Antifungal Immunotherapy and ... - Wiley Online Library

REVIEW doi: 10.1111/j.1365-3083.2008.02101.x ..................................................................................................................................................................

Antifungal Immunotherapy and Immunomodulation: A Double-hitter Approach to Deal with Invasive Fungal Infections M. Hamad

Abstract Department of Biology and Biotechnology, Hashemite University, Jordan and Taif University School of Medicine, Taif, Saudi Arabia Received 14 January 2008; Accepted in revised form 18 February 2008 Correspondence to: Dr Mawieh Hamad, Taif University School of Medicine, P.O. Box 888, Taif, Saudi Arabia. E-mail: taqiwmohanad@ yahoo.com

In recent years, the incidence of life-threatening fungal infections has dramatically increased. Despite significant developments in antifungal chemotherapy, its efficacy remains limited by the inability to sterilize infected organs and the tendency to induce resistance and cause side effects. In response to these challenges, it is now recognized that several aspects of antifungal immunity can be modulated to better deal with fungal infections. Extensive work was carried out on the development and testing of preventive and therapeutic antifungal vaccines. The potential use of cytokines, adoptive T-cell transfer, monoclonal antibodies (MoAb) and antimicrobial peptides (AMP) as solo or adjunctive therapies is also receiving much attention. Although each of these immunebased treatment strategies has many advantages and some shortcomings, none on its own, has proven satisfactorily effective to deal with invasive fungal infections. Appropriate combinations that optimize the advantages and minimize the disadvantages of immune-based antifungals are still lacking mainly due to the immense difficulty in sorting out candidate combinations given the long list of choices. In this review, immune-based antifungals are divided into two general categories on the basis of the intended target being the host (immunomodulation through vaccines, cytokines, adoptive T-cell transfer or MoAb) or the pathogen (immunotherapy through MoAb or AMP). Potential advantages and disadvantages of immunotherapy and immunomodulation are tentatively discussed so as to facilitate the design of future studies that aim at devising more potent immune-based antifungal treatment combinations.

Introduction The risk of fungal infections in immunocompromised hosts and in ICU patients has significantly increased over the last two decades. In clinical practice, Candida spp. and Cryptococcus spp. are the most frequently isolated yeasts and Aspergillus spp are the most commonly encountered filamentous fungi. The frequency of isolation of Fusarium spp., Scedosporium spp., Penicillium spp. and zygomycetes is also on the rise [1–3]. The rising rates of invasive fungal infections are attributed to the widespread use of immunosuppressants, antibiotics, antineoplastics and prosthetics as well as invasive surgery, burns, neutropenia and HIV infection [4]. Furthermore, better management of transplant patients and patients with cancer, AIDS or diabetes has significantly expanded the

number of immunocompromised hosts susceptible to fungal infections. Although conventional antifungal therapy or chemotherapy (mainly polyenes, azoles and 5-fluorocytosine) that targets the pathogen remains the treatment of choice for most fungal infections, it is becoming less and less capable of coping with current and projected trends of fungal infections. Side effects that associate with the use of antifungal agents, appearance of resistant fungal strains, varied spectra of activity and failure to sterilize infected organs seriously limit the efficacy of antifungal chemotherapy. On a more positive note, however, liposomal and lipid formulations of polyenes (amphotericin B and nystatin) and new azole derivatives (voriconazole, ravuconazole and posaconazole) are proving more effective and less harmful than previous generations [5]. Pathogen-tailored antifungal therapeutics are also

2008 The Author Journal compilation 2008 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 67, 533–543

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534 Antifungal Immune-based Treatment M. Hamad .................................................................................................................................................................. gaining some appeal; the capacity of the 19-mer2¢-OMe hairpin oligonucleotide to inhibit the growth of Candida albicans is a case in point [6]. Notwithstanding these developments, so long as combating fungal infections focuses only on the pathogen, resistance and adverse side effects are bound to persist. Combinatorial and adjunctive therapies that use two or more agents have proven more effective than solo therapies [7]. Additionally, approaches that target the host by conditioning or modulating its immune response are proving helpful in complementing deficiencies and minimizing some of the drawbacks of conventional therapy. For example, the inability of conventional antifungals to sterilize infected organs is considered as a disadvantage, hence the incessant efforts to introduce more potent agents that can eradicate fungi without taking into account the commensal non-pathologic and often mutually beneficial relationship between host and fungi as part of the normal flora. In this context, preservation of commensalisms can, in theory, be accommodated by cytokines that favour the differentiation and activation of Treg cells. Granted that conventional antifungals will not disappear any time soon, use of adjunctive therapy could make smaller doses of conventional antifungals given over shorter periods of time more effective and less harmful [7]. Therefore, there is a growing need for the development of combinatorial therapeutics that couple immunotherapy ⁄ immunomodulation with antifungal chemotherapy.

