CLINICAL ANDVACCINEIMMUNOLOGY, Apr. 2007, p. 474–476 Vol. 14, No. 4
1556-6811/07/$08.00⫹0 doi:10.1128/CVI.00458-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
T-Cell Recognition of
Paracoccidioides brasiliensis
gp43-Derived Peptides
in Patients with Paracoccidioidomycosis and Healthy Individuals
䌤
Leo Kei Iwai,
1,3,5† Ma´rcia Yoshida,
4Aya Sadahiro,
4Washington Robert da Silva,
1Maria Lucia Marin,
1,3Anna Carla Goldberg,
1,3‡ Maria Aparecida Juliano,
5Luiz Juliano,
5Maria Aparecida Shikanai-Yasuda,
4Jorge Kalil,
1,2,3Edecio Cunha-Neto,
1,2,3and Luiz R. Travassos
6*
Laboratory of Immunology, Heart Institute (InCor),1Division of Clinical Immunology and Allergy,2and Institute for Investigations
in Immunology,3Millennium Institutes, Sa˜o Paulo, Brazil; Department of Infectious and Parasitic Diseases, University of
Sa˜o Paulo Medical School, Sa˜o Paulo, Brazil4; Department of Biophysics5and Discipline of Cell Biology, Department of
Microbiology, Immunology and Parasitology,6Federal University of Sa˜o Paulo, UNIFESP, Sa˜o Paulo, Brazil
Received 6 December 2006/Returned for modification 5 January 2007/Accepted 13 February 2007
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 mono-nuclear cells from 29 paracoccidioidomycosis (PCM) patients to peptides of the immunodominant gp43 antigen ofParacoccidioides brasiliensis, the causative agent of PCM.
Paracoccidioidomycosis (PCM) is caused by the fungus
Paracoccidioides brasiliensisand is the prevalent human sys-temic mycosis in Latin America (4, 14, 19, 20). Current che-motherapy 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 forP. brasiliensisinfection (17, 18; re-viewed in reference 23) because it is recognized in virtually all sera from infected patients with active PCM, using different se-rological methods (6, 21). Although gp43 may be a virulence factor under some experimental conditions involving cell adhe-sion (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 immuno-dominant epitope in lymphocyte proliferation assays. The immu-nization of mice with either purified gp43 or P10 peptide was protective against subsequent intratracheal challenge with viru-lentP. brasiliensisagents (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 pep-tides 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 obser-vation that a great diversity of HLA-DR molecules is associated with the recognition of each peptide (Table 1) confirms the ab-sence 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. Addition-ally, 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) re-sponded to at least one peptide and that the ideal peptide con-centration 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 theP. brasiliensisgp43 protein with simi-larly low SI (2.0 to 2.4) responses (Table 2). The fact that PBMC from some healthy individuals with typical urban lives, and
there-* Corresponding author. Mailing address: Disciplina de Biologia Celular, Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sa˜o Paulo, Rua Botucatu 862, 8 andar, Sa˜o Paulo, SP 04023-062, Brazil. Phone: 50842991. Fax: 55-11-55715877. E-mail: travassos@ecb.epm.br.
† Present address: Section on Immunology and Immunogenetics, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215. ‡ Present address: Chemistry Institute, University of Sa˜o Paulo, Sa˜o Paulo, Brazil.
䌤Published ahead of print on 28 February 2007.
fore not exposed toP. brasiliensis, recognized gp43 and some of its derived peptides suggests that these individuals may have been fortuitously exposed toP. brasiliensison a trip to reserve areas of the fungus or that they have developed a cross-recognition re-sponse 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--1,3-D-glucanases from Saccharomy-ces cerevisiaeandCandida albicans(5). Using the Internet-avail-able FASTS tool (11) to search for the exo--1,3-D-glucanase region from several different microorganisms presenting quence homology to these recognized peptides, we identified se-quence positions 181 to 201 of the exo--1,3-D-glucanase region fromBlumeria graminis{GenBank accession number Q96V64;
VPNTLRAIQALAERYAPQTDV [named herein bg(181–201)]} and sequence positions 251 to 265 from the glucanase-like protein ofAspergillus nidulans{a hypothetical protein within the endo-glucanase region; GenBank accession number XP661656; EQTI LAFETLAQRYL [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--1,3-D-glucanase region fromSaccharomyces castelli{GenBank accession number Q875Z0; IDDHVKVACSWGTGVL [named herein sc(122– 137)]} has sequence homology with theP. brasiliensisgp43(283– 298) peptide (data not shown). Moreover, TEPITOPE analysis showed that these peptides from different fungi could be pre-sented by several HLA-DR molecules (data not shown), among which were those HLA-DR molecules carried by healthy individ-uals whose T lymphocytes recognized the peptides, strongly sup-porting the results obtained. These data suggest that these indi-viduals may have had previous contact with fungi carrying the related exo--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-glucanases, presented by antigen-present-ing cells to T lymphocytes.
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 mul-tiple B- and/or T-cell epitopes in a pool or as a polypeptide with multiple epitopes showed an increase in immunogenicity (1, 15).
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 (M) 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
(1g) 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
aPBMC (105cells/well) were stimulated by peptides at concentrations of 0.1, 1.0, and 10.0M as described. Only positive stimulation index (SI) values of
ⱖ2 are shown. Dashes (-) correspond to SI values of⬍2. Positive control, phytohemagglutinin (PHA). Blank cells, not done.
b*, 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.
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 pep-tide 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.
This study was supported by grant 00-08404-3, and L.K.I. was sup-ported by fellowship grant 99/15319-6 from the Sa˜o Paulo State Sci-ence Funding Agency (FAPESP). E.C.-N. is the recipient of produc-tivity grant 520533/97-6, and L.R.T. is a career fellow with the Brazilian National Research Council (CNPq).
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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 (M) 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 (1g) 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
aPBMC (105cells/well) were stimulated by peptides at concentrations of 0.1, 1.0, and 10.0M as described. Only positive stimulation index (SI) values of
ⱖ2 are shown. Dashes (-) correspond to SI values of⬍2. Positive control, phytohemagglutinin (PHA).
