Introduction

The increased number of fungal infections in recent years is directly related to the immunocompromised populations, with a serious impact on the health care systems [1, 2]. In the past, HIV-positive individuals were the main risk group of fungal infections. Nowadays, patients undergoing chemotherapy as in cancer, organ transplantation, premature neonates, and individuals in intensive care units, are also risk groups of invasive mycoses [3].

Pathogenic thermal-dimorphic fungi are agents of subclinical respiratory infections to systemic mycoses, being endemic in certain regions. Among these fungi, are members of genera Paracoccidioides, Histoplasma, Coccidioides, and Blastomyces. These microorganisms are present in the soil, plant remains, and/or animal excreta. The infection occurs through the inhalation of propagules found in the environment and the fungal cells can spread to the entire body. These microorganisms are responsible for high rates of mortality and high morbidity in endemic areas, by infecting thousands of people each year. Epidemiological data are not, however, accurate, as these mycoses are underreported (Reviewed in [4]).

Paracoccidioides spp. form a complex of species that are responsible for paracoccidioidomycosis (PCM), with the main etiological agents being P. brasiliensis and P. lutzii. Recently, three other species have been suggested to be included in this genus: P. restrepiensis, P. americana, and P. venezuelensis [5]. PCM is a systemic mycosis throughout Latin America, with great incidence in Brazil, Venezuela, Colombia, and Argentina [6]. According to the literature, more than 80% of cases of PCM are reported in Brazil, where the main infected population comprises rural workers [7, 8].

Histoplasmosis is an endemic mycosis, caused by Histoplasma capsulatum, frequent in the USA, Mexico, and South America, with case reports in China and India [9, 10]. It is an opportunistic mycosis, and HIV positive individuals are those mainly affected by this fungus in Latin America [11]. Infection occurs by inhalation of microconidia from the environment. In the absence of some form of immunosuppression, the infection is usually contained [12].

Coccidioidomycosis is a fungal infection caused by dimorphic, soil-dwelling Coccidioides immitis and C. posadasii, which are endemic in regions of Arizona, California, New Mexico, Nevada, Utah, Washington, Texas, and Mexico. There are also cases in some areas in Venezuela, Brazil, Argentina, and Paraguay. According to the literature, cases of Coccidioides spp. have been reported in increasing numbers in recent years probably due to changes in environmental conditions, human activities in endemic areas, immunosuppressed individuals, and the development of more accurate diagnostic tests (reviewed in [13]. The infection starts by inhalation causing a broad spectrum of disease, including mild febrile illness, severe lung infection and disseminated disease [14].

Blastomycosis can be caused by the agents Blastomyces dermatitidis and B. gilchristii, with high incidence in Canada and Eastern USA. Clinical manifestations are diverse, ranging from asymptomatic infection to extrapulmonary spread, reaching almost all organs of the body [15].

Currently, antifungal treatment options are limited, especially in developing countries. Although effective in most cases, the treatment of fungal infections is toxic, expensive, and must often be extended to a couple years [16]. In addition, the emergence of resistant strains and increase of relapsing disease are common clinical concerns [17].

The choice of antifungal drugs and treatment time in systemic mycoses depend on the severity of the infection [18]. Amphotericin B (AmB) is indicated to treat severe cases whereas azoles are preferred for the moderate ones [19]. In the case of PCM, the use of trimethoprim-sulfamethoxazole is also acceptable, presenting relative success. Echinocandins are a class of antifungal agents that cannot be used to treat mycoses caused by dimorphic fungi, since they have a natural resistance to these drugs [4, 20].

In order to achieve effective antifungal treatment against dimorphic fungi, therapy generally has to be administered for long periods, and even then, the infection may not be eliminated. Treatment in less severe cases can last up to 1 year, but in the cases of disseminated infection or in cases of disease in immunocompromised patients, therapy must be held beyond this period [4].

In recent years, concern about the increased incidence of serious infections caused by fungi, together with a difficult management therapy, stimulated the search for new therapeutic options [21]. Thus, the use of immunotherapeutic strategies has gained interest and the development of vaccines that can protect populations at risk or assist in antifungal treatment, reducing the time of therapy and drug toxicity, is a goal that many studies aim to achieve.

This review addresses some studies on the development of vaccines to systemic mycoses, including PCM, histoplasmosis, coccidioidomycosis, and blastomycosis.

