Biomedicine & Pharmacotherapy 109 (2019) 333–344

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Review

Fungal vaccines, mechanism of actions and immunology: A comprehensive T

review
Sanam Namia, Rasoul Mohammadib, Mahshid Vakilic, Kimia Khezripourd, Hamed Mirzaeie,
Hamid Morovatia,f,
⁎

a
Department of Medical Mycology and Parasitology, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
b
Department of Medical Parasitology and Mycology, School of Medicine/Infectious Diseases and Tropical Medicine Research Center, Isfahan University of Medical
Sciences, Isfahan, Iran
c
Department of Medical Mycology and Parasitology/Invasive Fungi Research Center (IFRC), School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran
d
Department of Pharmacotherapy, School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
e
Research Center for Biochemistry and Nutrition in Metabolic Diseases, Kashan University of Medical Sciences, Kashan, Iran
f
Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

ARTICLE INFO ABSTRACT

Keywords: Fungal infections include a wide range of opportunistic and invasive diseases. Two of four major fatal diseases in
Fungal infections patients with human immunodeficiency virus (HIV) infection are related to the fungal infections, cryptococcosis,
Vaccine and pneumocystosis. Disseminated candidiasis and different clinical forms of aspergillosis annually impose ex-
Immune response pensive medical costs to governments and hospitalized patients and ultimately lead to high mortality rates.
Therefore, urgent implementations are necessary to prevent the expansion of these diseases. Designing an ef-
fective vaccine is one of the most important approaches in this field. So far, numerous efforts have been carried
out in developing an effective vaccine against fungal infections. Some of these challenges engaged in different
stages of clinical trials but none of them could be approved by the United States Food and Drug Administration
(FDA). Here, in addition to have a comprehensive overview on the data from studied vaccine programs, we will
discuss the immunology response against fungal infections. Moreover, it will be attempted to clarify the un-
derlying immune mechanisms of vaccines targeting different fungal infections that are crucial for designing an
effective vaccination strategy.

1. Introduction regimes, and more importantly, postnatal steroid therapies. Most pre-
valent IFIs in VLBW infants are, Candida species (spp), Malassezia spp,
Nowadays, the importance of preventive and treatment methods for Aspergillus spp, and Zygomycetes [6]. It has been shown that Candida spp
fungal infections is highlighted by increasing number of the high-risk are the fourth and first causative agents of nosocomial bloodstream
groups exposed to invasive fungal infections (IFIs), including cancer infections in the US and the European countries, respectively [7].
patients under chemotherapy, bone marrow transplantation, acquired Furthermore, despite an experimental therapy, the mortality rate of
immune deficiency syndrome patients (AIDS), and all other diseases invasive candidiasis is about 30–40% [7]. HIV/AIDS patients show a
with immune deficiency following long-term hospitalizations [1,2]. IFIs high mortality rate following opportunistic fungal infections. Crypto-
could also be found in patients treated with a wide range of antibiotics coccus neoformans (C. neoformans) is also a most common yeast that
and intravenously or intra-arterially catheter treatment methods [3], in infects these patients [8]. The outbreak of cryptococcosis in HIV/AIDS
premature infants, and the hospitalized patients in intensive care units patients was increased at the beginning of the 1990s in the US, before
[4]. Furthermore, 40% of patients with hematologic malignancies are the utilization of highly active antiretroviral therapy. Using this therapy
exposed to IFIs [5]. Very low birth weight (VLBW) infants are at high between 1993–2000, a 92% decrease in the outbreak rate of this in-
risk to IFIs [6]. To prevent the expansion of IFIs, these infants require fection in HIV/AIDS patients was reported [9]. Generally, IFIs are re-
extensive therapies, such as intravenous catheters, long-term antibiotic sponsible for 50% of the mortality cases which encompasses 1.5 million

⁎
Corresponding author at: Department of Medical Mycology and Parasitology, School of Medicine, Tabriz University of Medical Sciences, Golgasht St., Tabriz, East
Azarbaijan, Iran.
E-mail address: morovatihamid1989@gmail.com (H. Morovati).

https://doi.org/10.1016/j.biopha.2018.10.075
Received 3 August 2018; Received in revised form 2 October 2018; Accepted 14 October 2018
0753-3322/ © 2018 Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
S. Nami et al. Biomedicine & Pharmacotherapy 109 (2019) 333–344

subjects per year. Such a high mortality rate is followed by nonspecific provide fundamental defense mechanisms through opsonization of
clinical signs and symptoms, scarcity of the preventive methods, ap- fungi [12,19,21]. For example, recognition of deposited complement
propriate diagnosis, and sufficient antifungal medicines [10]. Con- particles on β-(1,6)- glucans of the fungus surface by complement re-
sidering an increasing population of immunocompromised patients and ceptor 3 (CR3; a heterodimer of CD11b and CD18 which is expressed on
application of immunosuppressive treatments, we have been facing different types of immune cells, such as neutrophils, monocyte/mac-
with extremely dramatic increase in the life-threatening infections even rophages, and natural killer (NK) cells) leads to elimination of patho-
by the coexistent species, such as Candida albicans (C. albicans) [11]. gens by phagocytic cells [19], a process called opsonophagocytosis.
Therefore, it is essential to review how the immune system controls the Defensins (which are secreted by the epithelium and paneth cells in gut)
fungal infection. and collectins are involved in opsonizing and also induction of in-
flammatory responses in collaborating with helper T (Th)-17 profile
cytokines (Fig. 2) [3].
2. Immunology of fungal infections

