Fungal Vaccines and Immunotherapeuticsperspectivesinmedicine.cshlp.org/content/4/11/a019711.full.pdf · Fungal Vaccines and Immunotherapeutics Evelyn Santos and Stuart M. Levitz Department

Fungal Vaccines and Immunotherapeutics

Evelyn Santos and Stuart M. Levitz

Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01655

Correspondence: [email protected]

Concomitant with the increased prevalence of immunocompromised persons, invasivefungal infections have become considerably more frequent in the last 50 years. High mor-tality rates caused by invasive mycoses and high morbidity because of intractable mucosalinfections have created an unmet need for innovative prophylactic and therapeutic strategiesagainst fungal pathogens. Several immunotherapeutics and vaccines are in development toaddress this need, although one has yet to reach the clinic. This review focuses on past andcurrent immunotherapeutic and vaccine strategies being tested to either prevent or treatfungal infections, as well as the challenges associated with their development.

THE CASE FOR IMMUNOTHERAPY ANDVACCINE DEVELOPMENT AGAINST FUNGI

The burden of fungal pathogens to humanhealth has substantially increased in thepast half century attributable, in large part, tothe burgeoning numbers of immunocompro-mised individuals. For example, in the early1980s, the endemic fungus, Histoplasma capsu-latum, caused the majority of invasive mycosisin the United States, with an estimated incidencerate of 13.9 cases per million per year. By 2003,opportunistic fungal infections rose to promi-nence causing more than 300 cases of invasivemycosis per million per year in the United States(Pfaller and Diekema 2010). This staggering in-crease was partly because of the emergence ofAIDS, which predisposes hosts to opportunisticpathogens such as Cryptococcus neoformans, themost common invasive fungal pathogen in thispopulation. In fact, the incidence rate of cryp-tococcosis peaked in the early 1990s in the

United States before highly active antiretroviraltherapy (HAART) became widely available bythe end of the decade. The effect of HAART onthe decrease in incidence of cryptococcosis isevident in cities such as Houston, TX, whichsaw a 92% decline in cases of cryptococcosis inits HIV-infected population from 1993 to 2000(Pfaller and Diekema 2010). However, in sub-Saharan Africa, where HAART is not as widelyaccessible, cryptococcal meningitis may surpasstuberculosis in mortality, killing an estimated530,000 people yearly (Park et al. 2009).

In most of the developed world, immuno-suppression caused by AIDS accounts for only aminority of invasive fungal infection (IFI) cases.Only 4% of all patients diagnosed with invasivemycoses in the United States have HIV (Pfallerand Diekema 2010). The vast majority of IFIsare contracted in a hospital setting, where Can-dida species (spp.) rank fourth among causes ofnosocomial bloodstream infections. In fact,�80% of candidemia cases occur in the absence

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of overt immunosuppression. In these patients,risk factors associated with infection are largelyrelated to medical interventions, such as the useof broad-spectrum antibiotics or placement of acentral venous catheter. The remaining 20% ofcases occur in classically immunosuppressedpatients who are at greatest risk for developingIFIs over their lifetime. These patients often suf-fer from hematologic malignancies, undergocancer chemotherapy or other immunosup-pressive therapy, or are recipients of organ orbone marrow transplants (Perlroth et al. 2007).

Although medical intervention contributesto the high IFI incidence, fungal infections aregenerally underdiagnosed. This is, in part, a re-sult of nonspecific clinical signs and symptomsassociated with these infections, as well as thelack of sensitive diagnostic tests for some of themycoses (Brown et al. 2012). Such obstacles canhinder the timely administration of antifungals,substantially contributing to high mortalityrates (Perlroth et al. 2007; Pfaller and Diekema2010).

Even if an accurate diagnosis is achievedearly in infection or empiric treatment startedfor symptomatic patients presenting with rele-vant risk factors, overall mortality rates for bothendemic and opportunistic IFIs are still quitehigh (Pfaller and Diekema 2010). Mortalityrates attributable to invasive candidiasis canreach 40% despite empiric treatment (Perlrothet al. 2007). Several factors contribute to suchhigh case fatality rates. First, most patients pre-senting with IFIs have serious underlying dis-eases (Pfaller and Diekema 2010; Dimopouloset al. 2013). Second, several of the antifungalscurrently available can cause severe side effectsand adversely interact with many of the otherdrugs routinely administered (Dimopouloset al. 2013). Last, susceptibility to these antifun-gals is highly variable across fungal species, andemerging resistance is an increasing concern(Pfaller and Diekema 2010; Brown et al. 2012).