Antifungal immunity The relative role of the various immune components in fighting fungal infections (extensively reviewed in Refs 8–10 and in many references therein) varies depending on fungal species and its morphotype being yeast, pseudohyphae or hyphae as well as the anatomic site of the infection [10, 11]. In general terms, several aspects of innate and acquired immunity converge to protect the host against fungal infections (Fig. 1). Intact epithelia and endothelia, microbial antagonism, defensin, collectin and other antimicrobial peptides (AMP) provide the very first line of defence against fungal infections. Professional phagocytic cells (neutrophils, monocytes, macrophages and dendritic cells or DC) reduce fungal burden by oxidative and non-oxidative killing of fungi and by restricting fungal growth and infectivity. For instance, neutropenia results in increased susceptibility to candidiasis, cryptococcosis and aspergillosis in humans and animals. Oxidative burst-dependent killing of fungi involves the nicotinamide adenine dinucleotide phosphate oxidase – the inducible nitric oxide synthase – and the myeloperoxidase-mediated pathways. Non-oxidative killing involves cell lysis by defensin and neutrophil cationic peptides that target and disrupt fungal cell membrane. Mediators released by phagocytes restrict fungal growth

and minimize fungal infectivity by means of iron sequestration, inhibition of dimorphism and resistance to phenotype switching. The activity of DC and, to a much lesser extent, macrophages links innate and acquired immunity in a well-orchestrated manner. The central role of DC stems from their ability to express a diverse repertoire of pattern recognition receptors (PRR) that recognize and bind with a wide array of fungal pathogenassociated molecular patterns (PAMP). PRR like the toll-like receptor (TLR) and IL-1R family, complement receptor 3 (CD11b ⁄ CD18) and mannose binding lectin (MBL) mediate distinct downstream signalling cascades that result in the production of cytokines, AMP and other signalling and effector molecules. DC, as professional phagocytes, can internalize and process a diverse set of fungal peptides. Additionally, DC are effective at presenting antigenic peptides to T cells owing to their capacity to express ligands for T-cell co-stimulatory molecules, the CD80 ⁄ CD86 (B7.1 ⁄ B7.2) ligand that binds with CD28 and CTLA-4 (CD152) on T cells is a good example. DC ⁄ T helper (Th) cell engagement results in the differentiation of Th cells into Th1, Th2, Th17 or Treg cells. Differentiation of Th cells into Th1 leads to the production of protective pro-inflammatory cytokines IFN-c, IL-6 and IL-12, which is essential for protection against fungal infections. Additionally, IL-10 secreted by DC can activate CD4+CD25+ Treg cells which play a significant role in antifungal resistance [9]. However, high levels of IL-10 can negatively affect IFN-c production and hence weaken antifungal cell-mediated immunity against chronic candidiasis and severe endemic mycoses. Release of the signature cytokine IFN-c enhances the production and function of antibodies (Ab) and amplifies innate immune responses; other cytokines released by phagocytes and Th cells may also engage cytotoxic T cells. The role of Ab is well evidenced by the fact that systemic candidiasis (C. albicans), cryptoccocosis (Cryptoccocus neoformans) and other fungal infections often result in the production of opsonizing and complementfixing anti-sera. Finally, acquired cell-mediated immunity plays a very prominent role in the defence against fungal infections like systemic candidiasis, vaginal candidiasis, chronic mucocutaneous candidiasis among others. Based on current understanding of antifungal immunity as summarized in Fig. 1, various aspects of host immunity can be used or manipulated to better deal with fungal infections. Namely, preventive and therapeutic vaccines, cytokines, adoptive transfer of sensitized T cells, monoclonal antibodies (MoAb) and AMP. Although the list of specific examples in each of these classes is extensive, collective data regarding the use of any specific class in isolation is less than promising. Understandably, it is difficult to devise a combinatorial approach that enlists specific examples from each of these classes. Therefore, a re-evaluation of existing classes of immune-based 2008 The Author

Journal compilation 2008 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 67, 533–543

M. Hamad Antifungal Immune-based Treatment 535 ..................................................................................................................................................................