2.1. Innate immunity 2.2. Acquired immunity

The frontline battlefield of the immune system with fungi pathogen In addition to recognizing different regions of fungal cells through
is the physical barriers, chiefly the skin and the mucosal epithelial different types of PRRs, antigen-presenting cells (APCs), including DCs,
surfaces, existing in mouth, upper airways, and the gastrointestinal and MQs, and B cells present the antigenic epitopes on major histo-
genitourinary tracts, which are constantly exposed to environmental compatibility complex (MHC) class II or class I molecules (which are
organisms [12]. Moreover, epithelial cells play pivotal roles in expressed on APC surfaces) to CD4+ or CD8+ T cells, respectively
launching the effective antifungal responses through discriminating [17,22,23]. This way, these cells stimulate acquired immune response
pathogenic and non-pathogenic fungal morphotypes [13,14]. (Fig. 2).
The critical step in the initiation of an immune response is the re- During this process, environmental cytokines produced at the site of
cognition of the specific components of fungi, called pathogen-asso- APC-T cell binding trigger the differentiation of CD4+ T cells into a
ciated molecular patterns (PAMPs), by pattern recognition receptors specific Th cell subtypes through activating different signaling path-
(PRRs) (Fig. 1). Different types of innate immune cells, including ways. STAT1/STAT4 transcription factors are needed for Th1 differ-
macrophages (MQs) and dendritic cells (DCs) express a vast repertoire entiation, while STAT3/ROR-γt are required for Th17 development,
of PRRs, such as Toll-like receptors (TLRs), C-type lectin receptors GATA3/STAT6 are involved in Th2 development (Fig. 2) [12,18,19,24].
(CLRs), NOD-like receptors (NLRs), and RIG-like receptors (RLRs) [15]. If DCs (known as the major APC) release interleukin (IL)-12, the CD4+
Among them, TLR2, TLR4, and Dectin-1 have prominent roles in de- T cells will be differentiated to Th1 cells. Different sets of immune re-
tecting fungal cell wall PAMPs. Previously we showed that TLR2 gene sponses will emanate from Th-triggered cytokines [19]. Th1 and Th17
expression increased in mice group with systemic candidiasis (SC) and cytokines, chiefly, gamma-interferon (IFN-γ) and IL-17, produce pro-
also in cyclophosphamide-dependent immunosuppressed mice with SC tective and protective-inflammatory responses, respectively [25,26].
[16]. However, our recently published data showed that TLR2 had no
More precisely, IFN-γ induces cell-mediated immunity through stimu-
significant role in launching the immune responses in im- lating phagocytes and Th17 cells release IL-17 and IL-22 cytokines that
munosuppressed mice [17]. Phagocytic cells, MQs, DCs, and neu-
initiate the neutrophilic response and release antimicrobial peptides
trophils, are able to recognize the fungi at the first stages of infection peptides like defensins to the site of infection [27]. Finally, at the end
through a variety of receptors (including PRRs) and combat with fungi
stages of immune responses, Foxp3+/CD4+ T cells, which are called
through phagocyting and releasing antimicrobial components, such as regulatory T (Treg) cells, release the transforming growth factor (TGF)-
oxygen radicals. Additionally, phagocytic cells are able to produce cy-
β and IL-10 in order to repress the elevated levels of inflammatory re-
tokines, which induce the maturation of CD4+ T cells toward specific sponses (Fig. 2) [28].
subtypes (Fig. 2) [18–20].
Complement system and other humoral factors, such as antifungal
peptides, mannose-binding lectins (MBLs), defensins, and collectins also

Fig. 1. Signaling pathway illustration

during fungal sensing and processing.
Following PAMP-PRR interaction, CTKs phos-
phorylate both central tyrosine of ITAM and
also protein adaptors which triggers the further
stimulation of downstream signaling mediators
and eventually leads to the production of pro-
inflammatory cytokines and other soluble
mediators through activation of TFs. CTK; cy-
toplasmic tyrosine kinase. ITAM; im-
munoreceptor tyrosine-based activation motif.
TFs; transcription factors.