Although invasive mycoses are the mostdeadly manifestation of fungal infections, cuta-neous mycoses are much more common (Brownet al. 2012). In particular, mucosal candidiasisaffecting the oral cavity and gastrointestinaland genitourinary tracts can significantly im-

pact quality of life and are often refractory toantifungals (Vecchiarelli et al. 2012). Recurrentvulvovaginal candidiasis, for example, affects�75 million women of childbearing age world-wide (Brown et al. 2012), and long-term use ofantifungal agents in this population is thoughtto be contributing to the emergence of resistantstrains (Vecchiarelli et al. 2012). Thus, there is asignificant need for new therapies targeting bothinvasive and mucosal mycoses.

Currently, there are no immunotherapeu-tics or vaccines approved for the treatment orprevention of fungal infections. Several candi-dates are in the preclinical stage of developmentand two vaccines against Candida spp. are un-dergoing clinical trials (De Bernardis et al. 2012;Schmidt et al. 2012). Reviewed here are thera-peutic and prophylactic strategies that rely onthe immune system or specific immune com-ponents (Table 1). Important concepts andchallenges involved in the eradication and res-olution of fungal infections are exemplified.

IMMUNOTHERAPY

Generally, fungal immunotherapy involves theadministration of exogenous immune agents,such as white cells, antibodies, and cytokines,to beneficially alter the course of infection (Danand Levitz 2006; Armstrong-James and Harri-son 2012). Reviewed below are monoclonal an-tibodies (mAbs) and dendritic cell (DC) thera-py, and vaccine strategies developed to treat orprevent fungal infections.

Antibody Therapy

Antibodies or immunoglobulins recognize di-verse antigens through the genetic rearrange-ment and somatic hypermutation of its vari-able regions (Schroeder and Cavacini 2010).Constant regions, designated by an immuno-globulins isotype, are recognized by Fc recep-tors (FcR) on immune cells and C1q, a factorinvolved in the complement cascade that canlead to bacterial but not fungal lysis. Fungi resistlysis by porting a rigid cell wall composed of askeletal framework of fibrillar polysaccharidescemented by amorphous polysaccharides dec-

E. Santos and S.M. Levitz

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orated with surface proteins. The innermost lay-er of the fibrillar framework is composed ofchitin cross linked to b-1,3-glucans that expandoutward. These two polysaccharides are com-mon among fungi, making them attractive ther-apeutic targets. The chemical identity of theamorphous polysaccharides differs among spe-cies, although it generally includes mannans ofvarying lengths and linkages (Latge 2010). Al-though fungi evade lysis by the complementsystem, deposition of complement components,such as C3b and iC3b on its surface, leads to itsopsonization, enabling phagocyte recognitionof fungal particles (Cutler et al. 2007).

In many instances, ingestion of pathogensopsonized by antibody and/or complementleads to killing and protection to the host; how-ever, in some cases, antibodies are not protec-tive. For example, H1C, an IgG1 mAb specificfor an uncharacterized 70-kDa protein on thesurface of H. capsulatum, showed no protectiveeffect when given 2 h before challenge, despite

enhancing phagocytosis by a murine macro-phage cell line (J774.16) (Lopes et al. 2010).In contrast, the same group treated mice in asimilar fashion with IgG1 mAbs against heatshock protein 60, and these mice were protectedfrom infection (Guimaraes et al. 2009). Thisdiscrepancy illustrates how the identity of theantigen targeted by an antibody is an importantfactor in determining its effectiveness.

Furthermore, the epitope recognized by anantibody can also play a role in its effective-ness during an infection. This was illustratedby Casadevall and coworkers, who characterizedmonoclonal IgMs produced by two hybridomacell lines derived from the same B cell (Mukher-jee et al. 1995; Nussbaum et al. 1997). Becauseof somatic mutations, the mAbs differed by 11residues in their variable regions and, there-fore, recognized different epitopes on the cap-sule produced by C. neoformans. Although theyshowed similar half-lives and were able to agglu-tinate fungal particles, one monoclonal IgM

Table 1. Therapeutic and prophylactic strategies that relyon the immune system or specific immune components

Strategy Potential pros Potential cons Design considerations

Passive antibodytherapy

Adjuvant to current antifungals Emergence of fungalresistance

Target accessible to theantibody

Direct antifungal/fungistatic activity Antibody isotype andIgG subclass

DC immunotherapy/vaccine

Adjuvants that are too toxic toadminister to humans can bedirectly delivered DCs ex vivo

DC vaccination maynot be feasible inmost populations

DC subtype selection

Costly Adjuvant selection

Attenuated/killedvaccine

Provides numerous antigens specificfor the pathogen

Risk of inducing aninfection ordysregulatedinflammatoryresponse

Attenuation must beirreversible

Protection can be inducedindependent of CD4þ T cells

Recombinant protein(subunit) vaccine

Specifically formulated to elicit aprotective response

T-cell responses toantigens may differas a function ofHLA haplotype

Antigen must be presenton all strains of thefungusSafer than attenuated vaccines,

especially in immunosuppressed

Conjugate vaccines Can be formulated to induceresponses against glycan and/orprotein antigens

T-cell responses toantigens may differas a function ofHLA haplotype

Antigen(s) must bepresent on all strainsof the fungus

Can be self-adjuvantingSafer than attenuated vaccines in

immunosuppressed

IgG, immunoglobulin G; DC, dendritic cell; HLA, human leukocyte antigen.


