Fungal immunity mod. for Diploma students by Mohamed Refai

Lecture 2 Fungal Lecture 2 Fungal immunityimmunity

ByByProf Dr. M. RefaiProf Dr. M. Refai

Local defence mechanisms against mucosal

infection

Salivary proteinssuch as lactoferrin, beta-

defensins, histatins, lysozyme, transferrin, lactoperoxidase, mucins, and

secretory immunoglobulin A.

These impair adhesion and growth of fungi in the oropharyngeal cavity.

Healthy oral epithelial cells These inhibit

blastoconidia and/or hyphal growth of fungi

ifif The fungus succeeds in bridging any The fungus succeeds in bridging any of the body surfaces either as a of the body surfaces either as a result of result of burns, trauma or maceration or burns, trauma or maceration or surgical interference or surgical interference or application of catheter etc., application of catheter etc.,

The fungus will faceThe fungus will face non-specific defensesnon-specific defenses humoral defenses humoral defenses cellular host defenses. cellular host defenses.

In general, man and In general, man and animals are resistant to animals are resistant to fungus infectionfungus infection

The intact skin and The intact skin and mucous membranes are not mucous membranes are not easily invaded by fungi. easily invaded by fungi.

None specific defence

Secretory Ig A+

Complement

OpsonizationNeutrophils, Monocytes Alveolar macrophages

Phagocytosis

Phagocytosis

Immune cellsImmune cells NeutrophilsNeutrophils Macrophages Macrophages Dendritic cells, Dendritic cells, Natural killer cellsNatural killer cells T cells T cells Epithelial and endothelial cells Epithelial and endothelial cells

Specific pathogen-associated molecular patterns (PAMPs)pattern-recognition receptor (PRR) immune cells.

immune cells

Pattern recognition of fungi

Signaling pathways are activated to release Transicription factor

to

regulate expression of Inflammatory cytokines

Strategies employed by fungal pathogens to evade host defense mechanisms

1. Shielding of stimulatory PAMPs,

2. Modulation of inflammatory signals,

3. Shedding of decoy components, 4. Persistence in intracellular environments,

5. Complement evasion.

surface proinflammatory b-(1,3)-glucans or non-stimulatory a-(1,3)-glucan cell wall

Shielding of stimulatory PAMPs

detection of the invading pathogen

C. albicans & dectin-1dectin-1 recognizes the ß-glucans at the level of budding scars in the C. albicans, but

it cannot recognize the ß-glucans in the hyphae, where they are shielded by a layer of mannans

Histoplasma capsulatumthe α -(1,3)-glucan present in its outer layer cell wall contributes to pathogenesis by shielding its immunostimulatory β -glucans.

Paracoccidioides brasiliensis

Paracoccidioides brasiliensis transforms into its pathogenic yeast form, a change in its cell wall glucan polymer linkage occurs from β -(1,3) to α -(1,3)-glucan.

Cryptococcus neoformans Cryptococcus neoformans masks its surface PAMPs through the production of an extracellular capsule of glucuronoxylomannan (GXM)

Coccidioides immitis Coccidioides is recognized by the host by its immunodominant spherule outer wall glycoprotein (SOWgp).

During endospore differentiation, the fungus secretes a metalloproteinase (Mep1) which digests SOWgp

This enables the fungus to evade phagocytosis and killing at its vulnerable endospore stage of development

Strategies employed by fungal pathogens to evade host defense mechanisms 1. Shielding of stimulatory PAMPs, 2. Modulation of inflammatory signals,



Preferential activation of TLR2 over TLR4 by certain fungal morphotypic PAMP stimulates a Th2 humoral response over a Th1-dependent antifungal proinflammatory cytokine production.