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S. Nami et al. Biomedicine & Pharmacotherapy 109 (2019) 333–344

Fig. 2. Immune responses induced by fungus or vaccine-related antigens. After the failure of epithelial surfaces, as the first defense line against fungal in-
fections, the immune response starts a new phase. Following the PRRs (TLRs, Dectins, Galectin-3, Mannos receptors, DC-SIGN, and Mincle) and PAMPs interaction,
some specific signaling pathways are stimulated in the APC, eventually leading to the production of different cytokines. In this regard, the protein adaptors, such as
MyD88, Syk, RAS, and TRIF are activated through connecting to the cytoplasmic stimulatory domain of PRRs (such as TIR for TLRs), which then trigger the
downstream adaptor proteins (CARD-9, BCL-10, MALT-1, IRAK-1, IRAK-4, TRAF-6, and RAF-1). This, in turn, ultimately activate the transcription factors (NF-kB, AP-
1, IRF-3, and IRF-7), resulting in the production of cytokines. Moreover, Dectin-1 is able to stimulate the inflammasomes (consisted of different adapter proteins) and
finally triggers Caspase-8 and Caspase-1, which catalyze the production of IL-1β from pro-IL-1β. At the next step, the processed antigen in the APC (chiefly DCs) are
presented to naive T cells. According to the cytokines resulted from PAMP-PRR interaction, the class of T cell is formed. For example, IL-23, IL-6, and TGF-β trigger
the stimulation of the Th1 profile (through T-bet, STAT1, and STAT4 transcription factors) that induce the Th17 cytokines, such as IL-17 and IL-21, which are the
main players of inflammatory response. B cell responds to fungal antigens through two different ways. One is through T-independent (TI) response against non-
protein antigens (polysaccharides, lipids, glycolipids, acid nucleic). Due to the absence of T cell responses, no immunological memory, secondary response, affinity
maturation, and isotype switching (usually conducted by cytokines of T cell) are occurred, eventually leading to the production of IgM isotype antibodies with low-
affinity and low half-life. B cell also responds to protein antigens through the T-dependent (TD) immunity. The processed antigen is presented to the CD4+ T cell by
MHC class II molecules and CD40-CD40 L (CD154) interaction. Following the activation of CD4+ T cells, the desired help package is released (consisting different
cytokines and molecules) to the B cells. Afterwards, B cell responds to the help through triggering the specific signal transduction, which eventually leads to the
production of various antibody isotypes (isotype switching) with high affinity (affinity maturation) and high half-life. This also includes the immunological memory
and secondary responses. Nowadays, TD immunization is followed by many research projects (subunit and conjugate vaccines) through binding fungal poly-
saccharides to the engineered/synthesized proteins.

3. Fungal vaccines/main categories 3.1. Live-attenuated vaccines

Based on the analysis of different kinds of vaccines against in- According to the similarity of live-attenuated vaccines with in-
fectious agents, it has been reported that vaccines annually prevent 6 fectious agents, they launch long-term and strong immune responses,
million deaths all around the world [29]. The aim of this review is which can be efficient in the immunocompetent patients. However,
providing the comprehensive review of antifungal vaccines and their consideration of the precautionary aspects seems to be necessary.
immune mechanisms. Here, different kinds of vaccines which are used Vaccinologists have designed several products of live-attenuated vac-
for prevention of fungal infections are classified into three main groups. cine strategies, which are very efficacious to combat with highly-in-
We discuss live-attenuated, recombinant, and conjugate vaccines fectious disease, mainly infectious viruses containing influenza, polio,
(Fig. 3). Finally, almost all of the studied anti-fungal vaccine programs mumps, rubella, measles, varicella, and rotavirus [30]. During infection
are gathered and presented in Table 1 to form an overall view. with different pathogenic fungi, such as Histoplasma capsulatum (H.
capsulatum), Blastomyces dermatitidis (B. dermatitidis), Paracoccidioides
brasiliensis (P. brasiliensis), Pneumocystis carinii (P. carinii), and C. neo-
formans, these strategies are highly effective through triggering

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S. Nami et al. Biomedicine & Pharmacotherapy 109 (2019) 333–344

Fig. 3. Three main category of vaccine against fungi.