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(13F1) penetrated the capsule, recognizing anepitope found throughout the length of the cap-sule (Fig. 1). The other monoclonal IgM (12A1)bound epitopes found mostly on the outersurface of the capsule and, when given beforeinfection, this mAb was found to be more pro-tective than 13F1 (Mukherjee et al. 1995; Nuss-baum et al. 1997).

The constant region of an antibody alsocontributes to its effectiveness. This region isdetermined by a B cell’s activation status andthe signals it receives from CD4þ T cells. Afterbinding an antigen recognized by a surface-bound IgD or monomeric IgM, B cells becomeactivated and start producing pentameric IgM(Cutler et al. 2007). As CD4þ T cells also be-come activated, they start producing cytokines,such as interleukin-4 (IL-4) or interferon-g(IFN-g), and up-regulate the expression ofCD40 ligand on their surface. These moleculesengage their respective receptors on the sur-face of B cells and induce the rearrangementof the constant region in the immunoglobulinproduced (Kehry and Hodgkin 1993; Kehryand Castle 1994). This rearrangement is knownas “isotype switching” and, depending on thecytokine signal, B cells will produce IgE, IgA, ora subclass of IgG instead of IgM. For example, in

humans, IL-4 will induce IgG4 or IgE produc-tion, whereas IFN-g induces IgG1. The IgG sub-class and isotype induced by each cytokine dif-fer between humans and mice. Therefore, inmice, IL-4 will induce IgG1 and IgE production,whereas IFN-g induces mainly IgG2a (Mestasand Hughes 2004; Murphy et al. 2012b).

A number of studies have looked at the im-pact of the constant region on the effectiveness ofmAbs. For example, when a human hybridomacell line (3E5) spontaneouslyswitched from pro-ducing a nonprotective anticapsular IgG3 anti-body against C. neoformans to producing anIgG1 antibody with the same variant regions,the IgG1 isotype was found to prolong survivalin mice after infection (Yuan et al. 1995).Another group used the same hybridoma cellline to test the efficacy of additional humanIgG subclasses. Beenhouwer et al. (2007) con-structed recombinant human IgG1, IgG2, IgG3,and IgG4 mAbs by cloning the variable regionexpressed by 3E5 cells into expression vectorscontaining the sequence for each IgG subclass.After challenge with C. neoformans, IgG2 andIgG4 were shown to provide better protectionthan IgG1 or IgG3.

The elements that recognize the constantregions of antibodies, namely, FcRs and C1q,

Figure 1. Epitope recognition pattern. (A) Anticapsular monoclonal IgMs 12A1, and (B) 13F1 on C. neoformansyeasts as shown by immunoelectron microscopy. (From Nussbaum 1997; reprinted, with permission, from TheRockefeller University Press # 1997.)



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are likely responsible for the discrepancy in theprotection afforded by the different IgG sub-classes. C1q and the seven human and five mu-rine FcRs have varying affinities for each IgGsubclass (Schroeder and Cavacini 2010; Bruhns2012). Additionally, FcRs and the eight comple-ment receptors that mediate phagocytosis areexpressed by different immune cell types andregulate a diverse array of cell functions, includ-ing degranulation and proliferation (Ricklinet al. 2010; Bruhns 2012). This complexity al-lows for the flexibility needed to respond to thewide variety of insults handled by the immunesystem.

Some mAbs possess antifungal propertiesthat are independent of immune cell activity.For example, when incubated with C. neofor-mans, 2G8(IgG2b) prevents capsule formationand reduces capsule size (Hole and Wormley2012). 2G8 has also been found to be fungistaticagainst Candida albicans and prevent its attach-ment to human epithelial cells in vitro (Phanet al. 2007; Torosantucci et al. 2009; Dwivediet al. 2011). The efficacyof 2G8 in vivo has showntherapeutic promise in mouse models of pulmo-nary cryptococcosis, as well as invasive and vag-inal candidiasis (Torosantucci et al. 2009; Capo-dicasa et al. 2011; Hole and Wormley 2012).