Modulation of inflammatory signals

humoral response

cytokine production

C. albicans C. albicans induce immunosuppression through TLR2-mediated IL-10 release,

this leads to generation of CD4+CD25+ T-regulatory cells

with immunosuppressive potential

Aspergillus fumigatus A. fumigatus evades immune recognition by germination into hyphae with subsequent loss of TLR-4 recognition,

The TLR2-mediated IL-10 pathways remain intact, thus shifting the balance towards a permissive Th2-type profile

Strategies employed by fungal pathogens to evade host defense mechanisms 1. Shielding of stimulatory PAMPs, 2. Modulation of inflammatory signals,3. Shedding of decoy components,

4. Persistence in intracellular environments,


Shedding of surface protein gpA by the fungus as decoy to competitively inhibit mannose receptor (MR).

Shedding of decoy components


1. Shielding of stimulatory PAMPs, 2. Modulation of inflammatory signals,



Upon internalization by immune or non-phagocytic cells, Candida or Aspergillus can resist intracellular killing, germinate and eventually escape

Persistence in intracellular environments

Aspergillus fumigatusAlveolar macrophages

The first defensive cells that inhaled conidia

The conidia are internalized by the macrophages and prevented from growth for several hours until the macrophage begins to destroy them.

At 24 h after internalization, 90% of the conidia are killed.

The conidia then germinate to hyphae The hyphae are too large to be engulfed

Aspergillus fumigatus neutrophils

Resting conidia are relatively resistant to killing by either reactive oxygen intermediates or neutrophil cationic peptides and

their ingestion triggers neutrophil degranulation and the respiratory burst only weakly.

Resting conidia activate the alternative pathway induce neutrophil chemotaxis

Aspergillus fumigatusPolymorphonuclear neutrophils

bind the surface without the need for complement or immunoglobulin

This binding triggers secretion of reactive oxidative intermediary agents that rapidly damage the hyphae;

50% of hyphae are destroyed in 2 h

Histoplasma capsulatumMacrophages Macrophages

represent the first line of defense represent the first line of defense during infection withduring infection with H. capsulatumH. capsulatum ,,

they rapidly phagocytose the inhaled they rapidly phagocytose the inhaled conidia and transforming yeast cells,conidia and transforming yeast cells,

the infected macrophage subsequently the infected macrophage subsequently activate effector T cells and enhance activate effector T cells and enhance the release of Th1-associated the release of Th1-associated proinflammatory cytokines (IL-12, proinflammatory cytokines (IL-12, IFNIFN--γγ , , and TNFand TNF--αα ((

Histoplasma capsulatum the main cytokines involved in the main cytokines involved in

HistoplasmaHistoplasma clearance from the host clearance from the host are IL-12, IFN-are IL-12, IFN-γγ, and TNF-, and TNF-αα

IL-12 through its ability to IL-12 through its ability to regulate IFN-regulate IFN-γγ production is production is critical in inducing a protective critical in inducing a protective immune response in primary infection immune response in primary infection with the pathogen. with the pathogen.

IFN-IFN-γγ is pivotal for the host's is pivotal for the host's innate resistance to systemic innate resistance to systemic infection with infection with H. capsulatum.H. capsulatum.


1. Shielding of stimulatory PAMPs, 2. Modulation of inflammatory signals,



Binding of complement-inhibitory C4 binding protein (C4BP)and Factor H on fungal surface.

Complement evasion

Immune response to fungi :

HIThe main recognized functions of antibodies in fungal infections include

prevention of adherence,toxin neutralization, antibody opsonization and antibody-dependent cellular cytotoxicity

CMI The type of CMI induced is critical in determining resistance or susceptibility to fungal infection.

In general, Th1-type CMI is required for clearance of a fungal infection,

Th2 immunity usually results in susceptibility to infection or allergic responses

CMICMITh1 cells produce predominantly cytokines such as IFN-g, and promote cell-mediated immunity and phagocyte activation.