protective immune responses via common pathways (Table 1) [30,31]. factor (TNF)-α, IFN-γ, and granulocyte/macrophage colony-stimulating
This type is the first vaccine used in human subjects. There are several factor (GMCSF). This study indicates that CD8+ T cells could also rely
studies in this field evaluating the efficiency of killed and attenuated on alternate mechanisms for robust vaccine immunity against experi-
fungi (Table 1). These vaccines will be applicable for endemic fungal mental fungal pulmonary infections with two agents, B. dermatitidis
pathogen prevention in the future in subjects with healthy immune and H. capsulatum [41]. In the same framework, the genetically en-
system who live in endemic areas [32,33]. One important finding is the gineered BAD-1 attenuated strain has also been tested that eventually
heat-killed Saccharomyces cerevisiae (HKS) vaccine, which plays an leads to the failure in binding or entry of yeasts into macrophages and
important role in protection against different fungal infections as a pan- adherence to lung tissue, and also reduction of virulence in mice. [42].
fungal vaccine plan (discussed below) [34,35]. Vaccination with HKS Another study showed that subcutaneous administration of the BAD-1
through a subcutaneous route has been shown to be effective in pro- live yeast without any adjuvant elevated the survival rate of mice from
tection against virulent strains of the endemic fungus Coccidioides po- lethal challenge of B. dermatitidis [43]. Mice immunized with re-
sadasii (C. posadasii) [32], C. albicans [35] and Aspergillus fumigatus (A. combinant BAD-1 yeasts, alone or in combination with IL-12 as an
fumigatus) [35]. In addition, a study reviewed the clinical efforts about adjuvant, showed acceptable efficacy in launching immune responses
developing of whole recombinant S. cerevisiae-based therapeutic (Fig. 2) [44,45].
method for the treatment of cancer and viral diseases together with Another attenuated vaccine strategy, which is named H99 g, has
cytotoxic drugs to achieve more clinical responses [36]. One major previously been shown to protect CD4+ T cell-deficient mice from in-
issue is the specificity of the vaccine, which limits the spectrum of its fection with a virulent strain of C. neoformans through inducing murine
effects [34]. IFN-γ and Th1 responses [46]. The H99 g strain is regarded as a live
Formalin-killed Coccidioides immitis (C. immitis) spherules (FKS) is vaccination plan which is a potent stimulator of host cytokine pro-
another vaccine type in this category. Previously, a placebo-controlled duction and, therefore, could not be usable in human subjects. A similar
phase III trial has been carried out to evaluate the efficacy of FKS that work reported the critical role of both CD4+ and CD8+ T cells in the
was unsuccessful to prevent the harshness of infection [36]. Later, a protection of mice against C. neoformans infection [47]. The safety of
study showed that vaccination (subcutaneously or by oral gavage with attenuated vaccines in the immunosuppressed hosts has not been
or without adjuvants) with HKS protected 100% of CD1 mice from a guaranteed. But these two recent strategies (BAD-1 and H99 g) may
lethal C. immitis challenge through prolonging survival and reducing immunize the CD4+ T cell-deficient subjects, particularly HIV/AIDS
fungal burden. Oral live Saccharomyces, but not HKS, prolonged sur- patients [48].
vival without reduction in fungal burden. Survival of mice given HKS As a first live attenuated plan, a vaccine was designed for the pre-
was equal with FKS. This study indicates that HKS was superior to a vention of ringworm caused by Trichophyton verrucosum (T. verrucosum)
successful recombinant vaccine with adjuvant [32]. Moreover, a study in cattle [30,49]. This study was carried out in a 5-year period on over
showed promising results of subcutaneous immunization of mice model than 400,000 cattle and demonstrated the efficacy of this immuniza-
with an attenuated strain of C. posadasii [33]. This strain was unable to tion-immunoprophylaxis strategy. The results of these studies clear the
transform to pathogenic spherule form and endosporulation process, perspectives about the future utilization of antifungal vaccines for
following deletion of two chitinase genes. subjects with CD4+ T cell deficiencies, such as patients with HIV in-
Deletion of Blastomyces adhesion 1 (BAD-1) gene presents an atte- fection.
nuated vaccine which has been shown to recruit multiple arms of the The main challenge is that the application of attenuated vaccines
host immune response (Fig. 2) [37–40]. A study tested an immunization should not lead to other kinds of diseases in immunosuppressed pa-
plan for the BAD-1 vaccine in CD4+ T cell deficient host like HIV/AIDS tients.
patients. In the absence of T helper cells, fungal PAMPs activate
memory CD8+ cells via interaction between MHC class I and CD8+ T
cell that leading to secretion of their cytokines, such as tumor necrosis

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 Table 1
Studies conducted in the design of antifungal vaccine strategy (Nd; non-determined).
Target Pathogen Antigen/Strain Adjuant/carrier/ Vehicle Vaccine Type Model Route of injection Underlying immune Human clinical Reference(s)
S. Nami et al.

mechanism trial

Candidisis Als3p Aluminium hydroxide Recombinant Mice/Human Oropharyngeal, IgA1, IgG Phase I [57], [58], [60,82], [83] [84],
Als1p (Alum) protein(NDV-3) vaginal and IL17 A, IFN-γ
intravenous
SAP2 Cholera toxin Recombinant Mice/ Human Intravaginally Protective antibodies Yes (delivered [60,85]
(CT)/Virosomal carrier protein by
intramuscular)
secreted aspartyl Cholera toxin Recombinant Rat Intravaginally Antibodies – [85]
proteinase protein, Sap2p (CT)
PEV-7
Tet-NRG1 (C. albicans Nd (Not defined) Genetically Nd Nd T-cell mediated [86,87]
strain) engineered/Live immunity
attenuated
C. albicans PCA-2 strain Nd Live-attenuated Mice Intravenously Increased PMNs and – [88]
macrophage activity
Cell wall surface proteins Liposomal adjuvant Subunit Mice Subcutaneously Antibodies, Th17 – [89]
(CWSP)
C.albicans Mannan – Mannan-protein Mice Intravaginaly Protective antibody – [90]
extracts conjugate responses
Laminarin (Lam) β-glucan Complete Freund’s adjuvant Lam- diphtheria Mice Priming dose: Passive protection by – [67,90,91,92,93,94]
(CFA) toxoid CRM197 Subcutaneously anti β-glucan
conjugate Booster: Intranasally antibodies
Cross protection against
A.fumigatus infection
C. dubliniensis mannan/ Nd Conjugate Rabbit Intravenously Th1/Antibodies: IgG – [95]