Paracoccidioidomycosis

Aiming at a vaccine against P. brasiliensis, yeast cells of Pb18 strain were gamma irradiated with doses between 0.5 and 8.0 kGy. Fungal cells after gamma radiation became less virulent, and their proliferation was inhibited. Their metabolic activity was still present and the antigenic profile was maintained [22]. When using gamma irradiation at 6.5 kGy, alterations in the yeast morphology were reversible after 48 h. Electron microscopy showed nuclear alterations without damage in both plasma membrane and cell wall [23]. Looking for a diagnostic and protective antigen in P. brasiliensis, the 43-kDa glycoprotein (gp43) was identified [24]. The epitopes in gp43 that elicited a specific antibody response in patients with PCM are peptidic in nature therefore reacted with the deglycosylated molecule [25].

The gp43 antigen is also able to elicit positive skin tests in guinea pigs and in humans implying the presence of an immunodominant epitope to a CD4+ T cellular immune response [26, 27]. This epitope was mapped by Taborda et al. [28] to a specific sequence of gp43, QTLIAIHTLAIRYAN, denominated P10. This peptide has a hexapeptide core (HTLAIR), as the essential domain of the IFN-γ-dependent Th1 epitope recognized by H-2b isogenic mice. Previous immunization with P10 or gp43 in the presence of complete Freund’s adjuvant (CFA), protected mice intratracheally infected with P. brasiliensis, significantly reducing the fungal burden in the lungs, liver and spleen [28]. In a study using TEPITOPE algorithm, the P10 emerged as a good candidate for a human vaccine, since this peptide was found to be promiscuously presented by MHC class II molecules from mice and humans [29, 30]. In recent years, several studies on the different ways of peptide delivery and other properties of P10, have contributed to validate this peptide as a potential candidate for a human vaccine [20, 31,32,33].

Marques et al. [34], aiming at improving the treatment of PCM mainly to avoid relapses of the disease, carried out a study using P10 immunization combined with antifungal drug treatment. Using two protocols, the use of P10 in combination with antifungal drugs showed an additive protective effect in the experimental model using BALB/c mice. Th1 and Th2 cytokines were present in successfully treated mice and that the combined treatment with P10 immunization reduced the CFUs and maintained preserved lung alveolar structure. In addition, the combined treatment prevented the spread of the fungus to other organs. Subsequently, in order to mimic the anergic state of some patients with acute and subacute forms, the effects of combined treatment of trimethoprim and sulfamethoxazole or itraconazole, with P10 immunization in immunocompromised mice, were investigated. Anergic mice infected with P. brasiliensis showed high fungal loads in the liver, spleen and brain after 45 days of infection, in addition to high dosing of IL-4 and IL-10 and low levels of IL-12 and IFN-γ. This pattern was reversed by P10 immunization and not with antifungal drugs. With the combined therapy, a high Th-1 type immune conversion was seen. In addition, the immunization with P10 significantly reduced the fungal burden in lung, spleen and liver of anergic mice [35].

The protective efficacy of IFN-γ in the PCM murine model was demonstrated, as mice deficient in IFN-γ or IFN-γ receptor evinced 100% mortality after infection with a virulent isolate of P. brasiliensis (reviewed in [36].

Amaral et al. [37] carried out a study using three strategies of combined therapy, in which animals infected with P. brasiliensis received daily, sulfamethoxazole/trimethoprim alone or in conjunction with P10 and Freund’s adjuvant or P10 in poly-lactic acid-glycolic acid (PLGA) nanoparticles. In addition to the effectiveness of combined treatment with P10 in PLGA, the required amount of P10 to obtain protective results lowered significantly. P10 incorporated into PLGA was protective for prolonged periods compared with the combined treatment using P10 emulsified in Freund’s adjuvant. Using mice treated with dexamethasone, immunization with P10 has been shown to efficiently modulate the immune response in immunosuppressed hosts, increasing animal survival, reducing lung fungal load, and also the pulmonary fibrosis in mice immunized with the peptide [38]. The use of gene therapy has also been described as being an alternative to PCM, being able to induce memory cells [39]. Tested in intratracheally infected mice, the pcDNA3 expression vector encoding P10 induced a significant reduction of fungal load in the lung. Additionally, when also using the plasmid encoding mouse IL-12 a more significant activity in the elimination of the fungus [40] was obtained.