Th2 cells produce predominantly cytokines such as interleukins 3 and 4 (IL-3 and IL-4) and tend to promote antibody production

Cell mediated immunityCell mediated immunity Resistance to several mycoses is Resistance to several mycoses is associated however with cell mediated associated however with cell mediated immunity, particularly phagocytosis immunity, particularly phagocytosis and killing of invading fungi by and killing of invading fungi by peripheral blood monocytes, peripheral blood monocytes, alveolar and peritoneal alveolar and peritoneal macrophagesmacrophages

polymorphonuclear leukocytespolymorphonuclear leukocytes..

i. Alveolar macrophage i. Alveolar macrophage defense defense

The first phagocytic cells to encounter The first phagocytic cells to encounter the inhaled or aspirated fungal particles the inhaled or aspirated fungal particles

have the capacity to kill Aspergillus have the capacity to kill Aspergillus conidia by both oxidative and non-conidia by both oxidative and non-oxidative mechanisms.oxidative mechanisms.

have relatively have relatively weak oxidative weak oxidative candidicidal activity candidicidal activity

fungi characterized by hyphal forms, are fungi characterized by hyphal forms, are too large to be ingested completely by too large to be ingested completely by phagocytic cells. phagocytic cells.

the alveolar macrophagesthe alveolar macrophagesHistoplasma capsulatum: Histoplasma capsulatum:

Macrophages are the primary host Macrophages are the primary host cellscells

The yeast form survive within The yeast form survive within macrophages and multiplies macrophages and multiplies intracellularly. intracellularly.

Thus the macrophage, which usually Thus the macrophage, which usually eliminateseliminates organisms from the organisms from the primary site of infection, becomes primary site of infection, becomes a site of continued fungal growth a site of continued fungal growth and aids in and aids in disseminationdissemination of the of the yeast to other tissues.yeast to other tissues.

ii. Peritoneal ii. Peritoneal macrophagesmacrophages

can phagocytize, inhibit can phagocytize, inhibit intracellular growth or inhibit intracellular growth or inhibit germination of conidia of germination of conidia of Aspergillus or Candida. Aspergillus or Candida.

Activated macrophages appear to Activated macrophages appear to play a role in resistance play a role in resistance against disseminated candidosisagainst disseminated candidosis. .

iii. Normal peripheral blood iii. Normal peripheral blood monocytesmonocytes

Monocytes can damage and apparently kill Monocytes can damage and apparently kill Aspergillus hyphae, even though hyphae Aspergillus hyphae, even though hyphae are too large to be ingested completely.are too large to be ingested completely.

Monocytes and macrophages have been Monocytes and macrophages have been shown to be important cells in host shown to be important cells in host defense against dimorphic fungi. When defense against dimorphic fungi. When activated they have increased activated they have increased phagocytosis and decrease growth of phagocytosis and decrease growth of intracellular yeasts.intracellular yeasts.

iv. iv. Polymorphnuclear Polymorphnuclear leukocytes (PMN)leukocytes (PMN)

PMN play a major role in host PMN play a major role in host defense against fungi. defense against fungi.

PMN can kill Candida PMN can kill Candida blastoconidia, Coccidioides blastoconidia, Coccidioides immitis arthroconidia and immitis arthroconidia and Blastomyces conidia. Blastomyces conidia.

can also damage the hyphal forms can also damage the hyphal forms of Aspergillus and Candida. of Aspergillus and Candida.

The important role of the PMN in the host defense against fungus infection becomes obvious after the fungus escapes control by macrophage and monocyte cell lines and start mycelial growth, where an impressive ability of PMN to damage these organism is evident

Application of immune Application of immune responseresponse

VaccinationVaccinationSerodiagnosisSerodiagnosis

Important points to consider in antifungal

immunityand its relevance to

vaccination usually fungi display only moderate virulence

antifungal immune responses are usually redundant.

almost all pathogenic fungi have mechanisms to evade or intoxicate immune responses

Most fungal diseases pose obstacles to the concept and

practice of vaccination coccidiomycosis, histoplasmosis,blastomycosis, and paracoccidioidomycosis

are geographically limited, and low-incidence deep-seated diseases

widespread illnesses e.g. aspergillosis, cryptococcosis, and candidiasis typically occur in the immunocompromised

theoretically—ineligible for active immunisation

Antibodies used for passive immunity

Fungal vaccines

Fungal vaccines

Dermatphyte vaccinesDermatphyte vaccines The inactivated vaccines stimulate the CMI, as demonstrated by skin tests and leukocyte migration inhibition tests.