337
Human serum albumin and IgA
(HSA)
Fructose bisphosphate CFA Subunit Mice Intraperitoneally/ Antibodies – [96] [97],
aldolase (Fba) (cytosolic Subcutaneously
and cell wall peptides)
C. albicans serotypes a and Nonencapsulated Klebsiella Recombinant/ Women with vulvovaginal Oral Nd phase II [98]
b ribosomes pneumoniae proteoglycan Conjugate capsule candidiasis (VVC)
Heat-killed C. albicans Detoxified Escherichia coli: Recombinant/ Mice Intranasally/ Antibody(IgG, IgA)- – [99]
(HK-CA) LT(R192 G) Conjugate Intravenously Th1
Glycolytic enzyme enolase complete Freund Recombinant Mice Subcutaneously Antibody- Th1/2 – [100]
65 kDa mannoprotein Plasmid: pRLV130-139-140- Subunit/ Mice Intravaginally/ Adhesin-neutralising – [101]
(Camp65p) 161-162-169 Glycoconjucates Intravenously antibodies
C. albicans cell surface Alum Recombinant N- Mice Subcutaneously Antibodies – [102]
protein Hyr1 terminus of Hyr1
protein (rHyr1p-N)
Combining β-mannan and CFA Subunit/Conjugate Mice Intravenously Th1, antibodies – [103]
peptide epitopes
(continued on next page)
Biomedicine & Pharmacotherapy 109 (2019) 333–344
 Table 1 (continued)

Target Pathogen Antigen/Strain Adjuant/carrier/ Vehicle Vaccine Type Model Route of injection Underlying immune Human clinical Reference(s)
S. Nami et al.

mechanism trial

Aspergillusis Aspergillus fumigatus crude Nd Sonicate and Mice Intranasally Th1 cells producing – [104]
culture filtrate Ags filtrate Ags IFN-γ and IL-2
Asp f3 Incomplete Freund’s Subunit/ Mice Subcutaneously Antibodies/CD4+ T [105]
adjuvant recombinant cells
Aspergillus fumigatus Nd Sonicate and Corticosteroid Intranasally/ Unknown – [106]
viable conidia filtrate Ags ImmunosuppressedMice subcutaneously
Aspergillus fumigatus Aspergillus fumigatus Recombinant Mice Subcutaneously Antibody & cellular – [107]
hyphal sonicate (HS) allergen Asp f 3 immunity responses
Heat killed yeast (HKY) of Nd Live-attenuated Antibody knockout mice Subcutaneously Th1, Th2, Th17 – [108,109]
S. cerevisiae
A. fumigatus epitope p41 Murine cytosine guanine Subunit Mice Intranasally/ 1. MHC II alleles that [110]
from the cell wall dinucleotide (CpG) Intragastricly induces memory
glucanase (Crf1) oligodeoxynucleotide(ODN) CD4 + TH1 cells.
2. cross-protection
against lethal infection
with C. albicans that is
mediated by the same
epitope as in humans
Asp 16 f Unmethylated CpG Recombinant/ Mice Intranasally Th1 – [111]
oligodeoxynucleotides subunit (DCs)
(ODNs) pulsed with
Aspergillus antigens
Asp 3 f TiterMax (TM) Recombinant/ Mice Subcutaneously Th1 – [107]
subunit
Proteins: Gel1p, Crf1p, CpG oligodeoxynucleotide Recombinant/ Bone marrow transplanted Intranasally Th1 – [112]

338
Pep1p, Cat1p, Sod1p, Subunit mice
Dpp5p, RNUp, Mep1p,
Polysaccharides: _1–3
glucan, _1–3 glucan, GM,
Glycolipids: GSL, LGM
Panfungal β-glucans of S. cerevisiae Nd Heat Killed Yeast Mice Subcutaneous Th1, Th17, Antibodies – [34]
(HKY) to glucan and mannan
Blastomycosis Adhesin BAD1 gene Nd Whole organism/ Mice (T CD4+ depleted) Subcutaneously CD8+ T cells, MHC I, – [41]
Live-attenuated Th1 immunity
Paracoccidioidomycosis gp 43 (P10) Plasmid vector DNA vaccine Mice Nd T-reg cells – [1,113]
(PCM) (pcDNA3-P10) Immunological memory
gp 43 (P10) S. cerevisiae expressing gp43 Recombinant Mice Intraperitoneally Th1 immunity/ – [114]
(yMAgp43) protein elevation of IL-12 and
IFN-γ
P10- FliC fusion protein CFA/MAP Recombinant Mice Intratracheally Th1 – [115]
rPb27 Corynebacterium parvum/ Recombinant Mice Subcutaneously Antibodies – [116]
aluminum
hydroxide Al(OH)3
Heat shock protein 60 Adjuvant containing Recombinant Mice Subcutaneously Th1 – [117]
(HSP60) monophosphoryl lipid A,
synthetic trehalose
dicorynomycolate, and cell
wall skeleton
Mycobacterium leprae- Vector pVAX1/ Recombinant DNA Mice Intramuscular Th1 – [118]
derived HSP65
(continued on next page)
Biomedicine & Pharmacotherapy 109 (2019) 333–344
 Table 1 (continued)

Target Pathogen Antigen/Strain Adjuant/carrier/ Vehicle Vaccine Type Model Route of injection Underlying immune Human clinical Reference(s)
S. Nami et al.

mechanism trial

Coccidiomycosis Killed spheroles Nd Whole organism/ Human Intramuscularly in Nd Phase 3 [81]

