The incidence of diseases caused by pathogenic and opportunistic fungi
has been increasing in the past years. The development of new immunological tools to integrate or replace non-effective and less-effective therapies is becoming more urgent nowadays.
The treatment of PCM is based on the prescription of sulfonamides, polyenes, (amphotericin B) and azoles derivates. However, this treatment is extremely dependent on the drugs’ availability and infection severity. PCM requires a long-term treatment and it is limited by toxicity
of substances being used and their high cost. Moreover, resistant strains have already been reported [3
]. It is important to highlight that the use of drugs does not guarantee the complete elimination of the fungus, which may result on reactivation of quiescent foci [5
There are some reports describing the role of immunogenic antigens on the induction of immune responses to fungal infections, particularly PCM. Studies with yeast cells of P. brasiliensis
, attenuated by gamma irradiation (LevRad), have been providing promising results. These yeast cells may promote a higher protection when used as a preventive vaccine, against PCM [18
]. There are several examples of highly effective vaccines derived from live attenuated infectious
agents (mainly viruses), such as, polio, measles, mumps, rubella, varicella, influenza and rotavirus [29
]. For bacteria, the live attenuated Bacille Calmette-Guérin (BCG) vaccine for tuberculosis, is widely used and remained the gold standard despite efforts for the development of other vaccine formulations for this disease [30
]. However, reports of vaccines preventing medically important fungal infections in humans are scarce. The only known report is related to formaldehyde-killed Coccidioides immitis
spherules, which were tested against coccidioidomycosis [31
]. However, the use of this vaccine was not possible, since wrong dosages caused toxic manifestations [32
]. A vaccine based on attenuated fungi against ringworm caused by Trichophyton verrucosum
has been successfully used in veterinary medicine [33
]. A recombinant attenuated strain of Blatomyces dermatitidis
has been promising as a potential vaccine against blastomycosis experimental [34
]. Currently, the safety, tolerability and immunogenicity
of this live-attenuated vaccine have been tested in dogs [36
The high complexity of eukaryotic systems, associated with the high degree of variability within the P. brasiliensis
group, may pose limitations to the success of vaccines composed by single antigens. This limitation is mainly due to the fact that only few fungi have a single immunodominant
epitope possible to be targeted. Some vaccines may only reach considerable effectiveness through the combination of defined antigens promoting a coordinated interaction of many constituents involved in the host’s immune response. In this context, the LevRad therapy could be a promising candidate against PCM or an experimental model for the development of new approaches concerning the prevention or treatment of other important fungal infections. It is usual that the living agent induces strong, broad response involving multiple arms of the immune response, which provides natural immunity to disease [6
] without the risk of a progressive infection. Although, it is important that the mechanisms which promote the attenuation of fungi are stable.
Previous studies have already reported that radioattenuated fungi suffer an extensive DNA fragmentation, beyond cell repairment mechanisms. This leads to an irreversible loss of yeast reproductive ability and virulence [16
]. The inability of LevRad to cause infection was confirmed in this study using immunocompromised (BALB/c*) and immunocompetent
(BALB/c) mice. No CFU or histopathological alterations were observed, 30 and 90 days after inoculation with LevRad, in the organs of the studied animals. Athymic mice (Nude) achieved 100% of mortality at the fifth day of experiment, while Nude mice inoculated with LevRad remained alive for 30 days.
Combined therapy of immunotherapeutic and antifungal agents has been tested as an adjuvant in potentializing the chemotherapy. This is an attempt to reduce toxicity of conventional
drugs, the period of treatment and to prevent relapses of the disease [5
]. Here we reported, for the first time, the immunotherapeutic potential of radioattenuated fungi associated with fluconazole in the treatment of PCM.
The immunization of infected BALB/c mice with LevRad associated with fluconazole (InRadMed) elicited a great therapeutic effect against highly infective yeast strains of P. brasiliensis
. A reduction of CFU in the lungs from the InRadMed group in relation to animals only infected (C+) was already observed 60 days after infection. There was no fungal dissemination to the liver or the spleen. No fungal colonies were recovered from any organs (lung, liver and spleen) at 120 days post infection. It is safe to report that the combined administration of LevRad and fluconazole
was capable of preventing relapse effects in this study. This was also confirmed by the histopathological analysis. Such analysis showed that the organs (lung, liver and spleen) preserved lung parenchyma as well as did not present yeast cells or granulomatous lesions. It is already well documented that resistance to P. brasiliensis
infection is determined mainly by the host’s ability to restrict fungal dissemination rather than the control of fungal growth in the primary infection site [37
]. Moreover, it is important to consider the elimination of possible fungal quiescent focus as demonstrated 120 days after infection. The fungal cells may establish a dormant state and spontaneously reactivate. This reactivation may also occur when the host`s immune system is impaired.
