Peptide Vaccine Against Paracoccidioidomycosis

As mentioned above gp43, the major diagnostic antigen of P. brasiliensis, elicits a vigorous IFN-y-mediated T-CD4+ response that is protective against the i.t. challenge by virulent yeasts of this fungus. The P10 sequence contains the T cell epitope that is presented by MHC class II molecules from three different mouse haplotypes (Taborda et al., 1998). The promiscuous nature of P10 was also shown with HLA-DR molecules, since this peptide and the analogous gp43 (180-194) without C-terminal asparagine residue (N-glycosylation site in the original gp43) and with N-terminal lysine, bound to the 9 most prevalent HLA-DR molecules (Iwai et al., 2003). Furthermore, four additional peptides from gp43 all identified by the TEPITOPE algorithm were also promiscuous with respect to HLA-DR binding. Gp43 (180-194) was recognized by 53% of patients with treated PCM and the other promiscuous peptides were recognized by 32-47% of patients; 74% of patients recognized the combination of 5 promiscuous gp43 peptides (Iwai et al., 2003). An expansion of this study with 10 more patients (total of 29) showed that 79% of PCM patients recognized at least 1 peptide, and by pooling these peptides, the recognition frequency increased to 86%. The TEPITOPE algorithm scanned 25 Caucasian HLA-DRs and P10 and neighboring peptides were predicted to bind to 90% or more of these molecules. These results are the basis for devising a peptide vaccine against PCM that could be used at first as an immunological adjuvant to chemotherapy. The latter may involve a prolonged time and relapses are frequent.

To test this hypothesis, immunization with peptide 10 and chemotherapy were used together in an attempt to improve treatment of experimental PCM and prevent relapses. Two protocols were used. Mice were infected intratracheally with yeast cells of P. brasiliensis and drug treatment was started after 48 h or 30 days of the infection. The treatment continued for 30 days, during which groups of mice received intraperitoneal doses of itraconazole, fluconazole, ketoconazole, sulfame-thoxazole, or trimethoprim-sulfamethoxazole every 24 h. Amphotericin B was administered every 48 h. Immunization with P10 was carried out weekly for 4 weeks, once in complete Freund's adjuvant and three times in incomplete Freund's adjuvant (Marques et al., 2006). There was a significant reduction in the fungal load (measured as CFUs) in both groups immunized with P10 only, or treated with antifungal drugs, but an additive protective effect was observed with the combination of both. In animals treated with sulfamethoxazole, an early protection was followed by relapse, but the association of sulfamethoxazole and P10 vaccination succeeded in controlling the disease. In the second protocol that aimed at reproducing the condition of established infection, the fungal burden was examined after 60 and 120 days of infection. An additive protective effect of P10 immunization and drug treatment was also observed, with 60-80% reduction of lung CFUs compared with untreated infected animals. Lung homogenates were examined for cytokines in all groups. Generally, chemotherapy led to a predominant Th2 response with increased production of IL-4 and IL-10. P10 vaccination stimulated a Th1 response, rich in IFN-y and IL-12 without suppressing the Th2 component of the immune response (Marques et al., 2006).

In most cases of acute or subacute forms of paracoccidioidomicosis (most aggressive forms of PCM) the specific humoral immune response is preserved with the patients presenting high antibody titers, accompanied by severe depression of cell-immunity (Del Negro et al., 1994). In an attempt to reproduce experimental acute or subacute disease, Balb/c mice were treated with dexamethasone-21 phosphate added in the drinking water. After 30 days of treatment animals were anergic, as shown by negative DTH reactivity. This was previously determined in infected animals, treated with 0.15 mg/kg of dexamethazone and injected with P. brasiliensis crude antigen. Mice were then infected with a virulent P. brasiliensis isolate. Fifteen days after infection, animals were treated with trimethoprim-sulfamethoxazole or itraconazole, were immunized with P10 or received the association of both treatments (drug and peptide). Mice were killed 45 or 90 days after infection and CFU determination showed that treatment with drugs or P10 immunization resulted in significant reduction of the fungal burden in the lung, spleen and liver. The association of drugs and P10 immunization conferred additional protection. There was a significant increase of IL-12 and IFN-y and decrease of IL-4 and IL-10 in groups of mice immunized with P10 alone or in association with antifungal drugs. These results suggest that P10 immunization can be effective in the case of anergic patients.