Inactivated the deltoideus
C. immitis spherule-phase pBK-CMV phagemid vector DNA(expression Mice Intramuscularly/ CD4+ and CD8+ T – [119,120]
genes library Intradermaly cells/T-helper 1 cells
immunization: ELI)
T-cell epitopes Antigen 2/ Adjuvant: CpG Recombinant Mice Subcutaneously Th1, Th17, Th2 – [121,122,123]
proline rich Ag (Ag2/ Vector: YEp-FLAG-1 protein
PRA)/Chimeric
polyprotein
Attenuated mutant (ΔT Nd Live-attenuated Mice Subcutaneously Th1, Th17, Th2 – [124]
vaccine strain)
Immunodominant T cell Adjuvant: CpG Recombinant Mice Subcutaneously Th1, Th17, Th2 – [125]
epitopes
C. posadasii Gel-1 (β 1,3 CpG ODN Recombinant Mice Subcutaneously Th1. Antibody – [126]
glucosyltransferase) protein
Urease (rURE) Plasmid vector :pSecTag2A/ Recombinant Mice Subcutaneously (Th1)/Th2 – [127]
Adjuvant: CpG ODN
Spherule phase of C. Monophosphoryl lipid A- Recombinant Mice Subcutaneously Elevated IgG titer – [128]
posadasii Peroxisomal stable emulsion (MPL-SE)
matrix protein (Pmp1) adjuvant
vector: YEp-FLAG-1
Chimeric protein-aspartyl Adjuvant: recombinant Mice Subcutaneously Nd – [129]
proteinase, phospholipase CpG ODN protein
B and α mannosidase Vector: pGEM-TE
Histoplasmosis Water-soluble CFA Inactivated- Mice Intraperitoneally Nd – [130]

339
ethylenediamine extract filtrated Ags/
from cell wall Soluble antigenic
fractions
Ribosomes or live yeast Incomplete Freund Live-attenuated Mice Intravenously/ Lymphoid cells – [131]
cells of H. capsulatum Subcutaneously
Cell wall and cell Incomplete Live-attenuated Mice Subcutaneously/ Nd – [132]
membrane of yeast-phase Freund Intraperitoneal/
H. capsulatum G217B Intravenously
Histone H2B–like protein Incomplete Freund’s Live-attenuated/ Mice Intraperitoneally Th1/Antibody – [133]
Recombinant
Heat Shock Protein 60 – Recombinant Mice Subcutaneously Th1 – [134,135]
(HSP-60)
HIS-62 Bovine serum Recombinant Mice Subcutaneously Cellular immune – [136]
albumin (BSA) protein response
rHIS-62
80-kilodalton antigen Incomplete /Complete Recombinant Mice Subcutaneously Antibody/Cellular – [137]
Freund's responses
Sec31 homologue Monophosphoryl lipid A, Recombinant Mice Subcutaneously T cell mediated – [138]
trehalose dicorynomycolate,
and cell wall skeleton
H antigen(H.capsulatom) Adjuvant containing Recombinant Mice Subcutaneously Th1.Th2/CD8+ – [139]
monophosphoryl lipid A, antigen
synthetic trehalose
dicorynomycolate, and cell
wall skeleton and bovine
serum albumin (BSA)
(continued on next page)
Biomedicine & Pharmacotherapy 109 (2019) 333–344
 Table 1 (continued)

Target Pathogen Antigen/Strain Adjuant/carrier/ Vehicle Vaccine Type Model Route of injection Underlying immune Human clinical Reference(s)
S. Nami et al.

mechanism trial

Cryptococcusis GXM Tetanus toxoid (GXM-TT) Conjugate/Soluble Mice Subcutaneously Anti-GMX antibodies – [66]
antigenic fractions (Active immunization)
GalXM Quil A/ Freund's complete/ Subunit/Conjugate Mice Subcutaneously/ Antibody: IgG, IgM – [140]
BSA Intraperitoneally
C. neoformans strain H99γ Nd Live-attenuated T-cell depleted mice Nasal inhalation CD4+ and CD8+ T cells – [47]
(serotype A, Matα)
+ + +
Mutant C. neoformans Plasmid pCR II- Live- attenuated- CD4 T cells depleted Intranasally CD4 and CD8 T cells – [141]
strain lacking the enzyme TOPO 4.0 kb recombinant /immunocompetent mice
sterylglucosidase 1 named
(Δsgl1)
CneF (culture filtrate Ags), Nd Subunit/ Mice Nd Th1, Antibodies – [62]
Mannoprotein Recombinant
GXM CFA GXM–protein Mice Intraperitoneally High-titer IgG – [142]
conjugate responses
P13 (a peptide mimetic of Tetanus toxoid (TT) or Conjugated Mice Subcutaneously Anti GXM antibody – [143,144,145,146,147,148]
GXM) diphtheria toxoid (DT) response(IgG2 & IgG4)
Laminaran Nd Subunit (algal β- Mice Nd Passive immunity [149]
glucan based)
Pneumocystosis Kexin genes Adjuvant: CD40L Kexin-CD40 L DNA CD4-deficient mice Intramuscularly Elevated IgG titers – [120]
Vector: CMV to express vaccine
Antigene
EF-1α
to express CD40L
P55 protein (major surface Titermax Recombinant Mice Subcutaneously Th1-Th2 responses – [150]
glycoprotein) protein