DNA vaccine based on HSP65 from Mycobacterium leprae against PCM was able to induce protection against infection by P. brasiliensis through the production of IFN-γ and IL-12 [41]. Subsequently, controlled release systems were used to evaluate the DNAhsp65 vaccine and the effects of this strategy on pulmonary PCM treatment. Mice that received DNAhsp65 vaccine released by different nanoparticles showed significant decrease in CFUs when compared with the treatment using only DNAhsp65. In addition, this effect was obtained using about four times less DNAhsp65, demonstrating the efficiency of the controlled release system. The protection of animals is related to increased production of protective type Th1 cytokines, IFN-γ and IL-12 [42].

P. brasiliensis affects healthy individuals and, generally, the defense against this infection in the lungs requires a good innate and adaptive immune response. Dendritic cells (DCs) are efficient inducers of T lymphocyte immune responses against Paracoccidioides antigens [43,44,45]. The administration of gp43-pulsed DCs to mice resulted in increased production of IL-2 and IFN-γ by CD4+ T cells isolated from regional lymph nodes [46]. Subcutaneous or intravenous administration of DCs pulsed with P10 has subsequently been shown to confer protective immunity against infection by P. brasiliensis in the murine model. Mice receiving DCs pulsed with P10 showed a mixed cytokine response pattern with predominance of Th1 activation and significant reduction of fungal burden compared with control animals [43, 45, 47].

In a study using rPb40 and rPb27 recombinant proteins, Fernandes et al. [48] analyzed the therapeutic activity of these proteins in combination with fluconazole (FLZ). A significant therapeutic effect was observed, to the point of no recovery of CFU from lungs, livers, and spleens of BALB/c mice, immunized after 4 months of P. brasiliensis yeast infection. Animals submitted to the combination therapy showed low IL-4 and TGF-β and increase in TNF-α after 70 days of infection and high levels of IFN- γ after 120 days of infection.

Investigators have attributed to TNF-α the control of dissemination and growth of the fungus, assisting in the regulation of inflammatory responses in PCM [49]. TNF-α acts by attracting and activating effector cells and is extremely important in the granulomatous inflammatory reaction [50, 51]. In contrast, IL-4 can inhibit the production of IFN-γ and hinder the activation of macrophages (reviewed in [48].

Holanda et al. [52] constructed two types of vaccines (P10/(His) 6-purified protein (rPbT) and recombinant P10/human type 5 adenoviral vector (rAdPbT)), based on chimeric virus-like particles (VLPs), containing the main CD4+ T cell-specific epitope from P. brasiliensis (P10) within the C-terminal portion of HBcAg. As a result, the authors first proved that the recombinant VLP protein carrying a specific CD8+ T cell epitope from murine cytomegalovirus (rCMV), did not protect the infected animals, demonstrating that the use of chimera is valid for the evaluation of P10-based vaccine efficacy through the specificity of the CD4+ T cell response. Mice were immunized with rPbT and rAdPbT, and the Pb18 strain was used for infection of animals. High levels of protective Th-1 cytokines were formed, inducing a CD4+ T response. In addition, when rAdPbT was pre-inoculated it prevented the spread of PCM in the infected mice.

Recently, the recombinant single-chain variable fragments (scFv), that mimic Ab2-β7B12 mAb against gp43 of P. brasiliensis, incorporated into PGLA nanoparticles, modulated the production of cytokines, which protected against PCM. The cytokine profile in the lung of animals receiving scFv-nanoparticles showed increased IFN-γ and IL-12 and low IL-4 and IL-10 production, resulting in decreased fungal burden in treated mice. The authors suggest that the treatment modulated the humoral immune response with production of IgG2a and IgG1. Despite the positive data demonstrated, the results obtained in the prophylactic treatment need to be further developed [53].

The use of monoclonal antibodies (mAb) was also tested against PCM caused by P. brasiliensis, where anti-gp43 and anti-gp70 demonstrated efficacy in the contention of the experimental infection.

In addition to gp43, gp70 is also recognized in the serum of most PCM patients. This glycoprotein inhibited the action of peritoneal macrophages, suggesting a mechanism of action similar to that of gp43. With this in mind, a study was carried out where mAb were produced against gp70. When studying the activity of anti-gp70 IgG1 mAb by passive immunization of mice infected with P. brasiliensis, significant reduction in the recovery of CFUs from the lungs of the animals was shown when the C5F11 and B7D6 mAb were combined [54].

The biological function of P. brasiliensis 75 kDa protein was verified using IgM and IgG mAb. In vitro assays showed that these mAb were able to inhibit fungal growth. After yeast opsonization, the phagocytosis by murine peritoneal macrophages was stimulated. In addition, the fungal burden decreased significantly when passive immunization with mAb was performed [55].