Figure 1 Brief summary of the major protective antifungal immune responses in the mammalian system. DC, via their extensive PRR repertoire and their capacity to process and present antigens to Th cells, play the central role in orchestrating innate and acquired protective antifungal immunity. Potential targets for immunomodulation ⁄ immunotherapy in the scheme were indicated by the symbol ¯. PMN, polymorphonuclear cells; MN, mononuclear cells, NADPH, reduced nicotinamide adenine dinucleotide phosphate; iNO, inducible nitric oxide.

antifungals with the hope of defining broader categories that share common features may help overcome this difficulty. In this review, various classes of immune-based antifungals are categorized based on the general mechanism(s) of action and hence the intended target being the host for immunomodulation or the pathogen for elimination (Table 1). Where appropriate, overlaps between the two general categories are noted.

Antifungal immunomodulation (targeting the host) The ability to specifically manipulate the immune system by vaccination and adoptive T-cell transfer or non-specifically by the administration of cytokines and other modulators has a long history of success in viral and bacterial infections, cancer, autoimmunity, inflammation and other disease states. In the case of fungal infections, however, neither protective nor therapeutic vaccines have been approved for clinical use thus far. Likewise, adoptive T-cell transfer or the administration of cytokines, in the best of cases, provided partial protection. Despite these setbacks, there is mounting clinical and experimental evidence to suggest that hot-targeting immunomodulation can be effective when used in combination with conventional antifungals. Host-targeting immunomodulation shares a number of advantages including board spectrum of activity, minimal toxic side effects and the inability to induce fungal resistance (Table 2). Competence of host

immunity as a prerequisite (for vaccines in particular) and inflammation as a potential consequence are among the common disadvantages of immunomodulation (Table 2). Fungal vaccines

Vaccination, by definition, aims at exposing the immune system of the host to a specific pathogen (in one form or another) so as to generate pathogen-specific immunologic memory which can kick in very effectively should subsequent infections by the same pathogen occur. Accordingly, vaccination is classified as a host-targeting immunomodulation strategy. Vaccination approaches against fungal infections involving live or attenuated fungi, cell wall or cytoplasmic extracts and the transfer of passive or adoptive immunity were tried with little success. The coccidioidomycosis vaccine clinical trial conducted in the early 1980s by the Valley Fever Vaccine Study Group did not show distinct differences between immunized and placebo subjects [12]. Based on an extensive fungal vaccine development and trial record, identification of immunogenic fungal components and the inclusion of effective adjuvants have emerged as important aspects in antifungal vaccine design. Despite the presence of significant similarities between fungal and human cells, the list of potential fungal cell antigens that are immunogenic to mammalian hosts is growing [5, 13]. Additionally, several adjuvants tested in recent years


536 Antifungal Immune-based Treatment M. Hamad .................................................................................................................................................................. Table 1 General mechanism(s) of action of the various classes of immune-based antifungal agents and strategies. Immune-based approach

Mechanism(s) of action

Host-targeting agents (immunomodulators) Vaccines Immunologic memory accelerates and amplifies immune responses to subsequent encounters with the vaccinated-against pathogen Cytokines Reverse the effects of neutropenia (GM-CSF, G-CSF and MCSF) Enhance Th1-dependent protective immunity (IFN-c, IL-6, IL-10 and IL-2) Adoptive T-cell transfer Secretion of cytokines (CD4+ and CD8+ T cells) Cytotoxic activity (CD8+ T cells) Monoclonal antibodies? Blocking of ligand ⁄ receptor interactions involved in innate and acquired cell signalling Cytokine inhibitors that could modify Th differentiation and function Antifungal peptides? (cationic AMP) Modulate cytokine production, alter host gene expression profile and minimize pro-inflammatory responses Pathogen-targeting agents (immunotherapeutics) Monoclonal antibodies Antifungal cytotoxicity via complement- or cell-mediated activity Opsonization Blocking adhesion of pathogen to cell surface Anti-idiotypic network perturbation (positive or negative input) Antifungal peptides Inhibition of growth or killing of fungi by lysis, binding and disruption of plasma membranes, pore formation and leakage Disruption of signalling cascades by interacting with cytoplasmic kinases and nuclear transcription factors

