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Immunization and dermatophytes

Bernard Mignona, Jeremy Tabarta, Aline Baldoa, Anne Mathya, Bertrand Lossona
and Sandy Vermoutb
a
Department of Infectious and Parasitic Diseases, Purpose of review
Parasitology, Faculty of Veterinary Medicine, University
of Lie`ge, Lie`ge and bFederal Agency for Medicinal and
Despite the availability of effective vaccines for certain animal species, vaccination
Health Products, Brussels, Belgium against dermatophytosis requires improvement and further development in both animals
Correspondence to Bernard Mignon, Universite de and humans. This review provides an update on the current situation and focuses on
Lie`ge, Faculte de Medecine Veterinaire, Departement recent advances in hostdermatophyte relationships that could have implications for
des Maladies Infectieuses et Parasitaires, section
Parasitologie, Boulevard de Colonster 20, 4000 Lie`ge, future vaccination against the most prevalent of the fungal diseases.
Belgium Recent findings
Tel: +32 4 366 40 99; fax: +32 4 366 40 97;
e-mail: bmignon@ulg.ac.be Numerous dermatophytic virulence factors have recently been isolated and
characterized at the molecular level, notably secreted proteases involved in the invasion
of the keratin network. Their precise roles in the different steps of the infectious process
Current Opinion in Infectious Diseases 2008,
21:134140 and in immunopathogenesis are being studied, while all aspects of the host immune
response against dermatophytes, including the innate response, are becoming
increasingly documented. In addition, new molecular tools are now available for
studying dermatophytes, which will accelerate research on this topic.
Summary
The growth of knowledge concerning all aspects of the hostdermatophyte relationship
should contribute towards sound strategies for the development of effective and safe
vaccines against dermatophytosis.

Keywords
dermatophyte, immune response, protease, vaccine, virulence factor

Curr Opin Infect Dis 21:134140


2008 Wolters Kluwer Health | Lippincott Williams & Wilkins
0951-7375

nogens and virulence factors. Also, in the wake of recent


Introduction discoveries in immunology, including the aspects of
Dermatophytosis is a common and clinically multifaceted immunity to opportunistic fungal infections [1,2], scat-
fungal skin disease affecting both humans and animals. tered but interesting progress in the understanding of the
Usually, infection is limited to the stratum corneum, hair immune response to dermatophytes has been achieved.
or nails. The responsible agents are pathogenic derma-
tophytes, a group of highly specialized filamentous fungi This review intends to summarize and comment on the
which are able to invade and digest keratin in vivo. most recent literature, addressing hostdermatophyte
Despite their superficial localization in skin, they induce relationships and focusing mainly on vaccination, fungal
in their hosts a specific immune response, which can virulence factors, their putative involvement in both the
result, though not automatically, in subsequent protec- innate and specific host immune responses and potential
tion against reinfection. Besides, the use of live attenu- implications for modern and safe vaccination against
ated vaccines has proved to be a valuable mean of control dermatophytosis.
and even eradication of dermatophytosis in cattle and
fur-bearing animals. The existence of efficient and com-
mercially available vaccines against dermatophytosis is Vaccination against dermatophytosis
unique in the field of medical and veterinary mycology. To date, there is no commercial or standardized vaccine
Indeed, though being the most prevalent fungal diseases, for preventing or eliminating any fungal infection in
dermatophytoses remain considerably less studied than humans, including dermatophytosis. There is, however,
opportunistic mycoses such as aspergillosis and candidia- intensive research aiming to develop safe and efficient
sis, including immuno-pathological aspects [1]. During antifungal vaccines, but essentially against opportuni-
the past decade, however, there has been a resurgence of stic fungal infections affecting immuno-compromised
interest in dermatophytes and dermatophytosis leading patients [3]. In contrast to these infections, dermato-
notably to the molecular characterization of fungal immu- phytoses are not life-threatening and occur most often in
0951-7375 2008 Wolters Kluwer Health | Lippincott Williams & Wilkins