Buissa-Filho et al. [56] analyzed the 19G, 10D, 32H, and 17D (IgG2a) and 21F and 3E (IgG2b) mAb against gp43 by passive immunization of mice infected with P. brasiliensis. All mAb tested increased the phagocytosis rate in vitro, but the 3E showed a more significant result. Through passive administration of the antibodies protective and non-protective mAb were identified and the protective ones reduced the fungal burden in the lungs. It has also been shown that the epitope for 3E mAb, NHVRIPIGYWAV, is homologous with β-1,3 glucanase sequences from other fungi. Since gp43 can be found on the cell surface of P. brasiliensis [57], the opsonizing effect of 3E mAb may involve the recognition of this peptide sequence on the cell wall of the fungus. Although PCM protection is associated with a potent cellular response, this work demonstrates that protective anti-gp43 antibodies can also assist in the infection treatment in mice.

P. lutzii infections did not respond to serological tests using gp43. Thomaz et al. [58] analyzed the effects on P. lutzii experimental PCM, of two mAb generated to the heat shock protein from H. capsulatum (Hsp60), 7B6 (IgG2b), and 4E12 (IgG2a), since there is homology among the Hsp families from dimorphic fungi. The authors observed that passive transfer of 7B6 and 4E12 mAb to Hsp60 decreased the fungal load in the lungs of infected mice. In murine histoplasmosis, IgG2a mAb is associated with a Th1 response whereas mAb IgG2b induced a Th2 response, exacerbating the disease. In murine PCM caused by P. lutzii, both mAb were protective suggesting the activation of a Th1 response. In addition, the authors argue that the effects caused by mAb on phagocytosis are due to the significant increase in IFN-γ production, as documented in studies with P. brasiliensis.

The vaccine candidates against PCM are summarized in Table 1, and the main monoclonal antibodies tested in Table 2.

Table 1 Vaccine candidates tested against systemic mycoses by thermally dimorphic fungi
Full size table
Table 2 Main monoclonal antibody tested against paracoccidioidomycosis, histoplasmosis, blastomycosis, and coccidioidomycosis
Full size table

Histoplasmosis

In the last years, several vaccine candidates against H. capsulatum have been studied such as: cell wall and membrane extracts from H. capsulatum yeast cells [59]; the 62-kDa protein, named HIS-62 [60]; rH antigen [63, 78]; native and recombinant Heat Shock Protein 60 [62, 79]; extracts containing cell-free antigens from H. capsulatum yeast [64]; and alkaline extract of H. capsulatum packaged in glucan particles [65].

In 1991, Gomez et al. [59] did experiments to analyze T cell antigens from H. capsulatum yeast cells. An extract of cell wall and membranes from H. capsulatum yeast cells was prepared and tested to evaluate the antigenicity and immunogenicity of the extract. The preparation induced a cell-mediated immune response both in vivo and in vitro [59].

In another work, a 62-kilodalton antigen, HIS-62, from the cell wall and cell membrane of H. capsulatum yeast cells, was used to prophylactically vaccinate mice and a protective effect was observed against a lethal intravenous challenge with yeast cells, during a 28-day observation period [60].

Gomez et al. [61] after demonstrating the protective effect of HIS-62, a member of HSP-60 family, cloned the gene encoding this antigen and studied the immunological activity of the recombinant antigen (rHIS-62). They observed that BALB/c mice vaccinated with the recombinant protein were protected against a lethal intranasal challenge with yeast cells and also observed that splenocytes from mice immunized with H. capsulatum showed a strong response to rHIS-62.

The H antigen is an important antigen used to discriminate active and remote infection. Deepe and Gibbons [63] have demonstrated that the recombinant H antigen (rH) is able to protect C57BL/6 mice immunized with the recombinant protein and challenged with lethal and sublethal inoculum (intranasally) of yeasts of H. capsulatum. Increased production of IFN-γ, and GM-CSF was observed in spleen cells from mice immunized with rH and stimulated in vitro with 25 μg of antigen/ml for 24 h.

Scheckelhoff and Deepe [62] have shown that immunization with recombinant heat shock protein 60 (rHsp60) from H. capsulatum or fragment 3 (F3), a specific region of the protein, conferred protection in mice challenged with the fungus. The experiments demonstrated that rHsp60 causes the expansion of murine T cells that predominantly express the Vβ8.1/82 region TCR and that the elimination of Vβ8.1/82+ cells causes loss of protection efficacy by rHsp60.

