Yeast Expressing Gp43 Protein as a Vaccine Against Paracoccidioides brasiliensis Infection.

Paracoccidioidomycosis (PCM) represents the most frequent systemic mycosis in Latin American. The disease is caused by the pathogenic thermally dimorphic fungus Paracoccidioides brasiliensis, and is initially characterized by pulmonary lesions, which can subsequently disseminate to other organs, resulting in secondary injuries. Although its high incidence, there is no commercially available vaccine against fungal diseases. A novel strategy, using Saccharomyces cerevisiae yeast as a vehicle for immunization against PCM, was recently successfully described. Herein, we describe strategies for the construction of the suitable S. cerevisiae vaccine, and protocols of administration and evaluation of the efficacy of the vaccine against experimental PCM.

Intrinsic MyD88-Akt1-mTOR Signaling Coordinates Disparate Tc17 and Tc1 Responses during Vaccine Immunity against Fungal Pneumonia.

Fungal infections have skyrocketed in immune-compromised patients lacking CD4+ T cells, underscoring the need for vaccine prevention. An understanding of the elements that promote vaccine immunity in this setting is essential. We previously demonstrated that vaccine-induced IL-17A+ CD8+ T cells (Tc17) are required for resistance against lethal fungal pneumonia in CD4+ T cell-deficient hosts, whereas the individual type I cytokines IFN-γ, TNF-α and GM-CSF, are dispensable. Here, we report that T cell-intrinsic MyD88 signals are crucial for these Tc17 cell responses and vaccine immunity against lethal fungal pneumonia in mice. In contrast, IFN-γ+ CD8+ cell (Tc1) responses are largely normal in the absence of intrinsic MyD88 signaling in CD8+ T cells. The poor accumulation of MyD88-deficient Tc17 cells was not linked to an early onset of contraction, nor to accelerated cell death or diminished expression of anti-apoptotic molecules Bcl-2 or Bcl-xL. Instead, intrinsic MyD88 was required to sustain the proliferation of Tc17 cells through the activation of mTOR via Akt1. Moreover, intrinsic IL-1R and TLR2, but not IL-18R, were required for MyD88 dependent Tc17 responses. Our data identify unappreciated targets for augmenting adaptive immunity against fungi. Our findings have implications for designing fungal vaccines and immune-based therapies in immune-compromised patients.

Immune modulators as adjuncts for the prevention and treatment of invasive fungal infections.

Invasive fungal diseases are increasingly important opportunistic infections that are intimately linked to immune-suppression in the context of cytotoxic treatment of neoplastic diseases, stem cell and solid-organ transplantation, and primary immune deficiencies. Mortality rates remain high despite the availability of novel antifungals that are both safe and highly active in vitro, suggesting that clinical outcomes may be improved through modulation of host immunity. Ongoing advances in our knowledge of fungal-host interactions facilitate rational design of novel immunotherapeutics. Thus, antifungal immunotherapy now includes age-old interventions such as granulocyte and immunoglobulin transfusions, as well as promising experimental techniques such as antifungal vaccines and adoptive immunotherapy. To realize the potential of these rapidly evolving technologies, transition from the bench to clinical-phase studies must occur at a more rapid pace.

TLR3 essentially promotes protective class I-restricted memory CD8⁺ T-cell responses to Aspergillus fumigatus in hematopoietic transplanted patients.

Aspergillus fumigatus is a model fungal pathogen and a common cause of severe infections and diseases. CD8⁺ T cells are present in the human and murine T-cell repertoire to the fungus. However, CD8⁺ T-cell function in infection and the molecular mechanisms that control their priming and differentiation into effector and memory cells in vivo remain elusive. In the present study, we report that both CD4⁺ and CD8⁺ T cells mediate protective memory responses to the fungus contingent on the nature of the fungal vaccine. Mechanistically, class I MHC-restricted, CD8⁺ memory T cells were activated through TLR3 sensing of fungal RNA by cross-presenting dendritic cells. Genetic deficiency of TLR3 was associated with susceptibility to aspergillosis and concomitant failure to activate memory-protective CD8⁺ T cells both in mice and in patients receiving stem-cell transplantations. Therefore, TLR3 essentially promotes antifungal memory CD8⁺ T-cell responses and its deficiency is a novel susceptibility factor for aspergillosis in high-risk patients.