Another antibody-mediated interventionbeing tested for the treatment of fungal infec-tions involves labeling an anticapsular IgG1mAb (18B7) specific for C. neoformans with ra-dioactive isotopes. The isotopes, 213bismuthand 188rhenium, which emit a and b particles,respectively, have been shown to significantlyprolong survival of infected mice (Dadachovaet al. 2003). Additionally, 213Bi-18B7 was shownto be superior to amphotericin in clearing dis-seminated C. neoformans (Bryan et al. 2010). Anadvantage of therapeutics, such as the fungistat-ic 2G8 antibody and radiolabeled antibodies, isthat their effectiveness is independent of a host’simmunological status, making them excellentcontenders in treating IFIs in immunocompro-mised patients. A drawback specific to passiveanticapsular antibody therapy, however, is thatCryptococcus spp. shed their capsule duringinfection. This phenomenon could lead to an-tibody forming immune complexes with circu-

lating capsule rather than promoting opsono-phagocytosis or direct antifungal activity intarget tissues (Hogan et al. 1996).

Other disadvantages, which could be poten-tially associated with passive antibody therapy,include (but are not limited to) the emergenceof resistance, inconsistent protection across pa-tient populations, and dependence on early di-agnosis for optimal protection. The emergenceof resistance to mAbs would most likely occurfor therapies that target fungal proteins, whichcan lose or mask the antibody-binding sitewhile maintaining function. Additionally, anti-bodies that require recognition by host factorsto be protective may be ineffective in patientpopulations in which these factors fail to opti-mally function with that specific antibody iso-type. These variations may be a result of singlenucleotide polymorphisms or severe immuno-deficiencies that may be contingent on that pa-tient’s risk factors in contracting the infection.For example, if the mechanism by which a spe-cific antibody delivers protection requires ahealthy neutrophil population, then neutrope-nic patients would not be expected to benefitfrom such treatment. Finally, antibody therapymay be most effective when the organism bur-den is low early in infection, which would re-quire better diagnostic tools than are availabletoday. However, the study of how mAbs specificfor fungi affects disease outcome not only con-tributes to the development of potential thera-pies but could also assist in the development ofbetter diagnostic tools and help provide clueson how to design a protective vaccine.

Dendritic Cell Immunotherapyand Vaccination

DC immunotherapy involves incubating or“pulsing” DCs ex vivo with select antigens orpathogens, then returning the cells to the hostto boost protection against an infectious agent.This therapeutic strategy can be contrasted toDC vaccination, in which the goal is to protectthe host against future pathogen exposure. Al-though the technical process is the same, themajor difference between the two strategiescomes down to the timing of the intervention.



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DC vaccination requires that the treatment beperformed before infection and DC immuno-therapy happens after diagnosis.

DCs are activated after sensing pathogen-associated molecular patterns through patternrecognition receptors (PRRs), which induce theup-regulation of cytokines and costimulatoryproteins (Toews 2001; Cassone 2008). Theseproteins engage their receptors on the surfaceof T cells that recognize antigens presented byDCs on major histocompatibility complex(MHC) molecules (Toews 2001). Dependingon the cytokines produced, CD4þ T cells willdifferentiate into a specific T helper (TH) sub-type (Murphy et al. 2012a). For example, if DCsproduce IL-12, CD4þ T cells will emerge as TH1cells. Each TH subtype will produce a set of cy-tokines that will induce a particular set of im-mune responses. The TH subtypes that correlatebest with protection against fungi are TH1 andTH17 (Hamad 2008; Carvalho et al. 2012; Ian-nitti et al. 2012). TH1 cells produce IFN-g,which induces cell-mediated immunity by acti-vating phagocytes. TH17 cells produce IL-17and IL-22, augmenting a neutrophilic responseand stimulating the production of antimicrobi-al peptides in the affected tissue. Finally, Tregcells produce transforming growth factor-b andIL-10 and help contain the inflammatory pro-cess instigated by the other cell types (Iannittiet al. 2012).

One peptide that has shown promise as anantigen for DC immunotherapy is P10, a 15-amino acid synthetic oligomer that has beenpreviously mapped to be a T-cell epitope foundon a highly immunogenic protein secreted byParacoccidioides brasiliensis (Taborda et al.1998). When bone marrow DCs pulsed withP10 were given subcutaneously to naı̈ve micethat were subsequently infected with P. brasilien-sis yeasts, researchers found an �2-log decreasein fungal burden in immunized mice comparedwith control groups. This strategy was alsosuccessful in decreasing fungal burdens in thelungs of mice infected 30 days before therapy,suggesting that this strategy can be therapeuticas well as prophylactic (Magalhaes et al. 2012).