Sá-Nunes et al. [64] standardized the production of extract containing cell-free antigens (CFAgs) and observed in vitro that the spleen cells from mice immunized and stimulated with these antigens produced high amounts of IFN-γ and that immunization with CFAgs was able to induce protective immunity, reducing fungal burden, blocking the dissemination of H. capsulatum and protecting infected mice from lethal inoculum.

In 2018, an alkaline extract of H. capsulatum packaged in glucan particles was evaluated in a murine model. The results demonstrated that the alkaline extract in glucan particles was able to protect mice against lethal and sublethal challenges with H. capsulatum yeast cells. Vaccine with alkaline extract induced Th1 and Th17 response in lungs and draining lymph nodes and a small population of IL-17+ IFN-γ CD4+ was detected [65].

In histoplasmosis, it has been observed that B cells are effective in the immune response against the fungus [80]. In 2006, a study showed that depletion of CD4+ and CD8+ T cells in B cells−/− mice, the fungal burden increased considerably when compared with the wild type [80]. In other studies, the efficacy of mAb therapies has been demonstrated [75, 76]. Nosanchuk et al. [75] performed passive administration in mice of mAb to a surface histone-like protein of H. capsulatum (IgM) and observed the reduction of fungal burden in organs and prolonged survival when mice were challenged with lethal inoculum of H. capsulatum. Increased IL-4, IL-6, and IFN-γ in the lungs of treated mice were associated to increased protection. In another study with IgG1 and IgG2a mAb to H. capsulatum HSP60, prolonged survival of mice was obtained when they were challenged with a lethal inoculum of 1.25 × 107 H. capsulatum yeast cells compared with control mice receiving either irrelevant mAb or PBS. An IG2b mAb, however, was not protective. In conclusion, IgG1 and IgG2a mAb to H. capsulatum HSP60 can modify the pathogenesis of murine histoplasmosis [76].

All of the information about the vaccine candidates against histoplasmosis and the main monoclonal antibodies tested is summarized in Tables 1 and 2, respectively.

Blastomycosis

Blastomycosis can be classified as an acute subclinical infection evolving to chronic pneumonia. Although blastomycosis has the primary site in the lung, infection can become widespread, often in immunocompromised individuals, and reach the skin, bone, genitourinary system, and CNS [81].

For a protective immune response, CD4+ T cells play a major role in mediating resistance to fungal infection. CD4+ T cells confer resistance through secretion of T-helper (Th) 1 or Th17 cytokines such as IFN-γ, TNF-α, GM-CSF, and IL-17A, respectively, which activate neutrophils, macrophages, DCs and inflammatory monocytes for fungal clearance. Protective antibodies are produced [82, 83].

Blastomyces adhesin 1 (BAD1) is an avirulent strain created from disruption of the WI-1 gene [66, 67, 84]. Vaccination with BAD1 was able to elicit a protective response in mice vaccinated and infected with B. dermatitidis, increasing the frequency of CD4+αβ T cells and production of Th1 cytokines, mainly TNF-α, and to a lesser extent IFN-γ [85]. In the murine infection model performed with BAD1, it was shown that CD4+ T cells, apparently, are not needed for a protective immunity to B. dermatitidis infection and that CD8+ T cells alone mediate and induce lasting and persistent resistance to the fungus. In B. dermatitidis infection in CD4+ T cell deficient mice, CD8+ T cells were able to produce proinflammatory cytokines (TNF-α, IFN-γ, and GM-CSF) linked to vaccine resistance and crucial in fungal control. Elimination of CD8+ T cells from BAD1 vaccinated (KO) mice during the vaccine expression phase greatly abolished their resistance [86]. The fact that CD4+ T cells are dispensable for the success of immunological vaccination against pulmonary mycosis (blastomycosis and histoplasmosis) is important, since the highest incidence of opportunistic fungal infections occurs in immunocompromised patients including patients with AIDS. Nanjappa et al. [87], using various experimental models of CD4+ T cell deficient mice vaccinated with BAD1 and infected with B. dermatitidis, proved that CD8+ T cells persist in number and function, even without the residual antigen, and that IFN-α producing CD8+ T cells express CXCR3 chemokines receptor, and its blocking prevents the influx of CD8+ T cells into the lungs.