Table 2 Comparative summary of host-targeting immunomodulation and pathogen-targeting immunotherapy. Category

Targeting the pathogen (immunotherapy) Current collection of antifungal monoclonal antibodies Antimicrobial (antifungal) peptides

Potential advantages

Species-specific (MoAb); however, non-host-derived AMP have broad spectrum of activity that could target host cells Independent of host immune status Passive activity (rapid) Can be effective as solo antifungal therapeutics Increased risk of resistance

Potential disadvantages

Increased risk of toxicity to the host

in conjunction with different fungal immunogenic moieties have proven useful. Active immunization with a tetanus toxoid conjugate of glucuronoxylomannan resulted in the production of protective Ab in animal models of cryptococcosis [14]. Diphtheria toxoid CRM197 conjugated with the algal antigen laminarin (Lam), a poorly immunogenic b-glucan from Laminaria digitata, was protective against various forms of systemic and mucosal C. albicans infections in mice [15]. A novel polysaccharide– protein conjugate vaccine that uses Lam was also shown to elicit the production of Ab against cell wall b-glucan moieties and to mediate protection against experimental candidiasis and aspergillosis [16]. Vaccine-induced Ab manifested direct antifungal activity suggesting that vaccine efficacy may not solely depend on cellular immunity. Thymosin a1 was shown to promote the activation of DC to produce IL-12p70 and IFN-c that then prime Th1 responses against aspergillosis suggesting that TLR

Targeting the host (immunomodulation) Fungal vaccines and pulsed DC Cytokines Adoptive T-cell transfer Monoclonal antibodies and antimicrobial peptides (yet to be fully explored) Decreased risk of toxicity

Decreased risk of development of resistance Broad spectrum of activity Competence of host immunity is a prerequisite for therapeutic activity (vaccines); hence, the time needed for full activity is relatively long (slow) Inflammation is a potential consequence Mainly effective as adjunctive therapies

ligands are effective adjuvants [17]. Recovery from disseminated C. albicans infection in mice was correlated with the production of protective Ab against the molecular chaperone heat shock protein 90 (hsp90). Ab against pathogen-specific hsp90 epitopes and those against epitopes common to both pathogen and host are frequently reported in patients recovering from systemic candidiasis [18, 19]. Vaccination with the recombinant N-terminal domain of A1s1p (rA1s1p-N) was protective against disseminated candidiasis caused by virulent C. albicans strains in BALB ⁄ c mice and in other outbred mouse strains [20]. Several DNA-based vaccination approaches have been introduced as alternatives to conventional vaccination. A recombinant strain of Blastomyces dermatitidis lacking the WI-1 adhesin gene was protective against pathogenic strains of the fungus through Th1-mediated responses [21]. An hsp90-expressing DNA vaccine prepared by 2008 The Author



cloning mRNA from clinical isolates of C. albicans into the vaccination vector pVAX1 [22] was protective against systemic candidiasis in BALB ⁄ c mice. The vaccine elicited a protective anti-candida-hsp90 IgG response. DC pulsed with fungi, fungal extracts or fungal RNA were also used to develop fungal vaccines [23]. It is worth mentioning in this context that fungal RNA can activate DC via nucleotide receptor signalling cascades as evidenced by the upregulation of several TLR on DC following pulsation with fungal RNA [24]. Th1-mediated protection was reported in mice challenged with DC pulsed with Aspergillus fumigatus extracts mixed with CpG-ODN adjuvant [25]. Administration of A. fumigatus RNA-transfected DC induced resistance to subsequent challenges with the pathogen via Th1-derived IFN-c [24]. Active immunization is understandably the preferred means of protection as it sensitizes the immune system and enhances sterilization of infected organs thus preventing dormant infections granted that the subject is immunocompetent. Unfortunately, however, groups at risk of developing serious fungal infections (AIDS patients, diabetics, cancer patients and transplant patients) are immunocompromised and hence unlikely to greatly benefit from such approaches. Therefore, badly needed approaches that take suppressed and ⁄ or compromised immunity into account are discussed below. Adoptive T-cell transfer