340
Major surface glycoprotein Titermax Recombinant Mice Subcutaneously Antibody and T-cell – [151]
(also known as gp120) protein dependent
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3.2. Recombinant (subunit) vaccines could be applicable to produce and commercialize pan-fungal vaccines.
This is very crucial for immunosuppressed patients which are at high
Subunit vaccines are the most investigated sorts of fungal vaccines risk for various form of IFIs [63,65].
that consist of one or more purified recombinant proteins or poly- The first fungal conjugate vaccine was designed against C. neofor-
saccharides of fungi. Genetic engineering and also increased knowledge mans that contained glucuronoxylomannan (GXM), a capsular poly-
in the microbial pathogenesis and fundamentals of immunology help saccharide, and tetanus toxoid (TT) [66]. These two particles are linked
scientists to develop efficient subunit vaccines. Scientific basis of this by a covalent bond and a monophosphoryl lipid A (MPL) is used as an
technology is transferring and expressing of a gene encoding an im- adjuvant in the vaccine complex. Immune mechanism of this vaccine is
munogenic antigen, in order to trigger the desired immune response. In based on the antibody (especially IgA and IgG) responses. Additionally,
fact, in this approach, a gene which is transmitted encodes a portion a pan-fungal vaccine, designed by conjugating a β-glucan poly-
related to virulence and pathogenicity of organism. These protein an- saccharide extracted from brown algae, to inactivated diphtheria toxin
tigens are often combined with an appropriate adjuvant or protein (CRM) and complete freund's adjuvant (CFA), showed effective roles in
carrier, mostly bacterial toxoids, to establish an efficient immune re- protection against invasive candidiasis and aspergillosis [67]. Another
sponse and prolonged immunization (Fig. 2) [31,48,50]. Alum (alu- conjugate anti-Candida vaccine was constructed by conjugating β-1,2-
minum salts, such as aluminum hydroxide and aluminum phosphate) is mannotriose to a peptide segment from fructose-bisphosphate aldolase
one of the most common adjuvants in this field which induces strong (Fba), which is the surface antigens of Candida spp. Various forms of
antibody responses [48,51,52]. Recombinant subunit vaccines has this vaccine have been applied in different studies (with or without
several advantages, such as absence of the pathogenic agent and, alum adjuvant) [68] (see Table 1).
therefore, application of these vaccines becomes safer particularly in
immunocompromised patients [48,53]. By merging the DNA en- 4. Novel strategies
gineering and recombination technologies, vaccines have been carefully
designed, purified, and produced, which leads to the engineering of 4.1. DNA vaccines
highly specialized antigens [54,55].
A study showed that an invasion protein, agglutinin-like sequence 3 By entering the cDNA encoding the desired antigen into a plasmid
(Als3p) conjugated with alum, which is called NDV-3, conferred anti- and transferring the gene containing plasmid to the host's APCs (mainly
Candida protection through preventing yeast-epithelial/endothelial DCs), the antigen is expressed and eventually generates a desired im-
attachment [56]. In addition, NDV-3 induced a cross-protection against mune response. Bacterial plasmids contain non-methylated CpGs,
highly infectious bacterial pathogens, such as Staphylococcus aureus (S. which are recognized by TLR9 (expressed on DCs), and further stimu-
aureus) due to the structural homology between Als3p and clumping late the acquired immune responses. In addition to the antigen coding
factor-A of S. aureus. Most importantly, NDV-3 successfully passed the gene, the gene which codes the co-stimulatory molecules and also cy-
phase I clinical trial and was found to be safe and protective in human tokines can join to the plasmids. This vaccine could also be applied
subjects through triggering the antigen-specific T cells that released without any adjuvant. However, despite the current theories on safety,
IFN-γ and IL-17 A cytokines [57]. This vaccine has also been approved immunogenicity, and efficacy, the application of this type of vaccine for
to elicit a protection in animal models of oropharyngeal, vaginal, and human faced with some major challenges [30,69]. Previously, DNA
invasive candidiasis [48,58,59]. vaccines have also been examined through transferring one or more
Another study showed that secreted aspartyl proteinase-2 (Sap-2), a antigen coding plasmids [70–72]. The first fungal DNA vaccine may be
highly expressed virulence factor secreted by different Candida spp, related to ringworm caused by T. verrocosum, which was discussed
displayed protective roles against recurrent vaginal candidiasis in a above [49].
virosome-based format of the vaccine [48,60]. This vaccine was applied
in the rat model of vaginal candidiasis and also a phase I clinical trial
and showed effective results [48,60]. However, there are several pro- 4.2. Immunotherapeutic products
blems in the commercialization of recombinant vaccines, such as
healthy status of a subject (both immunocompromised and im- Two novel vaccine strategies share immunotherapeutic nature
munocompetent hosts), economic issues in targeting the human subject based on the application of fungus antigen-primed DCs and/or fungus-
(high costs of application in clinical trials), and also the method of specific T cell clones [31,73,74]. In these programs, fungus-protective
synthesis of the vaccine, such as glycosylation, which directly affects antigens were identified, regenerated, primed to the DCs ex vivo, and
the immunization circumstances [48,61,62]. eventually infused to the host in order to selective priming-activation of
DCs and formation of highly specific T cell clones. Immune responses
3.3. Conjugate vaccines produced by these strategies have clearly potent effects and precision.
Romani and colleagues described the benefits of these approaches
A conjugate vaccine is produced by covalent attaching of a poor [31,73,74].
antigen to a strong antigen, commonly polysaccharide to protein, re-
spectively. This is carried out in order to generate a potent immune 4.3. Pan-fungal vaccine strategy
response [63]. B cells, in confronting with polysaccharide antigens,
develop antibody responses without contribution of T cells, which is Fungal cell wall contains common epitopes. By inactivating and
called T-independent immune response. In fact, polysaccharide epi- conjugating the fungal common polysaccharides with different im-
topes are recognized by B cell receptors, but for the presentation of munogenic peptides, we are able to form a type of vaccine which
antigens to T cells, they should bind to peptides (hapten-carrier system) protects the host from different types of fungal infections. Nowadays,
and the peptide is required to be presented by MHC complexes ex- two pan-fungal vaccine plans have been developed. Killed S. cerevisiae
pressed on the APCs. Immunity stimulated by T cells is a strong and triggers the protective immune response against glucans and mannans,
durable. Through conjugating a polysaccharide to a protein carrier, which are common fungal polysaccharide epitopes. This vaccine also
MHC molecules are able to bind proteins and eventually induce the T launches the cross-reactive immune responses against homologous
cell responses (Fig. 2) [48,64]. proteins which exist on the fungal cell walls [34]. Another universal
One major advantage of conjugate vaccine strategy is that these vaccine strategy is made up from conjugating β-glucans to an in-
vaccines are based on targeting the polysaccharide epitopes, which are activated version of diphtheria toxin (CRM), which was mentioned
common in all fungi, especially β-glucans. Therefore, this technique above [67].