The question of antigen delivery using formulations that do not require CFA has been addressed in two ways. A plasmid DNA construct including the gp43 gene was used to immunize mice followed by i.t. challenge with P. brasiliensis yeast forms. A mixed Th-1/Th-2 immune response was elicited which protected Balb/c mice against fungal infection (Pinto et al., 2000). When lymphocytes from mice, which had been immunized with plasmid over a 60-day period were tested in vitro with gp43 or ConA, only IL-2 and IFN-y but not IL-4 and IL-10 were produced. Moreover, the lung clearance observed with plasmid immunization was very similar to that obtained with P10 immunization. Immunization with plasmid DNA with the P10 minigene, associated or not with a plasmid carrying a murine IL-12 DNA insert, was tested in Balb/c mice intratracheally infected with a virulent isolate (Pb18) of P. brasiliensis. Similarly, as described above, such immunization elicited significant reduction of fungal burden in lung, spleen and liver. Co-immunization with P10/IL-12 plasmids rendered increased protection with virtually no detection of dissemination to spleen and liver. A mixed Th1/Th2 immune response was also detected. A significant production of IL-12 and IFN-y and reduction of IL-4 levels in lung homogenates were observed with DNA vaccination with plasmid-P10 or -IL-12 individually or combined.

Another approach to deliver the T cell epitope involved a multiple antigen peptide (MAP) construction. Four equal LIAIHTLAIRYAN chains were synthesized on a branched lysine core containing glycine at the C-terminal position and the product was called M10 because of its analogy with P10. Control MAPs had the same lysine core attached to truncated peptides derived from P10, with 5-10 amino acids long. Proliferation of lymph node cells from P10 or M10-sensitized mice was identical when stimulated in vitro with either P10 or M10. Mice immunized once with M10 with no adjuvant and challenged intratracheally with P. brasiliensis showed significantly fewer lung, spleen and liver CFUs and few or no yeasts in lung histopathological sections (Taborda et al., 2004).

Further association of P10 with a gp43 peptide eliciting protective antibody response is still being considered because no defined linear sequences have been found so far reacting with antibodies. A report on the characterization of anti-gp70 mAbs protective against experimental PCM (Mattos Grosso et al., 2003) stimulated the parallel study with gp43. Both gp43 and gp70 are markers monitoring the regression of PCM through their decreased antigenemia and specific antibody response in patients submitted to chemotherapy (Marques da Silva et al., 2004; Silva et al., 2004).

The effect of anti-gp43 mAbs on the i.t. infection by P. brasiliensis was examined in Balb/c mice injected intraperitoneally with 6 IgG2a and IgG2b different antibodies 24 h before the challenge. Analysis of the CFU in the lungs of animals 15 days after infection showed that all tested antibodies, except mAb 32H, were partially protective. Thirty days after infection, however, mAbs 19G, 10D, and 3E still showed a protective effect, with reduced lung CFUs, while mAb 32H, 17D, and 21F allowed full infection, with lung CFUs similar to control animals treated only with irrelevant mAbs. In a phagocytosis assay with peritoneal and alveolar macrophages, all monoclonal antibodies, except 32H, increased significantly the phagocytosis index (Buissa-Filho et al., 2005). Studying the reactivity of mAb 3E, the most efficient in the reduction of fungal burden and phagocytosis, against a panel of gp43-derived peptides it is suggested that the epitope recognized by mAb 3E lies within the sequence NHVRIPIGYWAV shared with A. fumigatus, A. oryzae and B. graminis internal sequences of P-1,3-glucanases.This peptide might play a role, together with P10, as another candidate for a peptide vaccine against PCM. A scheme showing the network association of data on gp43 and P10 aiming at a human vaccine is shown in Figure 11.3.

Figure 11.3 Network associating the diverse studies with gp43 and derived peptide P10, aiming at a human vaccine. All interactions and materials used are described in the text. APCs, antigen presenting cells; DCs, dendritic cells; MAP, multiple antigen peptide; GR, granuloma; HLA-DR, human lymphocyte antigen, DR alleles; Hex13GlcNAc2, N-linked oligosaccharide of gp43, Hex13 = 12 a-mannosyl and 1 P-galactofuranosyl, residues, NAc2Glc = 2 N-acetylglucosaminyl residues

Figure 11.3 Network associating the diverse studies with gp43 and derived peptide P10, aiming at a human vaccine. All interactions and materials used are described in the text. APCs, antigen presenting cells; DCs, dendritic cells; MAP, multiple antigen peptide; GR, granuloma; HLA-DR, human lymphocyte antigen, DR alleles; Hex13GlcNAc2, N-linked oligosaccharide of gp43, Hex13 = 12 a-mannosyl and 1 P-galactofuranosyl, residues, NAc2Glc = 2 N-acetylglucosaminyl residues

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