At the risk of being redundant, it is worthwhile to emphasize the major role of Th1-dependent immune responses against fungal infections. The pivotal role of CD4+ T cells against fungal infections is well established [26–29]. Depletion of CD4+ T cells results in increased susceptibility to Pneumocystis pneumonia and other mycotic infections in rodents [29]. Differentiation of Th to Th1 represents the determining factor of resistance to fungal infections [11]. Induction of Th1-mediated responses by the secretion of IFN-c, IL-6, IL-10 and IL-12 confers significant protection against different forms of mycoses [30–32]. Additionally, positive lymphoproliferative responses characterized by overproduction of IFN-c in cultured cells were noted in healthy individuals receiving cellular extracts of: (i) A. fumigatus; (ii) A. fumigatus 88-kDa dipeptidase antigen; or (iii) A. fumigatus 90-kDa catalase antigen [31]. Adoptive transfer of CD4+ splenocytes from mice sensitized with A. fumigatus into naı¨ve mice resulted in prolonged survival following intravenous challenge with viable A. fumigatus conidia [33]. This and similar findings provide the rationale for further work on immunomodulation with antigen-specific T cells that could restore protective antifungal immunity in immunocompromised hosts. The combination of IL-12 and antiCD40, which prevents CD40 on B cells from binding

with CD154 on Th cells, significantly prolonged the survival of mice infected with C. neoformans [34]. Protection was positively correlated with decreased fungal burden in kidney and brain tissues and increased serum concentration of IFN-c and tumour necrosis factor alpha (TNF-a). Protection in this model seems to be dependent on IFNc as evidenced by the lack of protection in IFN-c knockout mice (IFN-c) ⁄ )) treated in the same manner. These findings show that adoptive Th cell transfer, as a hosttargeting immunomodulator, is of value in treating certain forms of mycosis. In contrast to the case of CD4+ T cells, classifying the adoptive transfer of CD8+ T cells as host-targeting immunomodulation is a harder bargain to sell; that unless the role of CD8+ T cells in fungal infections is furnished. In general terms, the role of CD8+ T cells in protection against infections (fungal or otherwise) is dependent upon CD4+ T-cell help. Although CD4 deficiency impairs CD8+ activation and function in non-fungal infections [35, 36], generation of fungi-specific effector CD8+ T cells capable of producing protective levels of IFN-c against pulmonary C. neoformans infections occurs in CD4-deficient (CD4)) mice [26]. Neutralization of IFN-c in CD4) CD8+ mice increased macrophage infection by C. neoformans. Depletion of CD4+ T cells in mice did not significantly decrease serum concentration of IFN-c induced by anti-CD40 ⁄ IL-2 combination treatment [34] suggesting that other T-cell subsets, such as the CD8+ subset, are involved. Based on these findings, the relative significance of a specific T-cell subset seems to heavily derive from the cytokine profile it produces during infection. Consequently, the direct use of pro-inflammatory, regulatory or suppressor cytokines to modulate the immune response during fungal infections seems the more logical. For one thing, the cytokine(s) to be administered and the optimal therapeutic dose at which to administer it to treat a particular fungal infection can be more easily determined and more effectively controlled. Cytokine therapy

Cytokines modulate the immune response of the host by acting as signalling molecules that specifically induce the proliferation, differentiation and activation or suppression (anergy or apoptosis) of different target cells. Specificity of cytokine activity is dependent upon the capacity of target cells to specifically express respective cytokine receptors. Cytokine ⁄ cytokine receptor engagement initiates a signalling cascade causing target cells to respond. Therefore, cytokines, as modulatory agents, act on the host, hence the classification host-targeting modulators. Administration of recombinant granulcocyte colony-stimulating factor (G-CSF), monocyte-CSF (M-CSF), GM-CSF or IFN-c was reported to shorten the duration of neutropenia (G-CSF and GM-CSF) and enhance phagocytic and