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Immunization and dermatophytes Mignon et al. 135

immuno-competent individuals. Except in the case of medicine. In fact, some inactivated vaccines are mar-
geophilic species (e.g. Microsporum gypseum), dermato- keted not only as a preventive but also as a treatment. We
phytes must be considered as obligatory parasitic fungi recently performed a random placebo-controlled double-
because they do not reproduce nor produce macroconidia blinded study to evaluate the curative efficacy of a
and microconidia in the environment, and do not form commercial inactivated vaccine against T. verrucosum in
part of the normal cutaneous flora. Because of this intrin- 17 naturally infected heifers. Both clinical and mycologi-
sic characteristic, the development of a useful vaccine cal follow-up was performed every 2 weeks for 5 months.
against human dermatophytosis could imply a more clas- There was no significant difference between vaccinated
sical approach than against opportunistic mycoses. This and control animals, the only significant factor affecting
view must, however, be tempered by the fact that some the time-course of the disease being time (B. Losson,
dermatophytes are so highly adapted to their hosts that personal communication). These results corroborate the
they may cause chronic, and even asymptomatic, infec- overall disappointing observations of many practitioners
tions, as sometimes observed for Trichophyton rubrum in on the global inefficacy of inactivated vaccines against
humans and Microsporum canis in cats. These mycoses dermatophytosis in cattle and horses.
result from fungal immunomodulation or the existence of
poor Th1 responders [4,5]. Efficient vaccines for cats are to be developed
Several well conducted studies were performed in the
Some live vaccines are effective in particular animal target species by DeBoer and Moriellos team using
species either an experimental cell wall vaccine [7] or a commer-
The need for a vaccine against human dermatophytosis cially available inactivated crude product of M. canis [8].
remains to be evaluated medically and economically. This These attempts were largely unsuccessful. Interestingly,
is not the case in animals. Indeed, both the high incidence their immunization protocols induced no, or a weak
of this contagious skin disease and its zoonotic and specific cell-mediated immune response, compared with
economic effects have rendered vaccination highly desir- that induced in self-curing natural infections. These
able, especially in cattle and cats. To be efficient, vaccina- results were interpreted by the authors as indicative of
tion must prevent the development of clinical lesions and the need to develop a subunit vaccine, with clearly
the transmission to other subjects and avoid contamination defined composition and immunologic properties, and
of the environment by infective fungal elements. In this affording a high safety degree. In this context, our lab
respect, a remarkable success was obtained in cattle, using produced a M. canis keratinolytic exo-antigen, which
a live vaccine containing microconidia of an attenuated proved to be immunogenic both in naturally infected
strain of Trichophyton verrucosum [6]. Large-scale vaccina- cats and experimentally infected guinea pigs. Moreover,
tion programmes carried out in Eastern Europe and in this model, the exo-antigen induced a specific DTH in
Scandinavia in the past 35 years using this vaccine have animals that had recovered from infection [9]. Sub-
dramatically reduced the incidence of both bovine ring- sequently the genes coding for two major proteases of
worm and concurrent human infections. Protection was the exo-antigen a keratinolytic subtilisin (Sub3) [10]
correlated with the induction of an immune response and a keratinolytic metalloprotease (Mep3) [11] were
comparable with that induced by natural infections, which isolated and recombinant proteins were produced. Exper-
is a cell-mediated immune response. In fact a Th1 imentally infected guinea pigs were then shown to
response, as demonstrated by a delayed-type hypersensi- develop a lymphoproliferative immune response against
tivity (DTH) skin reaction, is induced both in naturally both recombinant Sub3 [12] and Mep3 [13]. Con-
and vaccinated animals. These results prove that vaccina- sequently, the protective properties of these immuno-
tion against dermatophytosis is a realistic strategy for gens mixed with a Freunds adjuvant were evaluated in
preventing dermatophytosis. This success story cannot this species. Although each tested immunogen elicited a
obviate the lack of knowledge about basic immunology significant specific cell-mediated immune response, the
of dermatophytosis, especially in comparison with the latter was transient, which could account for the absence
constantly increasing level of understanding of immune of protection conferred by the vaccines against an
mechanisms involved in other fungal infections [1]. epicutaneous challenge [14,15]. In this respect, new
adjuvants should be used to stimulate a strong and
The efficacy of inactivated vaccines is questionable long-lasting Th1 response. The route of administration
Inactivated vaccines have also been developed and are should also be reconsidered. Indeed, not only the nature
currently marketed for use notably in cattle, horses and and the concentration of the immunogen itself, but also
cats. In cattle and horses, data concerning their efficacy the molecular and cellular context in which it is presented
and the duration of the induced immune response are not to the dendritic cells are instrumental to generate an
firmly established. One of their theoretical advantages appropriate Th1 cell immune response inducing active
over live vaccines is their potential use in already dis- cellular effectors, macrophages and neutrophils, on
eased animals, which is particularly relevant in veterinary the infection site [1]. Nevertheless, in view of further