Vaccines containing T. verrucosum conidia inactivated with formalin have been described for use in cattle

inactivated vaccine plus adjuvant

An inactivated vaccine plus adjuvant containing conidia and mycelium of two T. equinum strains has been used in the immunization of horses

The vaccine does not prevent the disease, but the lesions are less severe in vaccinated animals in compared to non-vaccinated animals.

Insol Dermatophyton inactivated vaccine

effective in horse, dog and cat, can be used as treatment of the disease, improving the clinical outcome.

It contains strains of T. verrucosum, T. mentagrophytes, T. sarkisovii, T. equinum,M. canis,M. canis var. distortum,

M. canis var. obesum, and M. gypseum.

The inactivated vaccine Dermatovac-IV

It contains an adjuvant and an optically standardized inactivated suspension of conidia and mycelium of the fungi M. canis, T. equimun, M. gypseum and T. mentagrophytes

The Ringvac bovis LTF-1301 live vaccine

The most effective and widely used

LTF-130 strain of T. verrucosum, has a characteristic high level of immunogenicity,

low virulence and great stability

has been used effectively in Russia and Norway

Vaccine-induced protection Vaccine-induced protection against 3 systemic against 3 systemic mycoses(mycoses(Coccidioides posadasiiCoccidioides posadasii , , Histoplasma capsulatumHistoplasma capsulatum , and , and Blastomyces dermatitidiBlastomyces dermatitidi endemic to endemic to North America North America

WWüüthrich et al. thrich et al. J Clin InvestJ Clin Invest 2011 121(2):554–568 ;2011 121(2):554–568 ;

Serological diagnosisSerological diagnosis

Serological tests are important Serological tests are important in the diagnosis of systemic in the diagnosis of systemic mycosesmycoses

The standard methods of The standard methods of serology are commonly used, i.e. serology are commonly used, i.e. agglutination, precipitation, agglutination, precipitation, Complement fixation, Complement fixation, immunoflourescence and ELISAimmunoflourescence and ELISA. .

Serological diagnosisSerological diagnosis the results often permit the results often permit differentiation between differentiation between colonization by fungi and a true colonization by fungi and a true mycotic infection. mycotic infection.

In all serological methods only In all serological methods only the titre dynamics are meaningfulthe titre dynamics are meaningful

A single serological result is of A single serological result is of no diagnostic value.no diagnostic value.

Serological diagnosisSerological diagnosis The choice of the test The choice of the test

Candida infectionCandida infection AntibodyAntibody developed against the polysaccharide developed against the polysaccharide component of surface glycoproteincomponent of surface glycoprotein

AntigenAntigen: extracted polysaccharides from C. : extracted polysaccharides from C. albicans serotype Aalbicans serotype A

Antibody typeAntibody type: -: -IgM in early infectionIgM in early infection. . the indirect the indirect haemagglutination test haemagglutination test

--IgG in late IgG in late infectioninfection

indirect indirect immunoflourescence. immunoflourescence.

AntibodiesAntibodies developed against developed against protein antigensprotein antigens of of the cell content are detected by precipiation the cell content are detected by precipiation tests.tests.


Candida infectionCandida infection Because of the ubiquitous nature of Because of the ubiquitous nature of Candida species , these serologic tests Candida species , these serologic tests are limited in discriminating between are limited in discriminating between normal and disease levels of antibodies. normal and disease levels of antibodies.

More specific tests are used for detection More specific tests are used for detection of of circulating C. albicans surface circulating C. albicans surface antigensantigens and and cytoplasmic proteinscytoplasmic proteins. .