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killing activities of neutrophils, monocytes and macrophages (G-CSF, M-CSF and GM-CSF) [37, 38]. In cancer patients on corticosteroids, neutropenia predisposes to invasive fungal infections [39]. By reducing the duration of neutropenia and enhancing the antifungal activity of granulcocytes, cytokine adjunctive therapy was reported to minimize the risk of invasive fungal infections and permit aggressive cytotoxic therapy. IFN-c, IL-12 and anti-IL-4 were also reported to be protective by enhancing Th-1-dependent immunity [40]. The efficacy of cytokine adjunctive therapy varies depending on the level and nature of immunosuppression as well as the antifungal agent used. In A. fumigatus-infected outbred ICR mice pretreated with hydrocortisone, administration of recombinant human G-CSF prior to infection antagonized the action of SCH56592 azole derivative [41]. Treated mice showed large lung abscesses with PMN cells and significant fungal burden. On the other hand, absence of G-CSF in similarly treated mice reduced lung fungal burden and resulted in longer survival rates. In mice with 5-fluorcytocine (5FC)-induced neutropenia, G-CSF augmented the antifungal activity of azoles. GM-CSF significantly enhanced both PMN fungicidal activity and the collaboration between PMN and voriconazole or fluoconazole to kill C. albicans [42]. However, given that voriconazole is a more potent (>10-fold) anti-candida azole than fluoconazole, the synergistic effect of GM-CSF was more evident when voriconazole was used in the combination. G-CSF was shown to reverse neutrophil dysfunction against Aspergillus hyphae in HIV-infected non-neutropenic patients [43]. Polyenes (AMB and NY) exhibit pro-inflammatory activities owing to their capacity to induce cytokine secretion. AMB, as a PAMP, is recognized by TLR-2 thus causing the release of several pro-inflammatory cytokines [44, 45]. Nystatin also exhibits pro-inflammatory activity mediated by TLR-1- or TLR-2-induced release of IL-1b, IL-8 and TNF-a [46]. The capacity of polyenes to engage innate immunity is reminiscent of fungal antigens that elicit TLR-based immune responses. For instance, different PAMP of A. fumigatus are recognized by TLR-2, TLR-4, TLR-9 and CD14. Following PAMP ⁄ TLR engagement on professional phagocytes, the resulting inflammatory response induces Th1-mediated protective immunity against A. fumigatus infections [17]. These findings provide new avenues to explore the ability of cytokine-based immunomodulation by using drugs that can influence cytokine release. The respective IL-4- and IL-23-directed differentiation of Th cells into Th2 and Th17 results in adverse consequences to the host as it prolongs and aggravates the infection. Unfortunately, however, strategies to counter this scenario, in the case of fungal infections, are yet to be thoroughly explored. One potential approach to consider here is the development of cytokine inhibitors.

Specific examples of cytokine inhibitors that bind cytokines with high enough affinity to block binding with their respective receptors were previously described. Infliximab and etanercept are inhibitors of the proinflammatory cytokine TNF; both drugs were approved by the FDA to treat Crohn’s disease, rheumatoid arthritis and other inflammatory diseases [47, 48]. In the case of fungal infections, the cytokine inhibitor anti-IL-4, which blocks the differentiation of non-protective Th2-mediated immunity, proved to be effective at enhancing the differentiation of protective Th1-mediated immunity [40].

Antifungal immunotherapy (targeting the pathogen) Soluble effector molecules that interact with and kill or neutralize the pathogen include opsonizing Ab, complement-fixing Ab and neutralizing Ab as well as fungicidal and fungistatic AMP. While the majority of MoAb function along these same lines, some have found use as hosttargeting immunomodulators through acting as cytokine inhibitors [47, 48] and ligand-receptor inhibitors [34], signalling molecules and so on. Thus far, most available antifungal MoAb act as fungal pathogen-targeting agents. Nonetheless, development of MoAb capable of modulating or redirecting the immune response is possible and desirable particularly in cases where the pattern of Th differentiation is non-protective (Th2) and in specific cases of selective immunodeficiency. As for AMP, besides their antimicrobial activity, some (ribotoxin-derived peptides, cathelicidins and other cationic peptides) can modulate cytokine production, alter host gene expression profile and minimize pro-inflammatory responses against microbial antigens. In general terms, however, Ab and AMP are more specific (have narrower spectrum of activity) when compared with host-targeting immunomodulators. They mediate their activity in a passive manner (rapid) independent of host immunostatus (Table 2). They are also prone to some of the limitations of conventional antifungals such as the risk of causing side effects and inducing fungal resistance.