Although DCs act mainly as antigen-pre-senting cells (APCs), they are a heterogeneous

population of cells that express different combi-nations of PRRs. Depending on the stimulussensed, distinctive DC subsets will induce dis-parate T-cell responses. Therefore, the subset ofa specific DC population must be taken intoaccount when designing DC immunotherapystrategies. Romani and coworkers (2006) usedtwo distinctive DC populations in a strategy thatmodeled bone marrow transplantation in mice.In this study, C57BL/6 mice transplanted withlymphocyte-depleted bone marrow from BALB/c mice were immunized with conventional den-dritic cells (cDCs) or plasmacytoid dendriticcells (pDCs) pulsed with Aspergillus fumigatusconidia. Mice then received a pulmonary chal-lenge with A. fumigatus 14 days after transplant.The group that received pDCs was 100% pro-tected, whereas mice that received cDCs gener-ally died within 10 days after challenge. pDC-immunized mice had higher levels of Treg cellsin draining lymph nodes after infection, where-as cDC-immunized mice had higher levels ofIFN-g, indicative of a strong TH1 response (Ro-mani et al. 2006). The outcome in this experi-ment illustrates how an overzealous inflamma-tory response induced by vaccination may resultin undesirable consequences for the host.

DC vaccination may not be economicallytenable for prophylaxis of the general popula-tion; however, it could be feasible in high-riskgroups such as bone marrow transplant pa-tients. DC immunotherapy may prove usefulas an adjunctive therapy in established fungalinfections. Additionally, understanding howDCs provide protection against specific patho-gens helps narrow down antigen componentsformulated in potential vaccines (Steinman andBanchereau 2007). Other fungal vaccinationstrategies target specific receptors on DCs, par-ticularly Dectin-1, a PRR that binds b-glucans.Dectin-1 has been shown to be indispensable forhost defenses against several fungal pathogensby linking fungal cell wall recognition to TH1and TH17 responses (Hardison and Brown2012). Antigen-loaded glucan particles derivedfrom Saccharomyces cerevisiae cell walls delivertheir cargo to DCs resulting in antigen-specificantibodies and a TH1/TH17-biased CD4

þT-cellresponse (Huang et al. 2012, 2013).



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VACCINES

As a prophylactic strategy, vaccination has hadunparalleled success in preventing morbidityand mortality from infectious diseases (Roushand Murphy 2007). However, currently thereare no fungal vaccines approved for humanuse. This is partly because of the stringent safe-ty and effectiveness criteria vaccines must meetto be licensed. Because the intended popula-tion for preventive vaccines is generally healthy,benefit to risk ratios for a new vaccine must bevery high. Therefore, vaccines have to be safe,inflict few side effects, and sustain long-termprotection in a population with varied geneticbackgrounds and MHC haplotypes (Levineand Sztein 2004). A less favorable side effectprofile may be tolerated for therapeutic vac-cines that provide benefits over existing treat-ments.

Despite these hurdles, there are two anti-Candida recombinant protein vaccines thatare currently in clinical trials. Additionally,there are several other fungal vaccines in pre-clinical development. These include killed andlive-attenuated whole cell vaccines, as well assubunit recombinant protein and polysaccha-ride-protein conjugate vaccines. Each of thesestrategies has unique advantages and disadvan-tages, which will be discussed below but mosttarget antigens that are specific for only onefungal genus. At least two strategies, however,take advantage of the common epitopes foundin the skeletal framework of the fungal cellwall. One uses killed S. cerevisiae to direct im-mune cells to recognize common fungal wallpolysaccharides including glucans and man-nans (Liu et al. 2011a). Another universal vac-cine design uses b-glucans conjugated to aninactivated version of diphtheria toxin to elicitresponses against different pathogenic fungi(Torosantucci et al. 2005). These aforemen-tioned studies are described at greater detailbelow.

Killed and Attenuated Vaccines

The first vaccines used to immunize humanswere developed empirically by exposing indi-

viduals to attenuated or killed pathogens to elic-it immunological memory. Several studies haveevaluated the effectiveness of killed and attenu-ated fungi as potential vaccines, including S.cerevisiae, which has the potential to protectindividuals from the major fungal pathogens.Immunization with heat-killed S. cerevisiaeyeasts (HKY) by subcutaneous injection hasbeen shown to be protective against C. albicans(Liu et al. 2012a), A. fumigatus (Liu et al.2011a), and the endemic fungus Coccidioidesposadasii (Capilla et al. 2009). Presumably, im-munization with S. cerevisiae offers protectionagainst a variety of fungi because it shares withthem common polysaccharide epitopes foundon its cell wall. Additionally, it may also elicitresponses that are cross-reactive to homologousproteins found on pathogenic fungi (Liu et al.2011a). Although protective against many dif-ferent fungi, HKY is less effective in prevent-ing mortality in models of coccidioidomycosisthan a more specific vaccination regimen usingformalin-killed Coccidioides immitis spherules(FKS). Vaccination with HKY or FKS protected70% and 100% of CD1 mice, respectively,from an otherwise lethal C. immitis challenge(Capilla et al. 2009). Inasmuch as S. cerevisiaeacts as a universal fungal vaccine, it may lacksufficient specificity to be as effective as vac-cines that contain pathogenic fungal antigens.Whether these studies could be translated tohumans, who have greater natural exposure tofungi than laboratory mice living in filteredcages, is unclear. Notably, a phase III placebo-controlled clinical trial evaluating the efficacy ofthe FKS vaccine found it to be ineffective in pre-venting coccidioidomycosis or mitigating se-verity of disease (Pappagianis 1993).

