Julián E. Muñoz

The Monoclonal Antibody against the Major Diagnostic Antigen of Paracoccidioides brasiliensis Mediates Immune Protection in Infected BALB/c Mice Challenged Intratracheally with the Fungus

ABSTRACT The protective role of specific antibodies against Paracoccidioides brasiliensis is controversial. In the present study, we analyzed the effects of monoclonal antibodies on the major diagnostic antigen (gp43) using in vitro and in vivo P. brasiliensis infection models. The passive administration of some monoclonal antibodies (MAbs) before and after intratracheal or intravenous infections led to a reduced fungal burden and decreased pulmonary inflammation. The protection mediated by MAb 3E, the most efficient MAb in the reduction of fungal burden, was associated with the enhanced phagocytosis of P. brasiliensis yeast cells by J774.16, MH-S, or primary macrophages. The ingestion of opsonized yeast cells led to an increase in NO production by macrophages. Passive immunization with MAb 3E induced enhanced levels of gamma interferon in the lungs of infected mice. The reactivity of MAb 3E against a panel of gp43-derived peptides suggested that the sequence NHVRIPIGWAV contains the binding epitope. The present work shows that some but not all MAbs against gp43 can reduce the fungal burden and identifies a new peptide candidate for vaccine development.

Therapeutic DNA vaccine encoding peptide P10 against experimental paracoccidioidomycosis.

Paracoccidioidomycosis (PCM), caused by Paracoccidioides brasiliensis, is the most prevalent invasive fungal disease in South America. Systemic mycoses are the 10th most common cause of death among infectious diseases in Brazil and PCM is responsible for more than 50% of deaths due to fungal infections. PCM is typically treated with sulfonamides, amphotericin B or azoles, although complete eradication of the fungus may not occur and relapsing disease is frequently reported. A 15-mer peptide from the major diagnostic antigen gp43, named P10, can induce a strong T-CD4+ helper-1 immune response in mice. The TEPITOPE algorithm and experimental data have confirmed that most HLA-DR molecules can present P10, which suggests that P10 is a candidate antigen for a PCM vaccine. In the current work, the therapeutic efficacy of plasmid immunization with P10 and/or IL-12 inserts was tested in murine models of PCM. When given prior to or after infection with P. brasiliensis virulent Pb 18 isolate, plasmid-vaccination with P10 and/or IL-12 inserts successfully reduced the fungal burden in lungs of infected mice. In fact, intramuscular administration of a combination of plasmids expressing P10 and IL-12 given weekly for one month, followed by single injections every month for 3 months restored normal lung architecture and eradicated the fungus in mice that were infected one month prior to treatment. The data indicate that immunization with these plasmids is a powerful procedure for prevention and treatment of experimental PCM, with the perspective of being also effective in human patients.

Poly(lactic acid-glycolic acid) nanoparticles markedly improve immunological protection provided by peptide P10 against murine paracoccidioidomycosis.

Background and purpose:  The present study reports on the preparation and testing of a sustained delivery system for the immunomodulatory peptide P10 aimed at reducing the in vivo degradation of the peptide and the amount required to elicit a protective immune response against paracoccidioidomycosis.

The role of adjuvants in therapeutic protection against paracoccidioidomycosis after immunization with the P10 peptide

Paracoccidioidomycosis (PCM), a common chronic mycosis in Latin America, is a granulomatous systemic disease caused by the thermo-dimorphic fungus Paracoccidioides brasiliensis. The glycoprotein gp43 is the main antigen target of P. brasiliensis and a 15-mer internal peptide (QTLIAIHTLAIRYAN), known as P10, defines a major CD4+-specific T cell epitope. Previous results have indicated that, besides having a preventive role in conventional immunizations prior to challenge with the fungus, protective anti-fungal effects can be induced in P. brasiliensis-infected mice treated with P10 administered with complete Freund’s adjuvant (CFA). The peptide elicits an IFN-γ-dependent Th1 immune response and is the main candidate for effective immunotherapy of patients with PCM, as an adjunctive approach to conventional chemotherapy. In the present study we tested the therapeutic effects of P10 combined with different adjuvants [aluminum hydroxide, CFA, flagellin, and the cationic lipid dioctadecyl-dimethylammonium bromide (DODAB)] in BALB/c mice previously infected with the P. brasiliensis Pb18 strain. Significant reductions in the number of colony forming units of the fungus were detected in lungs of mice immunized with P10 associated with the different adjuvants 52 days after infection. Mice treated with DODAB and P10, followed by mice treated with P10 and flagellin, showed the most prominent effects as demonstrated by the lowest numbers of viable yeast cells as well as reductions in granuloma formation and fibrosis. Concomitantly, secretion of IFN-γ and TNF-α, in contrast to interleukin (IL)-4 and IL-10, was enhanced in the lungs of mice immunized with P10 in combination with the tested adjuvants, with the best results observed in mice treated with P10 and DODAB. In conclusion, the present results demonstrate that the co-administration of the synthetic P10 peptide with several adjuvants, particularly DODAB, have significant therapeutic effects in experimental PCM.