[Therapeutic or prophylactic antifungal vaccination: problems and solutions]. / Therapeutische oder prophylaktische Vakzination bei Pilzinfektionen: Probleme und Lösungsansätze.

Due to a growing number of immunosuppressed patients after organ transplantation or antineoplastic chemotherapy, the significance of fungal infections has increased worldwide. In the future a large number of clinical studies with regard to genetically engineered vaccines - based on already performed pre-clinical investigations - will be conducted. However, classic vaccines might be of limited efficacy in fungal infections since fungi elicit primarily cellular immune responses.

Therapeutic efficacy of a conjugate vaccine containing a peptide mimotope of cryptococcal capsular polysaccharide glucuronoxylomannan.

Vaccination with P13, a peptide mimotope of the cryptococcal capsular polysaccharide glucuronoxylomannan (GXM), has been shown to confer protection against a subsequent lethal Cryptococcus neoformans challenge. In this study, we sought to investigate whether P13-based vaccines could be effective in an already-established infection. To address this question, we developed a systemic chronic cryptococcal infection model. We vaccinated chronically infected mice with P13-protein conjugates and monitored their survival. Compared to the controls, the conjugates prolonged the survival of chronically infected mice. The degree of protection was a function of the mouse strain (BALB/c or C57BL/6), the carrier protein (tetanus toxoid or diphtheria toxoid), and the route of infection (intraperitoneal or intravenous). Serum GXM levels were correlated with the day of death, but the correlation was driven by the carrier protein and mouse strain. The passive transfer of heat-treated sera from P13 conjugate-vaccinated mice conferred protection to naïve BALB/c mice, indicating that antibody immunity could contribute to protection. The measurement of peripheral blood cytokine (gamma interferon [IFN-gamma], interleukin-10 [IL-10], and IL-6) gene expression showed that P13 conjugate-vaccinated BALB/c and C57BL/6 mice mounted a strong Th2 (IL-10)-like response relative to the Th1 (IFN-gamma)-like response, with the degree depending on the mouse strain and carrier protein. Taken together, our data suggest that a vaccine could hold promise in the setting of chronic cryptococcosis, and that vaccine efficacy could depend on immunomodulation and augmentation of the natural immune response of the host.

[Carbohydrates and vaccines: from purified polysaccharides to semi-synthetic glycoconjugate vaccines]. / Sucres et vaccins: du polysaccharide purifié au glycoconjugué semi-synthétique.

Over the last decades, capsular polysaccharides have been successfully used as antibacterial vaccines. Marketing several polysaccharide-protein conjugate vaccines filled the gap in many areas of children and infant vaccination. By facilitating access to structures of increasing complexity, recent progress in glycochemistry has enabled the design of more and more precisely defined glycoconjugate vaccines using synthetic saccharide components which mimic epitopes naturally implicated in protection. This strategy was recently validated in humans. It opens the way to new perspectives in vaccine research devoted to prophylactic and/or therapeutic applications against bacterial, fungal, parasitic or viral infections, and certain cancers.

Prospects for development of vaccines against fungal diseases.

Despite recent additions to our antifungal drug armamentarium, success rates for many mycoses remain unacceptably low and antifungal drug therapy is often limited by toxicity, resistance and high cost. To circumvent these difficulties, alternative approaches to prevention and treatment are being developed, including vaccines and passive immunotherapy. Here, we review the progress of current research in this field, discuss some of the potential obstacles to developing and marketing a protective antifungal vaccine, and summarize two clinical trials of monoclonal antibodies as adjunctive treatment of established mycoses. In animal models of fungal infections, protective responses have been elicited with vaccines composed of whole organisms, soluble cell free fractions, purified proteins, glycans and nucleic acids. Methods to boost the immune response to vaccination include the use of adjuvants and antigen-loaded dendritic cells (DCs). A significant challenge to the development of effective vaccines will be to elicit immune responses in immunocompromised individuals who are most at risk for invasive fungal infections.