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5. Vaccine based immunity against fungal infections coccidioidomycosis, and paracoccidioidomycosis [2]. In the 1980s,
clinical trials were conducted on the coccidioidomycosis vaccines and
One of the most interesting and fascinating topics is to predict the these trials have continued until now [81]. Apart from all these efforts
immune responses before and after exposure to the vaccine agent in for producing a suitable vaccine with active or passive immunization,
both healthy and infected hosts. In other words, each vaccine stimulates none of the fungal vaccines have been confirmed by FDA.
the immune responses through different and more-specified ways, The first step in designing an effective strategy for vaccination
which scrutinizing them is one major point in progressing the vaccine against fungal infections is to improve our knowledge of the immune
strategies against fungal infections. We previously discussed the im- system. Following the profound knowledge of the mechanisms of im-
mune responses against fungi. Totally, T cell-mediated responses are mune responses to fungal infections, it is possible to design and apply
the main arms of the immune response in combating fungi. the immunological products which termed immunotherapy. Immune
Furthermore, there are several cellular (macrophages, neutrophils) and adjuvants, especially TLR-ligands, along with monoclonal antibodies
soluble (antimicrobial peptides, cytokines, and chemokines) tools. are the most important products of immunotherapeutics. Monoclonal
Vaccines not only launch but also amplify the immune responses, par- antibodies, in spite of high costs, shows acceptable results in conjunc-
ticularly the vaccines used in conjugation with an adjuvant. Some of the tion with vaccines. Nowadays, this strategy is expanding by many re-
antifungal vaccines enhance antibody responses and others mostly in- search projects. Targeting the pan-fungal antigens also presented ac-
tensify Th responses. But most of them simultaneously boost both types ceptable results in the production of universal fungal vaccines.
of immune responses (Fig. 2). The immune responses used for each However, application of new techniques, such as DNA vaccines and
vaccine are extracted and listed in Table 1. Here, we review the me- immunotherapeutic products, with significant advances in the fields of
chanisms of some important vaccines. genomics, vaccinomics, and proteomics will be useful to open new
avenues for the success of vaccine strategies in clinical trials.
5.1. Antibody mediated vaccine responses
Conflict of interest
Studies showed that specific antibodies cause protection against C.
neoformans by triggering classical opsonophagocytosis, complement The authors declare that there is no conflict of interest.
activation, and direct neutralization of adhesins or enzymes, which are
totally humoral immunity [62,75]. There are several studies about Acknowledgements
antibody-mediated immunity to fungal infections induced by passive
vaccination strategies (Table 1). The immune mechanism emerged from The authors are grateful to Department of Medical Mycology and
NDV3 and above discussed conjugate vaccine for cryptococcosis [66] Parasitology- laboratory of Medical Mycology, School of Medicine,
are based on IgG and IgA antibodies. Alum adjuvant is also a potent Tabriz University of Medical Sciences and also dear Dr. Behzad
stimulator of antibody responses (discussed above). Baradaran, the director of Immunology Research Center, Tabriz
University of Medical Sciences for critical reading of the paper.
5.2. Th mediated vaccine responses
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