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136 Skin and soft tissue infections

experiments, it must be remembered that the in-vivo usefulness cannot be ruled out. Indeed, in some models
experimental vaccine assays are more relevant when of other fungal infections, such as disseminated candi-
performed in the target species, the cat for M. canis. diasis, protection was shown to be elicited by the pro-
Furthermore, to mimic as much as possible a natural duction of specific antibodies, while experiments using
challenge, infection commingling should be considered. other models of the same disease pointed out the Th1
cell-mediated immune response to be the critical arm of
protection [16]. In fact, there is increasing evidence that
Immunity to dermatophytosis both protective and non-protective inhibiting/blocking
Even if available data are rather limited, the most recent antifungal antibodies coexist [17], and that protection can
literature on active immunization against dermatophytosis be conferred by the induction of an appropriate antibody
shows that there is a trend to develop safer and more response or by the use of specific monoclonal antibodies
efficient vaccines than those, attenuated or inactivated, (mAbs) [3]. This is exemplified by the recent develop-
belonging to the first generation. Although live vaccines ment of an original glyco-conjugate vaccine, based on a
are efficient against some dermatophytes in particular single b-glucan of algal origin, which was protective
animal species, they are proscribed for use in humans or against both candidiasis and aspergillosis in rodents,
in contact pet animals notably because of a possible through the induction of anti-b-glucan antibodies [18].
reversion to virulence. As a consequence, scientists essen- The importance of protective antibodies against specific
tially focus their studies on isolation, characterization and fungal antigens, including secreted proteases known as
subsequent use of major fungal immunogens able to elicit a virulence factors, was also demonstrated in a model of
specific Th1 response and to stimulate strong memory vaginal candidiasis in rats [19]. This study showed
T-cell responses. This requires the elucidation of the notably that genetically engineered antibody variable
innate and specific immune pathways that are associated domains against the secreted aspartyl protease Sap2, an
both with the various clinical manifestations resulting from enzyme which is involved in adherence to and invasion of
infection of a particular host by a particular dermatophyte the host tissues, had protective effects by inhibiting yeast
species and with protective vaccination. Additionally, the adherence to vaginal epithelial cells. In dermatophytosis,
identification of the fungal virulence factors and the under- further studies should also be performed to assess if
standing of their role in the first steps of infection, adhesion naturally or vaccine induced antibodies can be protective.
and invasion of epidermis, constitute, in our opinion, an As dermatophytes invade the most superficial layers of
important facet, from a fungal perspective, of the immu- the skin, their secreted antigens, such as keratinolytic
nopathogenesis of dermatophytosis. proteases [20], could be the most important targets of the
host immune system. Besides, M. canis infection in cats or
A Th1 cell immune response is needed for protection guinea pigs induces antibodies against the proteases
In the past decades, numerous studies have shown that Mep3 [13] and, to a lesser extent Sub3 [21]. Interestingly,
infections by dermatophytes induce a specific immune mAbs against Sub3 significantly decreased the adherence
response with humoral and cellular components, and that of M. canis arthroconidia (A. Baldo, unpublished data) in a
the efficient and protective response is a cell-mediated model of feline dermatophytosis using a recently devel-
response of the DTH type, characterized mainly by the oped in-vitro reconstructed feline epidermis (RFE) [22].
action of macrophages and neutrophils as effectors, and Though preliminary, these studies indicate that the role
thus by elevated activity of the key cytokines IL-12 and of antibodies in dermatophytosis must be revisited, as
IFN-g from the Th1 axis. The immune response that is this is progressively performed in other fungal infections.
raised, however, including the degree of inflammation,
varies according to the dermatophyte species, the host A pivotal role for innate immunity in dermatophytosis
species, and the physiopathological status of the host. During recent years, research about innate antifungal
Chronic and recurrent infections with T. rubrum and immunity was very productive leading to important dis-
M. canis are quite common in humans and cats respect- coveries, again concerning mainly opportunistic infec-
ively, without any associated immunodeficiency. Most tions [23,24]. A comprehensive overview of the innate
often, chronic human infections are correlated with poor immune mechanisms involved in the different clinical
specific DTH, elevated specific IgE and IgG4, and IgE- presentations of dermatophytosis would, however, be
mediated immediate hypersensitivity. Clinical and exper- instrumental, notably because the receptors, the path-
imental arguments supporting this consensual view about ways and the cells which are involved have an instructive
the duality of the immune response to dermatophytes have role on cells of the adaptive immune system, and thus on
been previously reviewed and discussed [4,5]. the induction or not of a Th1 cell mediated protective
response [1,23]. It is largely beyond the scope of this
A role for humoral immunity? brief review to give details about general or specific
Even if the role for specific antibodies in host defence concepts of innate immunity. Some recent studies, how-
against dermatophytes is questionable, their potential ever, dealing with this topic and dermatophytosis are