Monoclonal antibodies Currently, there is consensus that Ab contribute to antifungal immunity along with the dominant roles of innate and Th1-mediated immune responses. It has long been known that administration of serum Ab can enhance the outcome of antifungal chemotherapy in cryptococcosis [49]. Fungal infections are marked by significant Ab responses; however, the extent to which they confer protection varies depending on Ig isotype [50] and MHC background of the host [51]. Variations in IgVH gene usage were reported to significantly influence the specificity and efficacy of Ab [52]. Additionally, Ab antifungal 2008 The Author



efficacy can be compromised if protective Ab are not made in quantities sufficient to alter the course of infection or if their action is countered by blocking or competing non-protective Ab [53]. The discovery of protective MoAb against C. albicans [54, 55] and C. neoformans [56–58] has brought further attention to the role of Ab against fungal infections. MoAb to the capsular polysaccharide of C. neoformans were shown to prolong survival and decrease fungal burden in mice [59, 60]. Monoclonal antibody therapy can accommodate many of the limitations imposed by vaccine therapy. For instance, it is possible to develop MoAb with predetermined isotype ⁄ subisotype and use them in pure form and at an optimal dose. Unlike vaccines, therapeutic MoAb provide immediate protection irrespective of the immune status of the host; this makes them good candidates for use in immunocompromised hosts. Derivation of effective MoAb as therapeutic antifungals relies on the identification of immunodominant fungal antigens against which MoAb can be raised. The fungal cell wall contains several molecular structures involved in fungal pathogenesis and virulence. MoAb A9, an IgG1 raised against cell wall extracts of A. fumigatus, can bind to surface peptides on hyphal and yeast forms of the fungus, inhibit hyphal development and reduce spore germination time. A9 protects against murine IA by reducing fungal burden and enhancing survival rates [61]. MoAb raised against the glucosylceramide (GlcCer) moiety N-2¢-hydroxyhexadecanoyl-1-b-D-glucopyranosyl9-methyl-4,8-sphingadenine of Fonsecaea pedrosoi was shown to reduce fungal growth and enhance phagocytosis and killing of fungal cells by murine macrophages [62]. Growth of Fusarium spp. was inhibited in the presence of a fusion protein consisting of recombinant chicken-derived single chain Ab specific against surface antigens and AFB [63]. Expression of the fusion protein in transgenic Arabidopsis thaliana plants was protective against Fusarium oxysporum. G15, a human IgM MoAb raised in xeno mice transgenic for human IgM, IgG2 and Igj chain can recognize an epitope on the capsular polysaccharide glucuronoxylomannan (GXM) of C. neoformans. This MoAb was shown capable of prolonging survival of D strain 24067-sensitized mice following a subsequent lethal dose of the fungus [52]. Monoclonal antibodies raised against a number of intracellular and cell surface fungal components also were developed and tested in animal models. Anti-gp70, a MoAb raised against the 70-kDa intracellular ⁄ secreted glycoprotein component of Paracoccidioide brasilienses abolished lung granulomas in infected mice [64]. Candida albicans strains susceptible to the yeast killer toxin (KT) activity take cover by Ab generated in the host against KT (competing Ab) or its receptor (KTR) on C. albican surface (blocking Ab). Anti-idiotypic MoAb that neutralize anti-KT or anti-KTR Ab activity allow KT to

