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Anais da Academia Brasileira de Ciências

Print version ISSN 0001-3765On-line version ISSN 1678-2690

An. Acad. Bras. Ciênc. vol.72 n.1 Rio de Janeiro Mar. 2000

http://dx.doi.org/10.1590/S0001-37652000000100018 

SYNTHESIS AND IMMUNOLOGICAL ACTIVITY OF A BRANCHED PEPTIDE CONSTRUCTION CARRYING THE T CELL EPITOPE OF Paracoccidioides brasiliensis MAJOR EXOCELLULAR ANTIGEN, THE GP43*

L. R. TRAVASSOS1, C. R. NAKAIE2, E. M. CILLI2 AND C. P. TABORDA1

1Disciplina de Biologia Celular, UNIFESP, 04023-062 São Paulo, SP
2
Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, Brazil.

 

The 43,000 kDa glycoprotein, gp43, is a major immunodominant antigen of Paracoccidioides brasiliensis. It has been used as a diagnostic reagent of paracoccidioidomycosis both in serological reactions and skin tests. The gp43 was cloned and completely sequenced [Cisalpino et al. 1996. J Biol Chem 271: 4553] and the structure of its single high-mannose carbohydrate chain determined [Almeida et al. 1997. Glycobiology 6: 507]. Besides carrying B cell epitopes the gp43 also mediates cellular immune reactions [Rodrigues & Travassos 1994. J Med Vet Mycol 32: 77]. Its T cell epitope was mapped to a 15-amino acid peptide (P10). Peptide 10 not only stimulates the in vitro lymphoproliferation of primed lymph node cells from sensitized or infected mice, but its immunization with CFA in mice followed by an intra-tracheal challenge with a virulent strain of P. brasiliensis is protective, significantly reducing the number of colony-forming units in the lung and abolishing dissemination to the liver and spleen [Taborda et al. 1998. Infect Immun 66: 786].

To increase the immuneprotection by P10, replace CFA as an adjuvant, and examine additional protocols including cytokines (e.g. IL-12), we are investigating different delivery systems for P10. Presently we synthesized an oligomeric multiple molecule derived from P10 and composed of four equal peptide chains attached to a three branched lysine core [LIAIHTLAIRYAN)4-(K)2-K-G-amide, denoted M10]. Due likely to significant steric hindrance predicted during this parallel chain assembly, the initial synthesis attempt failed when the conventional protocol was applied. This occurred even when low substituted resin (methylbenzhydrylamine-resin, 0.2 mmol/g), recommended for minimizing chain aggregation inside resin beads and the common 50% dichloromethane (DCM)/dimethylformamide (DMF) solvent for crucial coupling steps were both used. Aiming to overcome this predicted difficulty, the synthesis protocol was altered following our previously proposed peptidyl-resin solvation approach [Cilli et al. 1996. J Org Chem 61: 8992]. By applying this strategy, 20% dimethylsulphoxide/N-methylpiperidinone mixed solvent was alternatively used for coupling reactions throughout all tridecapeptide sequence growth. With this approach the target M10 was obtained although with low overall yield. A progressive decrease in the synthesis yield was observed as the chain length increased: 70%, 51% and only 27% for 1 - 5, 1 - 10 and 1 - 13 fragment syntheses, respectively). Despite this reduced final yield, we herein demonstrated  that by applying the appropriate chemical strategy it is possible to synthesize extremely challenging macromolecules such as the lysyl branched-multiple tetrapeptide M10 composed of a total of 56 amino acid residues and reaching a molecular weight of more than 6 kDa.

Immunization of Balb/c mice with as little as 1 mg of M10 in CFA induced after 7 days a potent cellular immune response with lymphoproliferations being obtained in vitro with both M10 and P10 at 0.25 - 1.0mg/ml. Immunization with 20 mg of P10 also elicited T lymphocytes proliferating with M10 and P10. On a molar basis the in vitro responses to 1.5 - 0.6 mM M10 were twice as intense as those to 6 -3 mM P10. No proliferation was observed with the truncated (IRYAN)-(K2)-K-G-amide and (IHTLAIRYAN)4-(K2)-K-G-amide controls. These results suggest that the tetra-13 aa-peptide construction with oligomeric branching lysine (M10) can be used to elicit protective cellular immune responses against P. brasiliensis.

— ( September 14, 1999 ).

* Sponsored by PRONEX and FAPESP.

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