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Immunization and dermatophytes Mignon et al. 137

mentioned below as they provide some insights, which including Microsporum andouinii and T. rubrum, and to
may be pieces of the puzzle to be solved for this field. bind preferentially to hyphae rather than conidia, leading
in fine to the upregulated secretion of proinflammatory
Keratinocytes are the first epidermal cells that the der- cytokines such as TNF-a [31]. In contrast, upon pha-
matophytes encounter during the infection process. gocytosis by macrophages, T. rubrum conidia induce the
These cells have a pivotal role in the modulation of secretion of the anti-inflammatory cytokine IL-10, while
the host immune response [25]. Indeed, upon exposure other factors related to enhancement of a relevant pro-
to dermatophytes they can produce a broad spectrum of tective immune response are downregulated: class II
cytokines [26], including IL-8, a potent chemo-attractant major histocompatibility complex, CD54 and CD80
for neutrophils, which can kill dermatophytes, and the co-stimulation molecules, nitric oxide and IL-12 [32].
proinflammatory TNF-a [27]. Dermatophyte species Campos et al. [32] also showed that phagocytosis of
differ, however, in their ability to induce cytokine pro- T. rubrum conidia by macrophages was inhibited by both
duction by keratinocytes. The zoophilic species stimu- cell wall mannans and exo-antigen. This is in agreement
late the production of pro-inflammatory cytokines by with previously reported postulates stating that the cell
human keratinocytes better than anthropophilic ones wall mannans possess immuno-inhibitory properties
[26,28], and this correlates with the clinical aspects potentially responsible for T. rubrum chronic dermato-
observed in both types of infection. Such a dichotomic phytosis in humans. In fact, it appears that some immu-
view, however, is probably too simplistic, as we observed nomodulation depends on which dermatophyte factors
that feline keratinocytes, which are exposed to live or are produced in the course of infection, but also how they
heat-inactivated arthroconidia of a single dermatophyte are detected by the host. As an example, a yeast cell wall
strain (M. canis IHEM 21239) produce different cytokinic derivative, zymosan, considered to be an inducer of pro-
responses (J. Tabart, personal communication). In fact, inflammatory cytokines, was recently demonstrated to
we have no idea yet about the dermatophyte components induce regulatory dendritic cells and immunological tol-
that interact with keratinocytes, nor the cellular receptors erance through a TLR2 and dectin-1 mediated IL-10
involved. This should be investigated both because release [33]. The relevance of these regulatory mech-
keratinocytes are known to express several types of anisms in vivo is strengthened by the recent demon-
pattern recognition receptors (PRRs), including Toll-like stration that, in a mouse model of systemic candidiasis,