become active against the fungus and hence circumvent the evasiveness of pathogenic C. albicans. An IgM antiidiotypic MoAb generated in mice immunized with the anti-KT MoAb (KT4) was reported to have a potent killing activity against KT-sensitive C. albicans strains [65]. A decapeptide, killer peptide (KP), containing the first three amino acids of the light chain CDR1 of a KT antiidiotype optimized by single residue replacements has shown strong anticandidacidal activity in vitro [66]. In rats with vaginal candidiasis caused by fluconazole-susceptible or -resistant strains of C. albicans, post-challenge local administration of KP resulted in rapid clearance of the infection. Administration of KP into BALB ⁄ c mice infected with lethal intravenous doses of C. albicans significantly prolonged survival (>60 days) compared with 3–5 days in control mice. The molecular chaperon protein hsp90, essential for yeast cell viability, represents an immunodominant antigen that elicits significant Ab responses in animals and humans. A human recombinant anti-hsp90 MoAb (Mycograb) which was derived from anti-hsp90 Ab isolated from patients recovering from recent invasive candidiasis [67] was reported to be protective against Candida tropicalis but not C. albicans, Candida krusei, Candida glabrata or Candida parapsilosis infections in mice [12]. Combination therapy consisting of Mycograb and AMB resulted in complete resolution of C. albicans, C. krusei and C. glabrata infections. In mice infected with C. parapsilosis, however, Mycograb–AMB combination therapy cleared the liver and spleen but not the kidneys [12]. MoAb G5, an antiC. albicans IgA, was shown to have potent candidacidal activity in vitro and potent prophylactic activity in vivo [68]. Recently, the ability of an IgM MoAb (C7) to prolong survival and enhance macrophage opsonization was demonstrated in C. albicans-infected mice [69]. C7 was previously shown to inhibit C. albicans germination and adhesion to HEP2 cells and oral epithelial cells [70]. Despite significant progress in the development and testing of antifungal MoAb in animal models; clinical evidence of efficacy and safety are still lacking with the exception of few specific examples. The safety and pharmacokinetics of MoAb 18B7, specific against the C. neoformans capsular polysaccharide, was tested as an adjunctive therapy in HIV patients with prior history of cryptococcal meningitis [71]. Serum antigen titers declined by a median of twofold at week 1 and threefold at week 2 post-infusion. Minor side effects in subjects receiving a single infusion of 1–2 mg ⁄ kg body weight of the drug were reported. The half-life of 18B7 in serum was 53 h, but CSF was free of 18B7. A multinational, double-blind, placebo-controlled clinical trial of Mycograb as an adjunctive drug in patients with culture-proven invasive candidiasis who are receiving LAMB was also recently conducted [12].


540 Antifungal Immune-based Treatment M. Hamad .................................................................................................................................................................. Antimicrobial peptides

More than 150 AMP exhibit antifungal activity; such AMP (or AFP for antifungal peptides) include caspofungin (promising and thoroughly studied), micafungin, anidulafungin, aureobasidins, nikkomycins and sordarins. AFP are derived from bacteria (iturin, bacillomycin, syringiotoxins and cepacidines), fungi (echinocandins), insects (cecropins A and B, drosomycin and dermaseptin) and plants (zeamatin, the cyclopeptide alkaloids amphibine H, frangufloline and nummularine) [72]. Additionally, the mammalian innate response secretes several potent AMP and AFP including a- and b-defensins, HNP peptide series, gallinacin and the evolutionarily conserved cathelicidin cationic peptides in humans (hCAP-18 and LL-37), mice (CRAMP), rabbits (CAP18) and pigs (protegrins) [72– 74]. While pathogens or inflammatory mediators (IL-6 and TNF-a) can induce the expression of cationic AMP, most such AMP are constitutively expressed [75, 76]. Cathelicidins and other cationic peptides modulate cytokine production, alter host gene expression and minimize inflammation [73]. Although AMP vary considerably in length, residue sequence and structure, many of them are moderately hydrophobic with a net positive charge. Therefore, they tend to adopt an amphipathic arrangement with the hydrophobic and positively charged faces in opposite orientations. Synthetic non-amphipathic cationic AMP (CAP) were shown to possess selective fungicidal activity against fluconazole-resistant strains of C. albicans, C. tropicalis and C. glabrata in the lM range [77]. Substitutions of amino acids that increase the hydrophobicity of cationic AMP tend to increase antifungal activity [78]. With regard to AFP, they are mostly non-cell selective, visa`-vis, toxic to fungi and potentially toxic to mammalian host cells. Owing to their structure and hydrophobicity, the prime target for most known AFP is the plasma membrane of fungal cells. Accordingly, most AFP kill fungi by binding and disrupting plasma membrane integrity and by pore formation and subsequent cytoplasmic leakage and cell lysis. Others can inhibit the growth of fungi by interacting with cytoplasmic or nuclear targets thus disrupting cell signalling cascades [72] (Fig. 2). The recombinant defensin Tfgd1 synthesized from a cDNA cloned from Trigonella foenum-graecum exhibits broad spectrum antifungal activity [79]. The synthetic peptide D4E1 is active against Aspergillus with a 50% lethal dose of 2.1–16.8 lg ⁄ ml [72]. MIC of the synthetic peptide halocidin analogue di-K19Hc against clinical isolates of C. albicans and Aspergillus spp. are