Both CD4+ and CD8+ T cells can mediate vaccine-induced protection against Coccidioides immitis infection in mice.

To determine which lymphocytes are required for vaccine-induced immunity to coccidioidomycosis, we used a temperature-sensitive mutant of Coccidioides immitis to immunize mice lacking subsets of lymphocytes or specific cytokines and infected the mice 4 weeks later with virulent C. immitis. After 2 weeks, we determined the number of fungi in their lungs and spleens. Vaccine-induced immunity required alpha beta T lymphocytes. beta -2 microglobulin knockout (KO) mice were protected by immunization, and we transferred protection using CD4+ T cells from immunized mice. However, vaccination also protected CD4+ KO mice, which suggests that CD8+ T cells played a role in vaccine-induced immunity, even though they were not required. We adaptively transferred protection using spleen cells from immunized CD4+ KO mice to nonimmune B6 mice, but CD8+ -depleted spleen cells did not protect against infection. Recipients of spleen cells from immunized CD4+ KO mice had 6 times more tumor necrosis factor (TNF)- alpha mRNA in their lungs than did mice that received nonimmune spleen cells, and TNF receptor-1 KO mice were not fully protected by immunization. These results show that both CD4+ and CD8+ T cells can protect against coccidioidomycosis and that TNF- alpha is a necessary component of the acquired immune response.

Dose-response relationship and mechanism of action of Saccharomyces boulardii in castor oil-induced diarrhea in rats.

For biotherapeutic agents, there is a lack of information on dose-response relationships and mechanism of action. The present study was designed to address these issues for Saccharomyces boulardii using the rat model of castor oil-induced diarrhea. A single dose of Saccharomyces boulardii at 12 x 10(10) CFU/kg of viable cells given 1 hr before castor oil administration significantly reduced the onset of diarrhea. Repeated ingestion of the yeast, twice daily between 1.2 and 12 x 10(10) CFU/kg for 5 days before castor oil, showed a dose-response relationship. The percentage of rats with diarrhea decreased and a stronger protection was afforded by the repeated treatment. The mechanism of action of Saccharomyces boulardii in this model was investigated with two classes of antagonists, naloxone and L-arginine. The effect of Saccharomyces boulardii was not inhibited by naloxone but was significantly reduced by L-arginine. This last result suggests a novel mechanism of action for Saccharomyces boulardii involving a possible inhibition of nitric oxide production by the yeast.

Combined effects of chemotherapy and host antitumor response in a murine histocompatible lymphoma model.

Combined effects of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and host antitumor immune response were studied in mice inoculated intraperitoneally with histocompatible LSTRA leukemia cells carrying virus-induced transplantation antigens. Marked chemo-immune collaborative activity was found to occur when selected schedules of BCNU administration were employed. Moreover, synergist effects were also detected between chemotherapy and both specific and non-specific immunotherapy.

Treatment of equine phycomycosis by immunotherapy and surgery.

Treatment of equine phycomycosis with a vaccine derived from ultrasonicated hyphae of Hyphomyces destruens was attempted in 30 cases of clinical hyphomycosis, 10 cases of hyphomycosis following unsuccessful surgery and 5 cases of basidiobolomycosis. Approximately 53% of animals with clinical hyphomycosis were cured after vaccination, while a further 33% clinically improved. All horses with hyphomycosis treated within 2 weeks of unsuccessful surgery were cured. There was no response to vaccination with a Hyphomyces preparation in horses with basidiobolomyucosis, while surgery alone resulted in a cure of approximately 69%. Animals tolerated the vaccination procedures well. In all cases there was a moderate to severe reaction at the site of subcutaneous injection which subsided within 96 hours. In approximately 30% of injections a sterile abscess formed at the site but responded promptly to treatment. Response to vaccination at the site of the lesion was observed within 7 to 10 days of first injection. There was progressive reduction of pruritus, drying of the surface of the lesion, expulsion of kunkers, fibrosis of the granuloma, and eventually complete epithelialisation.