 5.0 | 243.0 | |||
| 09* | 2.0 | - | - | - | 2.1 | - | 2.0 | - | - | - | 2.1 | 3.0 | 2.6 | 3.5 | 2.2 | 2.0 | 2.5 | - | 4.1 | 2.2 | - | - | - | 2.7 | 40.8 | 1,181.7 | |||
| 04 | - | 2.3 | 5.0 | - | - | - | 2.3 | - | - | - | - | - | 3.6 | 2.0 | - | - | - | 2.5 | - | - | 2.2 | 2.1 | - | 2.3 | 19.1 | 946.5 | |||
| 21 | 2.8 | - | - | 2.1 | 5.3 | - | - | - | 2.3 | 5.2 | - | - | 15.8 | - | - | - | 4.8 | - | 3.1 | 4.5 | - | 2.3 | - | 4.1 | 41.5 | 418.9 | |||
| 28 | 2.0 | - | - | - | 2.0 | - | - | 2.0 | - | - | - | 2.1 | - | - | - | - | - | - | - | - | - | - | - | - | 8.4 | 358.6 | |||
| 08* | - | - | 6.8 | - | 2.4 | 2.6 | - | - | - | - | - | - | - | 2.3 | 3.5 | - | - | - | - | - | - | - | - | 4.2 | 25.6 | 121.6 | |||
| 07 | - | - | - | 3.9 | 2.6 | - | - | 2.1 | - | - | - | - | 2.1 | 4.5 | 2.5 | - | - | 2.6 | - | 3.1 | 3.0 | - | 5.2 | 4.0 | 20.7 | 169.0 | |||
| 13* | - | - | - | 3.9 | - | - | 12.0 | 2.6 | - | - | - | - | - | 3.4 | - | - | 6.2 | - | - | - | - | - | - | - | 19.4 | 761.3 | |||
| 14* | 4.2 | 4.5 | - | - | - | - | - | - | - | - | - | - | 10.6 | 4.5 | - | - | 2.8 | - | 3.4 | 7.8 | - | 2.6 | 6.1 | - | 88.3 | 710.7 | |||
| 06* | 2.0 | - | - | - | - | - | - | - | - | - | - | - | - | 2.8 | - | - | - | - | - | 2.2 | - | - | 7.7 | - | 29.8 | 803.8 | |||
| 11* | - | - | - | - | - | 2.0 | - | - | - | - | - | - | - | 2.3 | 6.8 | - | 7.7 | - | - | - | 10.2 | - | - | 5.2 | 30.6 | 977.9 | |||
| 13* | - | - | - | - | 2.2 | - | - | - | 2.3 | - | - | - | - | 3.4 | - | - | 6.2 | - | - | - | - | - | - | - | 29.8 | 340.1 | |||
| 26 | - | - | - | - | 2.2 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 3.8 | - | - | 2.9 | - | - | 40.5 | 205.4 |
| 25 | 2.3 | 3.7 | 3.2 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 2.6 | 7.1 | - | 15.2 | 188.9 |
| 22 | - | - | - | 2.6 | 2.3 | 2.0 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 3.4 | - | - | 8.8 | 149.6 |
| 15* | - | - | 3.4 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 8.8 | 2,205.8 | |||
| 17* | - | - | - | - | - | - | - | - | - | - | 2.2 | 5.4 | - | - | - | - | - | - | 2.5 | - | - | - | - | - | 23.4 | 293.1 | |||
| 01* | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 2.6 | - | - | - | - | 2.2 | - | 2.8 | 7.8 | - | 11.6 | 261.6 | |||
| 20* | - | - | - | - | - | - | - | - | - | - | - | 2.1 | - | - | - | - | - | - | - | - | - | - | - | - | 3.8 | 236.7 | |||
| 23 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 2.0 | - | - | - | - | 3.7 | - | 3.6 | 4.9 | 2.0 | 26.6 | 172.4 |
| 16* | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 18.1 | 740.0 | |||
| 10* | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 15.0 | 162.4 | |||
| 12* | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 2.4 | - | 19.9 | 261.4 |
| 05* | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 3.2 | 53.4 | |||
| 19* | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 2.3 | 106.5 | |||
| 29 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 2.2 | - | - | - | - | 7.2 | 151.2 | |||
| 24 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 7.2 | - | 76.9 | 248.4 |
PBMC (105 cells/well) were stimulated by peptides at concentrations of 0.1, 1.0, and 10.0 μM as described. Only positive stimulation index (SI) values of ≥2 are shown. Dashes (-) correspond to SI values of <2. Positive control, phytohemagglutinin (PHA). Blank cells, not done.
*, data previously shown in the study by Iwai et al. (10), except for peptides gp43(183-197), gp43(179-199), and gp43(181-195). ID, identification.
TABLE 2.
Proliferation responses of PBMC from healthy individuals to gp43 and the eight gp43 peptides selected by a TEPITOPE algorithma
| Control subject no. | SI value for indicated concn (μM) of peptide:
|
SI for control
|
||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| gp43 (45-59)
|
gp43 (94-108)
|
gp43 (106-120)
|
gp43 (283-295)
|
gp43 (180-194)
|
gp43 (183-197)
|
gp43 (179-199)
|
gp43 (181-195)
|
gp43 (1 μg) | PHA | |||||||||||||||||
| 0.1 | 1 | 10 | 0.1 | 1 | 10 | 0.1 | 1 | 10 | 0.1 | 1 | 10 | 0.1 | 1 | 10 | 0.1 | 1 | 10 | 0.1 | 1 | 10 | 0.1 | 1 | 10 | |||
| 02 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 232.1 |
| 05 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 234.1 |
| 06 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 439.2 |
| 07 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 353.2 |
| 09 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 545.3 |
| 12 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 216.4 |
| 04 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 2.0 | 160.0 |
| 13 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 2.9 | 265.9 |
| 14 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 4.5 | 403.9 |
| 15 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 2.5 | 216.0 |
| 16 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 4.3 | 47.9 |
| 10 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | 2.2 | - | - | 4.4 | 189.1 |
| 01 | - | - | - | - | - | - | - | - | - | 2.4 | - | - | - | - | - | - | - | - | - | 2.0 | - | - | - | - | 4.1 | 233.1 |
PBMC (105 cells/well) were stimulated by peptides at concentrations of 0.1, 1.0, and 10.0 μM as described. Only positive stimulation index (SI) values of ≥2 are shown. Dashes (-) correspond to SI values of <2. Positive control, phytohemagglutinin (PHA).
Recent data from several groups have suggested that vaccines based on a unique epitope are not potent enough to induce a complete protective immune response. The combination of multiple B- and/or T-cell epitopes in a pool or as a polypeptide with multiple epitopes showed an increase in immunogenicity (1, 15). In this work, we also observed an increase in the recognition from 48% to 86% of patients when we used the combination of five promiscuous peptides selected by TEPITOPE analysis. The peptide combination approach opens the possibility for a successful therapeutic peptide-based vaccine that would work as an adjuvant to the currently used fungal chemotherapy, thereby improving its efficacy and reducing the time of the treatment.
Acknowledgments
This study was supported by grant 00-08404-3, and L.K.I. was supported by fellowship grant 99/15319-6 from the São Paulo State Science Funding Agency (FAPESP). E.C.-N. is the recipient of productivity grant 520533/97-6, and L.R.T. is a career fellow with the Brazilian National Research Council (CNPq).
Footnotes
Published ahead of print on 28 February 2007.
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