receptors (TLRs) and mannose receptors, and because TLR2 has a direct role in the control of immune
the dermatophytic virulence factors are currently being responses, and also the issues of infections, through
characterized at the molecular level. It should be remem- the control of expansion and function of regulatory
bered that in most dermatophyte infections, the fungal T cells [34].
elements are confined to the cornified skin layers. Con-
sequently, though living keratinocytes are used in most Besides keratinocytes and dendritic cells, neutrophils are
in-vitro experiments, they are in contact in vivo with important cells in innate antidermatophyte immunity. In
secreted fungal antigens rather than structural ones. This mice, they were shown to accumulate as early as 46 h
is the reason why we are currently studying the role of after experimental infection with T. mentagrophytes [35],
keratinocytes in the induction of the anti-M. canis after adherence of conidia to the corneocytes, during the
immune response using a fully differentiated, horny layer germination phase. Additionally, it is generally admitted
containing RFE [22] and the available recombinant that neutrophils are, in addition to macrophages, the
secreted fungal proteases such as Sub3 and Mep3 effector cells allowing in fine, via the Th1-dependent
[13,14]. Besides cytokines, human keratinocytes also inflammatory response, the elimination of a dermatophy-
secrete antimicrobial peptides (AMP), cathelicidins and tic infection. The role of neutrophils in Th1 cell acti-
defensins with potential antifungal activity. Their role in vation through several mechanisms including dendritic
skin defence against dermatophytes was suggested by cells instruction [36,37], however, remains to be
several authors who showed that human b-defensin [29] addressed in dermatophytosis. Such a role for neutrophils
and cathelicidin LL-37 [30] are either fungistatic or has been demonstrated in oral candidiasis, another super-
fungicidal in vitro against T. rubrum and that their expres- ficial fungal infection [38].
sion is upregulated in vivo in tinea corporis.

There is no doubt that dendritic cells, especially epider- Virulence factors of dermatophytes
mal Langerhans cells, are antigen-presenting cells As mentioned above, the characterization of fungal
that are instrumental for both initiation and modulation pathogenic factors has been one of the main fields of
of the adaptative antidermatophyte immune response. research on dermatophytes during the last 5 years. It
Recently, dectin-2, a C-type lectin-like receptor consti- undoubtedly constitutes an important topic to further
tutively expressed by mature dendritic cells, such as study the immuno-pathogenesis of dermatophytosis and
Langerhans cells, was shown to be a PRR for fungi, to develop prophylactic strategies. Indeed, virulence

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138 Skin and soft tissue infections