Therapeutic use of a cationic antimicrobial peptide from the spider Acanthoscurria gomesiana in the control of experimental candidiasis

BackgroundAntimicrobial peptides are present in animals, plants and microorganisms and play a fundamental role in the innate immune response. Gomesin is a cationic antimicrobial peptide purified from haemocytes of the spider Acanthoscurria gomesiana. It has a broad-spectrum of activity against bacteria, fungi, protozoa and tumour cells. Candida albicans is a commensal yeast that is part of the human microbiota. However, in immunocompromised patients, this fungus may cause skin, mucosal or systemic infections. The typical treatment for this mycosis comprises three major categories of antifungal drugs: polyenes, azoles and echinocandins; however cases of resistance to these drugs are frequently reported. With the emergence of microorganisms that are resistant to conventional antibiotics, the development of alternative treatments for candidiasis is important. In this study, we evaluate the efficacy of gomesin treatment on disseminated and vaginal candidiasis as well as its toxicity and biodistribution.ResultsTreatment with gomesin effectively reduced Candida albicans in the kidneys, spleen, liver and vagina of infected mice. The biodistribution of gomesin labelled with technetium-99 m showed that the peptide is captured in the kidneys, spleen and liver. Enhanced production of TNF-α, IFN-γ and IL-6 was detected in infected mice treated with gomesin, suggesting an immunomodulatory activity. Moreover, immunosuppressed and C. albicans-infected mice showed an increase in survival after treatment with gomesin and fluconazole. Systemic administration of gomesin was also not toxic to the micConclusionsGomesin proved to be effective against experimental Candida albicans infection. It can be used as an alternative therapy for candidiasis, either alone or in combination with fluconazole. Gomesins mechanism is not fully understood, but we hypothesise that the peptide acts through the permeabilisation of the yeast membrane leading to death and/or releasing the yeast antigens that trigger the host immune response against infection. Therefore, data presented in this study reinforces the potential of gomesin as a therapeutic antifungal agent in both humans and animals.

DNA vaccine encoding peptide P10 against experimental paracoccidioidomycosis induces long-term protection in presence of regulatory T cells

Paracoccidioidomycosis is a granulomatous systemic mycosis endemic in Brazil and other Latin America countries. A DNA vaccine encoding the immunoprotective peptide 10 (P10) significantly reduced the fungal burden in mice when given prior to or after intratracheal challenge with Paracoccidioides brasiliensis. Presently, the generation/expansion of CD4+ CD44hi memory T cells as well as Foxp3+ Treg cells in mice immunized with the DNA vaccine (pcDNA3-P10) before and after infection with P. brasiliensis was investigated. Memory CD4+ CD44hi T cells simultaneously with Foxp3+ Treg cells increased in the spleens and lungs of pcDNA3-P10 immunized mice on day 0, 30, 60 and 120 postinfection. Histopathology of the lung tissue showed minimal inflammation in immunized mice compared with the unimmunized group, suggesting a role for regulatory T cells in controlling the immunopathology. The DNA vaccine shows that the repeated immunization generates memory cells and regulatory T cells that replace the initially protective pro-inflammatory T cells conferring a long term protection while preserving the integrity of the infected tissue.