At the same time, the groups of infected animals immunized with LevRad (InRad) and treated with fluconazole (InMed) presented a significant reduction in the number of CFU, when compared to C+. This reduction was not observed between the InRad and the InMed groups though. The lungs obtained from the InMed and InRad groups presented organized well discrete focal neutrofilic and linfohistiocytic granulomatous
lesions with a few number of yeast cells. Immunization of infected animals with LevRad was effective, although at first it did not present benefits when compared with fluconazole treatment. However, it is important to highlight that the infected animals were immunized only twice with LevRad (no adjuvants), with a fortnight interval between events. On the other hand, fluconazole was administered for 30 days with no interruptions. Besides, animals from the InRad group were first evaluated 15 days after the medication was suspended whereas the animals from the InMed were evaluated immediately after the end of treatment.
The infected mice immunized with LevRad presented early expression (60 days after infection) of high TNF-α transcript levels in relation to the other groups (C+, InMed and InRadMed) which were not enough to ensure the fungi
elimination. Moreover, it is plausible to believe that IL-4 may has contributed to the colonization and reproduction of the fungus in the lung, as well as its dissemination to the other organs (liver and spleen) as observed at 60 days post infection in InRad, InMed and C+. In contrast, we observed at 120 days after infection high transcript levels of IFN-γ and iNOs enzyme especially in the InRad and InRadMed groups, with low IL-10 and IL-4 levels. This observation occurred concomitant with a complete resolution
of the disease in InRadMed (120 days after infection). A significant decrease in the number of CFU and histopathological lesions was also observed in InMed and InRad. It is well known that IFN-γ plays an important role in resistance to P. brasiliensis infection due to increase in the clearance of fungal cells, its regulation by cell-mediated immune responses and its regulatory effects on specific humoral immune responses [38
]. NO may be considered a potent microbicide factor and its secretion is related with the fungus elimination and consequent host protection [39
]. Regarding TGF-β, the high expression verified at 120 days post infection may be due to the regulation of inflammatory response induced by proinflamatory cytokines [7
In the present study, we also evaluated the specific antibody levels. High levels of IgG in the InMed, InRad and InRadMed groups were observed due to a response to the presence of the exoantigen of Cryptococcus neoformans
(Mexo). This suggests that this antigen may also contain B cell epitopes
. A significant production of IgG1 was observed in all groups (C+, InMed, InRad and InRadMed) from 60 to 120 days of infection. High production of IgG2a, IgG2b and IgG3 in the InRad group was registered, as well as of IgG2b and IgG3 in the InRadMed group, after 60 days of infection. At 120 days, IgG2a and IgG2b levels were high in the InRad, InMed, InRadMed and C+ groups. However, IgG2a titers were higher in infected mice (immunized and treated) than in the C+ group. Protection against PCM has been attributed to a vigorous cellular immune response especially associated with Th1, whereas the high IgG levels and depressed cellular immune response was associated with progressive disease. Recently, however, some evidences have demonstrated the antibody-mediated protection against pathogenic fungi, such as antibodies to Cryptococcus neoformans
polysaccharides or to Candida albicans
cell wall components [44
] as well as anti-gp70 Mabs [47
] and some anti-gp43 MAbs [48
] in experimental PCM. Besides, some IgG isotypes are associated with a Th1 response profile as well as works effectively on opsonization and activation of the complement system. IFN-γ is considered the major inducer of a switch to IgG2a secretion
, whereas IL-4 has been associated with isotypes switching to IgG1 and TGF-β functions as an important Ig2b switch factor [11
The high expression of IFN-γ, iNOS and TGF-β, and low expression of IL-4 and IL-10 (observed 120 days after infection) associated with high production of IgG2a and IgG2b, as well as, the low fungal load, the preserved tissues and low mortality
rates suggest the predominant establishment of Th1-type immune response observed in InRadMed.
Finally, the results obtained in the present study provide important information concerning fungal immunotherapy demonstrating, by using yeast attenuated cells, that it is achievable for PCM. It was observed an additive effect of LevRad immunization to chemotherapy in PCM experimental that could be relevant mainly for studies dealing with drug resistance