The role of Th17 signaling is controversial in the immune response against fungal infection [88, 89]. To elucidate its role in the protection induced by vaccination with engineered, live, attenuated BAD1 vaccine, Th17 response and cytokine IL-17A were evaluated. The murine experimental model has shown that both response and cytokine may be engaged in vaccination to confer resistance against multiple dimorphic fungi. Neutralization of IL-17A using mAb and vaccination of IL-17−/− mice showed that the cytokine is required for vaccine resistance, even in mice with normal Th1 cells, and that Myd88 adaptor protein is critical in inducing a Th17 response and vaccine immunity. The lack of it, therefore, impaired the induction of the Th17 antigen-specific Th1 response [68].

Studies with Dectin-1, a standard recognition receptor (PRRs) for fungi, involved in the innate Syk kinase signaling pattern [90, 91] and Card9 adaptor [92], critical in inducing the Th17 response to fungal infections, showed that the receptor is also dispensable in the development of BAD1 vaccine resistance and Th17 response induction [68]. This is in contrast to the vaccine-protective response induced against H. capsulatum and C. posadasii where Dectin-1 is required for the development of the Th17 immunogen response [93]. The differentiation of the Th17 immune response in blastomycosis is via Myd88, and Myd88−/− (KO)-mice failed to control yeast vaccinations and were unable to recruit antigen-specific Th17 and Th1 cells to the lung to combat infection [68].

On the other hand, the Card9 adaptor protein is essential for the antifungal Th17 protective response in BAD1 vaccination in B. dermatitidis infection model [69]. Card9 (KO)-mice infected with B. dermatitidis, and unvaccinated, were more susceptible to infection [94]). Card9 is required for differentiation of Th17 cells, governing the expression of instrumental cytokines in the targeting of the Th17 and Th1 protective response. Thus, the Dectin-2/FcRγ/Syk/Card9 signaling pattern is indispensable for the differentiation of Th17 and Th1 cells in resistance acquired infection by B. dermatitidis [69].

The BAD1 vaccine model also evaluated the use of IL-1β cytokine as an adjuvant in the murine model of blastomycosis infection. IL-1β is a proinflammatory cytokine that enhances the expansion and differentiation of CD4+ and CD8+ T cells [95, 96]. IL-1β as an adjuvant in BAD1 vaccination owes its protective effect to the decreased CFU of mice vaccinated with BAD1 and infected with B. dermatitidis; to the frequency and number of IL-17 producing cells. The use of the cytokine in the vaccination helped to promote differentiation of Th17 antigen-specific and reduced the generation of Th1 and Th2 antigen-specific cells [97].

In search for a protective immune response against infection by B. dermatitis, a 120-kDa surface protein was identified, named WI-1 [66]. It is an adhesin that binds the fungus to complement and an immunodominant antigen recognized by infected patients [98, 99]. The immunization with WI-1 elicits immune response in a murine model and enhances resistance to pulmonary infection, with increased IFN-γ and decreased CFU, while eliciting an IgG humoral response [77, 100]. Utilization of monoclonal antibodies to WI-1, in a study of five mAb (BD6-BC4, DD5-AD3, DD5-AD9, DD5-AD11, DD5-AE5, DD5-CB4, CA5-AA3, and CA5-BC12), no improvement in the immune response was observed against B. dermatitidis.

All of the information about the vaccine candidates tested against systemic mycoses and the main monoclonal antibodies tested against blastomycosis are summarized in Tables 1 and 2, respectively.

Coccidioidomycosis

Coccidioidomycosis, commonly known as San Joaquin Valley fever or Valley fever, is a fungal infection with high morbidity and potential mortality affecting persons in endemic areas [101]. Dimorphic etiologic agents, Coccidioides immitis and Coccidioides posadasii, are morphologically identical and their predicted proteins are more than 90% homologous [102]. The fungal mycelium can produce arthroconidia (spores) by hyphal segmentation in the soil, which are aerosolized and capable of causing infection by inhalation. The inhaled spores first convert to spherule initial forms, which grow in the lungs to multinucleate spherules [101, 103]. The vast majority of infections are mild and self-limited, usually leaving no residual damage greater than a pulmonary granuloma [104]. In contrast, a small percentage of infections do not resolve spontaneously, giving rise to chronic cavitary pulmonary infections or extrapulmonary infection foci, with high antibody titers. The higher these titers, the worse the prognosis of the infection [93].

Vaccine-induced immunity against coccidioidomycosis seems feasible because symptomatic second infections with Coccidioides spp. are extremely rare [105]. Successful vaccination requires a T cell-mediated immune response with both Th1 and Th17 playing a role in the vaccine-mediated protection [106]. There is no evidence that antibody response is protective in humans as there is no association between inherited or acquired immunoglobulin deficiencies and disseminated coccidioidomycosis, but in mouse models, there is conflicting evidence whether B cells are needed for vaccine-induced immunity [107, 108].