A Naturally Occurring Antibody Fragment Neutralizes Infectivity of Diverse Infectious Agents

A phosphorylated peptide, named K40H, derived from the constant region of IgMs was detected in human serum by liquid chromatography coupled to high-resolution mass spectrometry. Synthetic K40H proved to exert a potent in vitro activity against fungal pathogens, and to inhibit HIV-1 replication in vitro and ex vivo. It also showed a therapeutic effect against an experimental infection by Candida albicans in the invertebrate model Galleria mellonella. K40H represents the proof of concept of the innate role that naturally occurring antibody fragments may exert against infectious agents, shedding a new light upon the posthumous role of antibodies and opening a new scenario on the multifaceted functionality of humoral immunity.

Antifungal Activity of the Biphosphinic Cyclopalladate C7a against Candida albicans Yeast Forms In Vitro and In Vivo

Vulvovaginal and invasive candidiasis are frequent conditions in immunosuppressed individuals caused by Candida albicans and non-albicans Candida spp. Fluconazole and Amphotericin B are the main drugs used to fight the infection. However, resistance to fluconazole and other azole antifungal drugs is an important clinical problem that encourages the search for new therapeutic alternatives. In this work, we evaluate the antifungal activity of the biphosphinic cyclopalladate C7a in the in vitro and in vivo model. Our results showed fungicidal activity, with low values of minimal inhibitory concentrations and minimum fungicidal concentrations, even for fluconazole and/or miconazole resistant Candida isolates. Fluorescence microscopy and transmission electron microscopy revealed that the compound was able to inhibit the formation of hyphae/pseudohyphae and, moreover, promoted morphological alterations in cellular organelles and structures, such as disruption of cell wall, apparent mitochondrial swelling, chromatin marginalization into the nuclei and increased numbers of electron-lucent vacuoles. C7a significantly decreased the biofilm formation and reduced the viability of yeast cells in mature biofilms when tested against a virulent C. albicans strain. In vivo assays demonstrated a significant decrease of fungal burden in local (vaginal canal) and disseminated (kidneys) infection. In addition, we observed a significant increase in the survival of the systemically infected animals treated with C7a. Our results suggest C7a as a novel therapeutic agent for vaginal and disseminated candidiasis, and an alternative for conventional drug-resistant Candida.

Antibodies Against Glycolipids Enhance Antifungal Activity of Macrophages and Reduce Fungal Burden After Infection with Paracoccidioides brasiliensis

Paracoccidioidomycosis is a fungal disease endemic in Latin America. Polyclonal antibodies to acidic glycosphingolipids (GSLs) from Paracoccidioides brasiliensis opsonized yeast forms in vitro increasing phagocytosis and reduced the fungal burden of infected animals. Antibodies to GSL were active in both prophylactic and therapeutic protocols using a murine intratracheal infection model. Pathological examination of the lungs of animals treated with antibodies to GSL showed well-organized granulomas and minimally damaged parenchyma compared to the untreated control. Murine peritoneal macrophages activated by IFN-γ and incubated with antibodies against acidic GSLs more effectively phagocytosed and killed P. brasiliensis yeast cells as well as produced more nitric oxide compared to controls. The present work discloses a novel target of protective antibodies against P. brasiliensis adding to other well-studied mediators of the immune response to this fungus.

Dendritic Cells Primed with Paracoccidioides brasiliensis Peptide P10 Are Therapeutic in Immunosuppressed Mice with Paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is an endemic systemic mycosis in Latin America, with the highest prevalence in Brazil, Colombia, and Venezuela. Fungi of the Paracoccidioides genus are etiologic agents of the disease. The 15 amino acid peptide P10 is derived from gp43, the main diagnostic antigen of Paracoccidioides brasiliensis. We previously reported that P10-pulsed dendritic cells (DCs) induce a protective response against P. brasiliensis. Presently, dexamethasone-treated BALB/c mice were intratracheally infected with P. brasiliensis Pb18 to establish the therapeutic efficacy of P10-pulsed DCs. Immunosuppressed and infected animals that received DCs had a reduction in their fungal burden, and this result was most pronounced in mice receiving DCs primed with P10. The efficacy of therapeutic DCs was significantly augmented by concomitant treatment with trimethoprim-sulfamethoxazole. Additionally, primed-DCs with or without the antifungal drug induced a beneficial Th1-biased immune response and significantly reduced tissue damage. In conclusion, these studies with immunocompromised mice demonstrate that P10-pulsed DCs with or without concomitant antifungal drugs are potently effective in combating invasive PCM. These findings support further translational studies to validate the use of P10-primed DCs for PCM in immunocompetent and immunosuppressed hosts.