In vivo activity of terpinen-4-ol, the main bioactive component of Melaleuca alternifolia Cheel (tea tree) oil against azole-susceptible and -resistant human pathogenic Candida species

BACKGROUND: Recent investigations on the antifungal properties of essential oil of Melaleuca alternifolia Cheel (Tea Tree Oil, TTO) have been performed with reference to the treatment of vaginal candidiasis. However, there is a lack of in vivo data supporting in vitro results, especially regarding the antifungal properties of TTO constituents. Thus, the aim of our study was to investigate the in vitro and the in vivo anti-Candida activity of two critical bioactive constituents of TTO, terpinen-4-ol and 1,8-cineole. METHODS: Oophorectomized, pseudoestrus rats under estrogen treatment were used for experimental vaginal infection with azole (fluconazole, itraconazole) -susceptible or -resistant strains of C. albicans. All these strains were preliminarily tested for in vitro susceptibility to TTO, terpinen-4-ol and 1,8-cineole for their antifungal properties, using a modification of the CLSI (formerly NCCLS) reference M27-A2 broth micro-dilution method. RESULTS: In vitro minimal inhibitory concentrations (MIC(90)) values were 0.06% (volume/volume) for terpinen-4-ol and 4% (volume/volume) for 1,8-cineole, regardless of susceptibility or resistance of the strains to fluconazole and itraconazole. Fungicidal concentrations of terpinen-4-ol were equivalent to the candidastatic activity. In the rat vaginal infection model, terpinen-4-ol was as active as TTO in accelerating clearance from the vagina of all Candida strains examined. CONCLUSION: Our data suggest that terpinen-4-ol is a likely mediator of the in vitro and in vivo activity of TTO. This is the first in vivo demonstration that terpinen-4-ol could control C. albicans vaginal infections. The purified compound holds promise for the treatment of vaginal candidiasis, and particularly the azole-resistant forms

Plant antibodies for human antifungal therapy

There is an increasing interest in the development of therapeutic antibodies (Ab) to improve the control of fungal pathogens, but none of these reagents is available for clinical use. We previously described a murine monoclonal antibody (mAb 2G8) targeting β-glucan, a cell wall polysaccharide common to most pathogenic fungi, which conferred significant protection against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans in animal models. Transfer of this wide-spectrum, antifungal mAb into the clinical setting would allow the control of most frequent fungal infections in many different categories of patients. To this aim, two chimeric mouse-human Ab derivatives from mAb 2G8, in the format of complete IgG or scFv-Fc, were generated, transiently expressed in Nicotiana benthamiana plants and purified from leaves with high yields (approximately 50 mg Ab/kg of plant tissues). Both recombinant Abs fully retained the β-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially β1,3-linked glucan molecules present at the fungal cell surface and directly inhibited the growth of C. albicans and its adhesion to human epithelial cells in vitro. In addition, both the IgG and the scFv-Fc promoted C. albicans killing by isolated, human polymorphonuclear neutrophils in ex vivo assays and conferred significant antifungal protection in animal models of systemic or vulvovaginal C. albicans infection. These recombinant Abs represent valuable molecules for developing novel, plant-derived immunotherapeutics against candidiasis and, possibly, other fungal diseases

A novel glyco-conjugate vaccine against fungal pathogens

To generate a vaccine to protect against a variety of human pathogenic fungi, we conjugated laminarin (Lam), a well-characterized but poorly immunogenic β-glucan preparation from the brown alga Laminaria digitata, with the diphtheria toxoid CRM197, a carrier protein used in some glyco-conjugate bacterial vaccines. This Lam-CRM conjugate proved to be immunogenic and protective as immunoprophylactic vaccine against both systemic and mucosal (vaginal) infections by Candida albicans. Protection probably was mediated by anti-β-glucan antibodies as demonstrated by passive transfer of protection to naive mice by the whole immune serum, the immune vaginal fluid, and the affinity-purified anti-β-glucan IgG fractions, as well as by administration of a β-glucan–directed IgG2b mAb. Passive protection was prevented by adsorption of antibodies on Candida cells or β-glucan particles before transfer. Anti-β-glucan antibodies bound to C. albicans hyphae and inhibited their growth in vitro in the absence of immune-effector cells. Remarkably, Lam-CRM–vaccinated mice also were protected from a lethal challenge with conidia of Aspergillus fumigatus, and their serum also bound to and markedly inhibited the growth of A. fumigatus hyphae. Thus, this novel conjugate vaccine can efficiently immunize and protect against two major fungal pathogens by mechanisms that may include direct antifungal properties of anti-β-glucan antibodies