The proline-rich antigen (PRA) of Coccidioides immitis was studied as a vaccine candidate for coccidioidomycosis [70]. It is a glycopeptide component of 194-aa [109]. Using recombinant PRA (rAg2/PRA) conjugated with monophosphoryl lipid A (MPL) adjuvant in prophylactic immunization, a reduction in the fungal load of infected lungs was obtained, with the production of high levels of IFN-γ in splenocytes of immunized mice re-stimulated with rPRA [70].

An attenuated lineage of Coccidioides posadasii was also used as a vaccine candidate for coccidioidomycosis. The lineage used was deficient in chitinases involved in the stage of differentiation of the endospore in the parasitic life cycle. Studies with live and attenuated lineage (cts2/ard1/cts3Δ strain) designated ΔT, showed that it could protect BALB/c mice susceptible to lethal infection with C. posadasii. Infected and non-vaccinated C57Bl/6 mice succumbed to infection 3 weeks after challenge, whereas another group vaccinated with the C. posadasii mutant had 100% survival 75 days post-challenge. The vaccine had fewer reatogenic effects than the previous formulation using formalin killed beads (FKS). The live and attenuated line used in the immunization tests elicited Th1 cytokines, with activation of CD4+ T cells and secretion of IFN-γ and IL-12p70 and marked decrease in the production of IL-6, a cytokine correlated with disseminated coccidioidomycosis [71].

The vaccine decreases proinflammatory cytokine levels (IL-1β, IL-1α, TNF-α, IL-6, and G-CSF) and also the number of neutrophils in the pulmonary infiltrate. The number of tissue and alveolar macrophages increased in the protected animals as also did that of CD4+ and CD8+ T cells in the pulmonary infiltrate of vaccinated mice. Chemo-attractants of macrophages, monocytes, and dendritic cells (CCL2/MCP-1 and CCL3/MPI-1α) were also found in high concentrations in the lungs. These data imply that an IL-17 signaling pattern contributes to initiation as well as to control the inflammatory response in Coccidioides infection since this cytokine controls neutrophil expansion via G-CSF and CXCL1 and induces CCL2/MCP-1 production as well as CCL3/MIP-1α. Vaccination also showed a population of CD8+ IFN-γ+ T cells in the lungs of immunized animals and that these cells can act synergistically with CD4+ T cells [106]. The importance of Th17 signaling was observed in the lung homogenate of mice vaccinated with ΔT. In fact, the period post-challenge showed a predominance of Th2 and Th17 cytokines, followed by a decrease in Th2 levels and a progressive increase in Th1 levels, with the end of Th17 predominance. The use of knockout mice helped to elucidate this issue: vaccinated IL-17−/− mice were partially protected against lethal lung infection by Coccidioides, while IL-4−/− and IFN-γ−/− mice were not protected, confirming that the vaccine should induce Th17 signaling pattern to be effective [106].

In search for a durable immune response, the ΔT vaccine was conjugated to the EP67 adjuvant, a biologically active agonist of the C-terminal region of the human complement, C5a. The decapeptide adjuvant induces release of Th1 cytokines from APCs and enhances processing and antigen presentation by DCs in humans [72]. The vaccine-EP67 conjugation showed small inflammatory infiltrate in the lung when compared with vaccine alone. Conjugation increased the expression of MHC class II molecules on the cell surface, especially of DCs, when compared with animals vaccinated only with ΔT, together with increased expression of costimulatory molecules (CD80/86 and CD40). In vitro tests with splenocytes from mice vaccinated with the conjugate and recall with Coccidiodes showed a significant increase of IL-2 and IFN-γ in the supernatant, when compared with splenocytes from animals vaccinated only with the ΔT vaccine. There was also high T-bet expression in the mice vaccinated with the conjugate, showing promotion of differentiation of the Th1 response and dominant CD4+ IL-17+ [110].