Xue et al. (2009) developed another wholefungal cell vaccination strategy against coccidi-oidomycosis. This group vaccinated BALB/c andC57BL/6 mice with an attenuated strain ofC. posadasii, which cannot endosporulate be-cause of the loss of two chitinase genes. Theyfound that two subcutaneous injections withthe attenuated strain 14 days apart protected75%–100% of the animals from an otherwiselethal pulmonary challenge with the virulentC735 strain of C. posadasii (Xue et al. 2009).



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Attenuated live vaccines generally have agood safety profile in immunocompetent indi-viduals; however, they may still cause an infec-tion or a dysregulated inflammatory response inimmunosuppressed individuals (Pirofski andCasadevall 1998). Therefore, this strategy maybe most appropriate in the case of endemic fun-gi, which can infect immunocompetent indi-viduals (Chu et al. 2006). However, there areexceptions to this rule depending on the typeof immunosuppression, as the Centers for Dis-ease Control and Prevention recommend thatcertain live vaccines be administered to a subsetof immunocompromised patients (ACIP 2011).An attenuated version of Blastomyces dermatiti-dis, also an endemic fungus, has been tested asa possible immunization strategy in settings oflow CD4þT-cell counts as encountered in AIDSpatients. Deletion of Blastomyces adhesin-1(BAD1) in this dimorphic fungus renders theorganism avirulent in mouse models of disease.Subcutaneous immunization with this mutant(DBAD1) protected 100% of CD42/2 micefrom a lethal inoculum (Wuthrich et al. 2003).

Yet, another live vaccine strategy that hasbeen successful in a CD4þ T-cell-deficientmouse model involved immunizing mice witha C. neoformans strain engineered to producemurine IFN-g (H99g). This strain has been pre-viously shown to protect mice from infectionwith the wild-type C. neoformans strain by in-ducing a TH1 response (Wormley et al. 2007). Ina more recent study, depletion of CD4þor CD8þ

T cells during the immunization phase (primaryinfection with H99g) or secondary lethal in-fection protected 100% of mice from mortali-ty. However, when both CD4þ and CD8þT cellswere depleted during immunization with H99gor secondary infection, mortality reached 100%(Wozniak et al. 2011). Although the H99g strainvaccine could not be used in humans, it doesserve as a proof of concept for live vaccinationstrategies, whereby pathogens are genetically en-gineered to produce cytokines that enhancehost-protective responses.

Killed and attenuated vaccines are highlyeffective in combating opportunistic and en-demic fungal infections. It is possible that theDBAD1 attenuated B. dermatitidis vaccine and a

humanized version of C. neoformans H99g vac-cine could protect individuals with low CD4þ

T-cell counts, such as HIV patients. However,assuring the safety of attenuated vaccines inthe immunosuppressed population has histori-cally been challenging, as previously discussed.In immunocompetent individuals, attenuatedvaccines against viral infections have been high-ly successful, and perhaps an attenuated vaccineagainst an endemic fungal pathogen may con-tribute to the eradication of these diseases wherethey are prevalent.

Recombinant Protein Vaccines

The increased knowledge obtained in the lastcentury in the fields of microbial pathogenesisand basic immunology has contributed to thedevelopment of acellular vaccines. In particular,recombinant protein vaccines would not bepossible without our increased understandingof the innate immune system, especially therole DCs play in shaping the adaptive immuneresponse. With some exceptions, protein anti-gens by themselves are not sufficient to activateAPCs and elicit an adaptive immune response.Therefore, acellular vaccines are frequently for-mulated with an adjuvant to potentiate the im-munogenicity of the antigen and skew adaptiveimmunity to the appropriate response. Manyadjuvants are microbial products that activateAPCs by binding PRRs. However, some are stillin preclinical evaluation and others are not ap-proved for use in humans because of high tox-icity (Tritto et al. 2009; Levitz and Golenbock2012).

One class of adjuvants that is approved forhuman use is aluminum salts such as aluminumhydroxide and aluminum phosphate. Com-monly known as “alum,” this adjuvant elicitsstrong antibody-mediated responses to ad-mixed antigens (Levitz and Golenbock 2012).As such, alum is commonly used in vaccinesthat are designed to promote complement-me-diated lysis and opsonophagocytosis, as well asneutralize protein toxins, viral particles, andother pathogens from attaching to host tissue(Tritto et al. 2009). NDV-3 (NovaDigm, GrandForks, ND), an anti-Candida vaccine that in-