Peptides of the Constant Region of Antibodies Display Fungicidal Activity

Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents

Protection by Anti-β-Glucan Antibodies Is Associated with Restricted β-1,3 Glucan Binding Specificity and Inhibition of Fungal Growth and Adherence

Anti-β-glucan antibodies elicited by a laminarin-conjugate vaccine confer cross-protection to mice challenged with major fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. To gain insights into protective β-glucan epitope(s) and protection mechanisms, we studied two anti-β-glucan monoclonal antibodies (mAb) with identical complementarity-determining regions but different isotypes (mAb 2G8, IgG2b and mAb 1E12, IgM). C. albicans, the most relevant fungal pathogen for humans, was used as a model

AIDS-related mycoses: the way forward.

The contribution of fungal infections to the morbidity and mortality of HIV-infected individuals is largely unrecognized. A recent meeting highlighted several priorities that need to be urgently addressed, including improved epidemiological surveillance, increased availability of existing diagnostics and drugs, more training in the field of medical mycology, and better funding for research and provision of treatment, particularly in developing countries

Anticandidal Immunity and Vaginitis: Novel Opportunities for Immune Intervention▿

The majority of human Candida infections occur at a mucosal surface. One of these infections, vulvovaginal candidiasis (VVC), is a widespread, common disease affecting a large proportion of otherwise healthy women. Some women undergo recurrent forms of VVC (RVVC); in these cases the quality of life is devastated, the associated cost of medical visits is high, there is substantial use of unprescribed therapy, and there is a possible increase in drug resistance. However, relatively little is known about the pathogenic and immunoregulatory mechanisms underlying VVC and RVVC. While there is consensus that local rather than systemic factors play a decisive role in controlling the infection, there is no consensus about the nature of the local factors. The results obtained with animal models and human investigations have not provided an overall consistent picture; rather, they have generated divergent interpretations about the role of innate and adaptive immunity against vaginal infection. Nonetheless, recent experimental evidence has resulted in some optimism concerning the challenge of clarifying the immunological basis of susceptibility to, and protection from, vaginal candidiasis through the development of appropriate immune interventions to integrate with or even replace antifungal chemotherapy. The aim of this review is to provide a short update of the evidence mentioned above and the resulting optimism. Here, we critically consider the linkage between the virulence traits of the fungus and the host responses to these traits, two interrelated aspects which have rarely been treated together in other reviews of the topic (36, 39-41, 48, 54, 56, 57, 65, 66, 74, 94, 102). On this basis, we suggest that despite substantial belief to the contrary, novel tools derived from adaptive immunity, in particular virulence-neutralizing antibodies (Abs), may become part of the anti-Candida armamentarium to fight vaginal infectio

Intravaginal and Intranasal Immunizations Are Equally Effective in Inducing Vaginal Antibodies and Conferring Protection against Vaginal Candidiasis

Oophorectomized, estrogen-treated rats were immunized by the intravaginal or intranasal route with a mannoprotein extract (MP) or secreted aspartyl proteinases (Sap) of Candida albicans, with or without cholera toxin as a mucosal adjuvant. Both routes of immunization were equally effective in (i) inducing anti-MP and anti-Sap vaginal antibodies and (ii) conferring a high degree of protection against the vaginal infection by the fungus. These data suggest that appropriate fungal antigens and adjuvant can be used to protect against candidal vaginitis, by either route