factors can be expressed all along the infection process to in-vitro infection models, such as RFE for M. canis
modulate the host immune response and therefore can be dermatophytosis [22], are also needed, especially as they
important immunogens. Structural antigens of dermato- will allow the evaluation of the role of the putative fungal
phytes, including cell wall glycoproteins, remain poorly virulence factors on the innate host immune system. As a
or even not characterized. Most efforts have focused result, new prophylactic strategies for the control of
primarily on secreted proteases. The keratinolytic, dermatophytosis could emerge.
immunogenic, in-vivo-produced Sub3 and Mep3 of
M. canis were the first to be fully characterized at the
molecular level [1013]. They belong to large protein Conclusion
families that were further characterized in several der- Unfortunately, dermatophytosis has been understudied
matophyte species [39,40]. Their role in fungal adhesion compared to opportunistic mycoses during the last
to keratinocytes was recently hypothesized [41] and is 20 years. There is, however, a resurgence of interest in
currently investigated in our laboratory (see above). both dermatophytes and dermatophytic infection. In
These endoproteases are thought to be involved in the particular, several potential fungal virulence factors have
digestion of hard keratinized substrates in cooperation been characterized at the molecular level. The elucida-
with exoproteases such as aminopeptidases and dipepti- tion of the role of the currently and future isolated
dyl-peptidases (Dpp) that have been isolated from pathogenic factors in the hostfungus relationship is
T. rubrum [42] and M. canis (S. Vermout, unpublished necessary. This ongoing work will undoubtedly contrib-
data). The action of all these proteases is likely to be ute to a better understanding of the immunopathogenesis
preceded by sulphitolysis of keratin, a mechanism which of dermatophytosis, as will the identification of host cells,
was recently shown to be controlled by a sulphite efflux cell receptors and cytokine pathways, notably those
pump encoded by the SSU1 gene [20]. Other regulatory involved in appropriate innate and thus protective
genes include DNR1 [43] of M. canis and PACC of specific immune responses.
T. rubrum [44] both encoding a transcription factor which
is involved in regulation of the keratinolytic activity.
Acknowledgements
There are several examples that the molecular charac- The authors are grateful to Professor M. Monod and Dr J.P. Bouchara
for their helpful comments on the manuscript. They also thank
terization of the putative dermatophyte pathogenic Dr D. OBrien for his advice and suggestions.
factors is an important step towards a comprehensive
understanding of the immunopathogenesis of dermato-
phytosis. Sub6 from T. rubrum [40] and the orthologue of References and recommended reading
T. rubrum Dpp5 [42] in Trichophyton tonsurans were Papers of particular interest, published within the annual period of review, have
been highlighted as:
demonstrated to be the previously described Tri r2  of special interest
[45] and Tri t4 [46] allergens respectively, two proteins  of outstanding interest
Additional references related to this topic can also be found in the Current
capable of inducing dual immune responses in humans World Literature section in this issue (p. 201).
[5]. Also, the well known different inflammatory reactions
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This is a clear, concise and well illustrated review on how neutrophils and dendritic
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41 Kaufman G, Horwitz B, Duek L, et al. Infection stages of the dermatophyte
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42 Monod M, Lechenne B, Jousson O, et al. Aminopeptidases and dipeptidyl-
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This elegant study showed that, in mice, activation of Langerhans cells by CpG via 43 Yamada T, Makimura K, Abe S. Isolation, characterization, and disruption of
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28 Tani K, Adachi M, Nakamura Y, et al. The effect of dermatophytes on cytokine
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140 Skin and soft tissue infections

46 Woodfolk J, Slunt J, Deuell B, et al. Definition of a Trichophyton protein 51 Wang L, Ma L, Leng W, et al. Analysis of the dermatophyte Trichophyton rubrum
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47 Giddey K, Monod M, Barblan J, et al. Comprehensive analysis of proteins This is a considerable work intended to be a comprehensive resource and
 secreted by Trichophyton rubrum and Trichophyton violaceum under in vitro platform to assist functional genomic studies in dermatophytes (see references
conditions. J Proteome Res 2007; 6:30813092. [5356]). Further transcriptome expression patterns analysis will be more
The authors present a powerful methodology to analyse and compare the secreted informative, as soon as the complete genome sequences from dermatophytes
protein profiles of different dermatophyte species in different culture media are available.
(see Giddey et al. [48]).
53 Liu T, Zhang Q, Wang L, et al. The use of global transcriptional analysis to
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