Reactive T cell recombinant antigens eliciting protective and durable immune response against lethal Coccidioides infection have been studied for a vaccine. The multicomponent extract of the C. posadasii cell wall was used in an attempt to identify these antigens. The C. posadasii spherules wall isolate was used as a vaccine, conjugated with the ODN CpG adjuvant. A robust protective response in C57Bl/6 mice was elicited against C. posadasii lethal lung challenge resulting in 90% of survivors 50 days after the challenge. An amino acid sequence of a polypeptide revealed similarity with aspartyl protease from other filamentous fungi and was termed Pep1 [73]). ELISPOT analysis of CD90+ T cells from the splenocytes of C57BL/6 mice and HLA-DR4 transgenic mice vaccinated with recombinant rPep1 and recall with rPep1, produced high levels of IFN-γ, a further evidence that the antigen stimulates a potent immune response for Th1 signaling. Most of the surviving mice had their lungs pathogen-cleared 90 days after challenge, indicating that the cellular immune response is durable [111].

Other proteins from the parasitic cell seemed also to have been revealed among them the putative proteins phospholipase B (Plb) and alpha-mannosidase (Amn1). Vaccination of the C57BL/6 mice with the multivalent recombinant Pep1, Plb, and Amn1 conjugated to ODN CpG resulted in enhanced survival of the mice infected with C. posadasii and showed improved clearance of the pathogen in this murine coccidioidomycosis model [112].

Using a computational analysis of the primary structure of each of the three T cell reactive proteins, a recombinant epitope-based vaccine (rEBV) was created. The vaccine was consisted of Pep-1-P1, Pep2-P2, Amn1-P10, Amn1-P11, and Plb-P6, five epitopes based on their affinity for HLA-DR4 molecules. Assays using splenocytes from mice vaccinated with rEBV and ODN CpG and infected, when a recall with rEBV was made, produced high levels of IL-2 and IFN-γ, Th1 cytokines, and high expression of IL-17A indicating the activation of Th17 signaling. The protective immune response was observed by the clonal expansion of Th1 (CD4+IFN-γ+) and Th17A (CD4+IL-17A+) cells in the lungs of mice, 9 days after the challenge and reduction of the CFU of mice vaccinated with rEBV and CpG ODN [74].

Glucan particles (GP) were used as an adjuvant to rEBV vaccine in the search for a protective and long-lasting immune response against coccidioidomycosis. Glucan particles, prepared from yeast cell wall (Saccharomyces cerevisiae) rich in β-1,3-glucan were effective as an adjuvant and efficient delivery agent [113]. These particles are hollow, small, can be filled with protein antigen and can be easily captured. Because they are rich in β-glucans they are recognized by Dectin-1, a receptor expressed on the surface of host phagocytes as DCs, macrophages, and neutrophils and induce cytokines involved in Th17 signaling pattern [113,114,115]. High levels of IL-17A and IFN-γ were observed in splenocyte cultures of mice vaccinated with rEBV plus GP plus OVA, increased number of CD4+ IFN-γ+ and CD4+ IL-17A+ T cells in the lungs of mice vaccinated with rEBV plus GP plus OVA and infected as compared with mice vaccinated with rEBV and ODN CpG. Better results were also obtained for fungal clearance, histopathology and animal survival [74].

All of the information discussed above is summarized in Table 1.

Final Remarks

The development of prophylactic and/or therapeutic vaccines faces many difficulties. In addition to several limitations of antifungal treatment, the major challenge is to find a vaccine that can induce an effective immune response in immunosuppressed patients. The emergence of strains resistant to antifungal drugs and the increase of immunosuppressed populations aggravate this situation. Over the last few years, many researchers have struggled to find vaccines that may reduce the morbidity and mortality of patients with fungal infections, but so far, few prototypes of successful treatment have emerged from clinical trials. Accumulated knowledge, however, on the pathogen-host relationship and the understanding of the immune response to these microorganisms leave us closer to this goal.

One of the major challenges for the development of vaccines against fungal infections is that these microorganisms mostly affect immunocompromised individuals. Generally, CD4+ T cells are essential to mediate protection against systemic mycoses, but patients deficient in these cells do not respond efficiently to antifungal treatment. Alternatively, CD8+ T cells are a promising strategy, since the protective immunity mediated by these cells has already been documented against different fungal infections, among them PCM, histoplasmosis and blastomycosis.

Another experimentally tested procedure is the use of vaccines in conjunction with antifungal therapy aiming at decreasing treatment toxicity and therapy period. As discussed previously, the prolonged time of antifungal therapy against systemic mycoses may lead to serious relapses, with great impact on health care. Studies focusing on the use of therapeutic vaccines associated with antifungal agents are, therefore, to be highlighted.

Finally, since many fungal infections are still largely neglected, development of therapeutic vaccines has to overcome scientific and health-policy obstacles to assure convenient financial investment allowing for progress in this area.