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cludes the invasin protein agglutinin-like se-quence 3 (Als3p) and alum in its formulation(Schmidt et al. 2012), prevents yeast attachmentand invasion of epithelial and endothelial cells.This strategy works by eliciting IgG and IgAagainst Als3p (Phan et al. 2007). In a phase Iclinical trial, NDV-3 was found to be safe andtolerable in research subjects, and shown to elic-it IFN-g- and IL-17A-producing antigen-spe-cific T cells (Schmidt et al. 2012). In animalmodels, this vaccine protected mice from oro-pharyngeal, vaginal, and invasive candidiasis(Spellberg et al. 2006; Ibrahim et al. 2013). Fur-thermore, because of high structural homologybetween Als3p and clumping factor A on thesurface of Staphylococcus aureus, NDV-3 hasalso been shown to be protective against thishighly virulent bacterial pathogen in animalmodels (Spellberg et al. 2008). If found effica-cious in humans, NDV-3 could have a high im-pact in driving down mortality from hospital-acquired infections by targeting two of the mostdeadly nosocomial bloodstream infectiousagents (Pfaller and Diekema 2010).

Adjuvanticity can also be achieved by attach-ing a relevant antigen to the shell of a virus. Notonly can the shell, or virosome, activate APCsbut it also acts as a carrier, delivering a desiredantigen to targeted cells. Pevion Biotech (Bern,Switzerland) is developing a virosome-basedvaccine against recurrent vaginal candidiasis,targeting secreted aspartyl proteinase-2 (Sap2).Sap2 is a virulence factor that is highly expressedby Candida spp. isolated from vaginal mucosa.This protease hydrolyzes both structural andimmune-associated proteins such as comple-ment factors and immunoglobulins (Cassoneet al. 2007). Rats immunized with recombinantamino-terminal region of Sap-2 attached toH1N1 virosomes showed accelerated clearanceof vaginal candidiasis, and were shown to pro-duce IgA and IgG against Sap2 in vaginal fluid(De Bernardis et al. 2012). Although results havenot yet been published, a phase I clinical trial hasbeen conducted (NIH 2010).

In contrast to whole cell vaccines, recombi-nant vaccines are generally much safer, partic-ularly for use in immunocompromised patients(ACIP 2011). However, designing recombinant

vaccines can be quite challenging because anti-gens have to be chosen on the basis of immu-nogenicity and potential effectiveness to con-serve human and economic resources neededto assess potential vaccines in clinical trials. Infact, both of the subunit vaccines mentionedabove were designed based on what was knownabout their respective antigens, particularly theantigen’s role in the pathogenesis of infection(Cassone et al. 2007; Phan et al. 2007). Whenformulating recombinant fungal vaccines, spe-cial consideration must also be given to themethod chosen for its synthesis, as native gly-cosylation is lost in bacterial expression systems.Lack of native glycosylation decreases the im-munogenicity of some fungal proteins (Lamet al. 2005; Levitz and Specht 2006; Spechtet al. 2007).

Conjugate Vaccines

Although epitopes on polysaccharides can berecognized by B- and T-cell receptors, for anti-gens to be presented to T cells, they must beloaded on MHC molecules, which only bindpeptides. To bypass this bias toward peptide an-tigens, vaccinologists have conjugated poly-saccharides to proteins thus creating antigensthat can be presented on MHC molecules andselect B and T cells that that are specific forglycan epitopes. The first conjugate vaccine de-veloped for fungal infections contained C. neo-formans capsular polysaccharide, glucuronoxy-lomannan (GXM) covalently linked to tetanustoxoid (TT). Given with monophosphoryl lipidA (MPL) as an adjuvant, this antigen elicitedhigh levels of IgG and IgA specific for GXMand protected 70% of intravenously challengedmice. Protection from mortality was at leastpartially conferred by elicited antibodies, asadoptive transfer of antisera from immunizedanimals also protected mice from a lethal inoc-ulum of intravenously administered C. neofor-mans (Devi 1996).

Torosantucci et al. (2005) have conjugatedlaminarin, a b-glucan polysaccharide derivedfrom brown algae, to inactivated diphtheria tox-in (CRM) to create a universal fungal vaccine.This conjugate, injected with complete Freund’s



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adjuvant (CFA) subcutaneously, protected 70%and �80% of CD2F1 mice from invasive can-didiasis and aspergillosis, respectively, whereas�20%–30% of mice given CFA or CRM alonesurvived either infection. The laminarin-CRMconjugate was also effective in treating vaginalcandidiasis in Wistar rats when given with chol-era toxin as an adjuvant (Torosantucci et al.2005).