Serological tests commonly used are latex Serological tests commonly used are latex agglutination, immunoflourescence agglutination, immunoflourescence


Cryptococcus infectionsCryptococcus infections AntibodiesAntibodies to Cr. neoformans are to Cr. neoformans are usually not detected at early usually not detected at early infection, but later or on recoveryinfection, but later or on recovery

AntibodiesAntibodies can be detected using. can be detected using. indirect immunoflourescent technique, indirect immunoflourescent technique, haemagglutination inhibition test or haemagglutination inhibition test or ELISA.ELISA.

If an infection of the CNS is suspected, If an infection of the CNS is suspected, cerebrospinal fluid must be submitted cerebrospinal fluid must be submitted for serological testing.for serological testing.


Cryptococcus infectionsCryptococcus infections During infection, During infection, capsular materialcapsular material is is solubilized in the body, and being an solubilized in the body, and being an antigen, it can be titrated with a antigen, it can be titrated with a specific rabbit anti-Cr. neoformans specific rabbit anti-Cr. neoformans antiserum using latex agglutination antiserum using latex agglutination test. test.

A positive agglutination is A positive agglutination is diagnostic for cryptococcosis. diagnostic for cryptococcosis.

This test is very useful in the This test is very useful in the early infection. early infection.


AspergillosisAspergillosis Complement fixation testComplement fixation test with conidia with conidia suspension,suspension,

Indirect immunoflourescenceIndirect immunoflourescence with with intact fungal elements, intact fungal elements,

AgglutinationAgglutination tests with extracted tests with extracted polysaccharides on latex particles or polysaccharides on latex particles or erythrocytes. erythrocytes.

PrecipitationPrecipitation test (Double test (Double immunodiffusion and immunodiffusion and immunoelectrophoresis )immunoelectrophoresis )


AspergillosisAspergillosis Detection of Aspergillus Detection of Aspergillus antigenaemia in patients with antigenaemia in patients with invasive aspergillosis is more invasive aspergillosis is more sensitive and specific. sensitive and specific.

ELISA using monoclonal ELISA using monoclonal antibodies is increasingly used antibodies is increasingly used in diagnosis nowadays.in diagnosis nowadays.


BlastomycosisBlastomycosis Serological tests are not useful in the Serological tests are not useful in the diagnosis or prognosis. diagnosis or prognosis.

Cross-reactions are common with other Cross-reactions are common with other diseases like histoplasmosis and diseases like histoplasmosis and coccidioidomycosis.coccidioidomycosis.

ParacoccidioidomycosisParacoccidioidomycosis Immunoduffusion test is extremely Immunoduffusion test is extremely useful.useful.

The CF test is quantitative and useful The CF test is quantitative and useful in assessing prognosis, but cross in assessing prognosis, but cross reactions occur reactions occur


HistoplasmosisHistoplasmosis 1. Latex agglutination, 2. Complement 1. Latex agglutination, 2. Complement Fixation, 3. Immunodiffusion, 4. ELISAFixation, 3. Immunodiffusion, 4. ELISA

Cross-reactions with blastomycosis and Cross-reactions with blastomycosis and other fungus infections are seen.other fungus infections are seen.

Serology for histoplasmosis is a little Serology for histoplasmosis is a little more complicated than for other mycoses, more complicated than for other mycoses, but it provides more information than but it provides more information than blastomycosisblastomycosis

CoccidioidomycosisCoccidioidomycosis Complement fixation, immunodiffusion and Complement fixation, immunodiffusion and latex agglutination testslatex agglutination tests


Skin tests Skin tests Histoplasmin.Histoplasmin. HistfarcinHistfarcin CoccidioidinCoccidioidin

intradermal injection of 0.1 mlintradermal injection of 0.1 ml evoke in infected individual a evoke in infected individual a positive delayed hypersensitivity positive delayed hypersensitivity reaction larger than 5 mm in reaction larger than 5 mm in diameter after 24-96 hoursdiameter after 24-96 hours

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Fungal immunity mod. for Diploma students by Mohamed Refai