A b-1,2-mannotriose conjugated to a pep-tide segment from fructose bisphosphate al-dolase (Fba) and TT has also been tested as apossible anti-Candida vaccine. Fba protein andb-1,2-mannose are antigens found on the sur-face of Candida spp. Different formulationsof this vaccine were tried in a BALB/c invasivecandidiasis model. One group received the con-jugate (b-man-Fba-TT) by itself, another re-ceived conjugate and alum, and the third groupreceived conjugate along with MPL. The conju-gate only and conjugate plus alum groups re-ceived the most protection as 100% of the micesurvived infection, compared with 0%–20% inthe control group, whereas �60% mice receiv-ing the conjugate and MPL formulation sur-vived the infection. In this strategy, couplingof the inactivated tetanus toxin to the glycan-peptide conjugate was crucial for the success ofthe vaccine, as 100% of the mice immunizedwith just b-1,2-mannotriose-Fba succumbedto infection (Xin et al. 2012).

Conjugating proteins to polysaccharides en-ables the immune system to recognize abundantfungal cell wall glycan components, increasingthe chance that antibodies will recognize thepathogen. Additionally, this strategy can beused to target saccharide epitopes that are com-mon to all fungi, particularly b-glucans, there-by creating one vaccine that is effective againsta broad range of pathogenic fungi. This maybe particularly advantageous to patients whoare iatrogenically immunosuppressed and athigh risk for developing disseminated infec-tions from multiple fungal pathogens. Althoughhighly needed, developing immunizations forthis population can be quite challenging, asthey are less likely to respond to acellular vac-cines and may be at risk for developing diseaseafter attenuated immunizations. However, acel-

lular vaccines may retain efficacy if given beforethe start of the immunosuppressive regimen(Bozza et al. 2009).

CONCLUSION

Largely because of medical advances and theemergence of AIDS, the number of immuno-suppressed individuals susceptible to fungal in-fections has exponentially increased in the last50 years. High mortality rates associated withIFIs have emergently precipitated the need fornew therapeutics. In addition, advancements inunderstanding host–pathogen interactions inmucosal infections and IFIs have brought sev-eral immunotherapy and vaccine strategies tothe preclinical stage of development. Out ofthe strategies that have been shown to be suc-cessful at this stage, a few have gone on to betested in clinical trials. Lack of awareness aboutthe severity of the human and economic tollfungal infections inflict worldwide has contrib-uted to a poor funding climate for medical my-cology (Brown et al. 2012). Despite these chal-lenges, some strategies described above havereached clinical trials and show some promiseas they advance in clinical development. Hence,there is hope that with the further developmentof immunotherapies and vaccines, at least somelethal fungal infections will become rare onceagain.

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2014; doi: 10.1101/cshperspect.a019711Cold Spring Harb Perspect Med Evelyn Santos and Stuart M. Levitz Fungal Vaccines and Immunotherapeutics

Subject Collection Human Fungal Pathogens

PathogensEvolutionary Perspectives on Human Fungal

John W. Taylor Pathogenicity TraitPolyphyletic Pathogens with a Convergent

−−Thermally Dimorphic Human Fungal Pathogens

Anita Sil and Alex Andrianopoulos

HumansBlack Molds and Melanized Yeasts Pathogenic to

de HoogAnuradha Chowdhary, John Perfect and G. Sybren

Mechanisms of Antifungal Drug Resistance

Howard, et al.Leah E. Cowen, Dominique Sanglard, Susan J.

within MacrophagesFungal Pathogens: Survival and Replication

MayAndrew S. Gilbert, Robert T. Wheeler and Robin C.

Cryptococcus and CandidaTreatment Principles for

Laura C. Whitney and Tihana Bicanic

Innate Defense against Fungal Pathogens

Hise, et al.Rebecca A. Drummond, Sarah L. Gaffen, Amy G.

The Human MycobiomePatrick C. Seed

PharmacodynamicsAntifungal Pharmacokinetics and

Alexander J. Lepak and David R. AndesInfectionsTreatment Principles for the Management of Mold

Dimitrios P. Kontoyiannis and Russell E. Lewis

EntomophthoralesHuman Fungal Pathogens of Mucorales and

al.Leonel Mendoza, Raquel Vilela, Kerstin Voelz, et

Adaptive Immunity to Fungi

al.Akash Verma, Marcel Wüthrich, George Deepe, et

GenomesFunctional Profiling of Human Fungal Pathogen

Alexi I. Goranov and Hiten D. Madhani

Pathogenic Species ComplexCandidaThe Siobhán A. Turner and Geraldine Butler

and Related SpeciesAspergillus fumigatus

R. Juvvadi, et al.Janyce A. Sugui, Kyung J. Kwon-Chung, Praveen

Fungal Morphogenesis

al.Xiaorong Lin, J. Andrew Alspaugh, Haoping Liu, et

http://perspectivesinmedicine.cshlp.org/cgi/collection/ For additional articles in this collection, see

Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved

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Fungal Vaccines and Immunotherapeuticsperspectivesinmedicine.cshlp.org/content/4/11/a019711.full.pdf · Fungal Vaccines and Immunotherapeutics Evelyn Santos and Stuart M. Levitz Department