Basis of Immunology and Immunophysiopathology of ...adrien.six.online.fr/IP-HCMC2005/Document/IP-HCMC2005_IAIa_SP... · Basis of Immunology and Immunophysiopathology of Infectious

Basis of Immunology and Immunophysiopathology of Infectious Diseases, Institut Pasteur in Ho Chi Minh City, Vietnam, January 24 – February 5, 2005

Basis of Immunology and Immunophysiopathology of Infectious Diseases

Jointly organized byInstitut Pasteur in Ho Chi Minh City and Institut Pasteurwith kind support from ANRS & Université Pierre et Marie Curie

January 24 – February 5, 2005at the Institut Pasteur in Ho Chi Minh City, Vietnam

Lecture :Immunity to parasite infection

Dr. Sylviane PiedJanuary 31, 2005


• Introduction to parasitism.• Immune effector mechanisms to

protozoa: the example of Plasmodium.• Immune effector mechanisms to

Metazoa: the example of helminths.• Immune evasion strategies used by

parasite.• Immunopathology associated to

parasite infection



What is a parasite (1) ?Living micro or macro organisms whose life cycle mainly relies on other living organisms (biotope).Need to establish a long-term interaction with theirhosts– for resource– to achieve their life cycle and transmission

Necessity to maintain host fitness (stable equilibrium between the two partners by restoringthe host homeostasis)

without triggering symptomatic process (clinicallydetectable)they exploit/subvertthey shape through dynamic processes


Complexe developmental life cycle involvingseveral host vertebrate and/or invertebrate

- different cell or tissue type - different biotopes

Generate different signals:

Friendly cross talk: AsymptomaticAsymptomatic infection (no clinically detectable symptoms)

ADAPTATION

Unfriendly cross talk: DiseaseDisease (symptomaticprocess of different severity)

What is a parasite (2)?


LeishmaniaTrypanosoma

Unicellular Microparasites

1. Protozoa

Plasmodium


Helminths are large

Hookworms live in the gut and lungs

They cling to their hosts

Ascaris live throughout the body, including

the gut

They can be49 cm long

2. MetazoaPluricellular Macroparasites


Infectious Process (1)1. Phase of latency : parasite growth (asexual

multiplication and differenciation (AsymptomaticAsymptomatic)

2. Early Immune response: (steadysteady--statestate perturbationperturbation) – Prevention of acute pathological process development– Absence of severe pathological process

– Induction of pathological process

3. Late Immune response: _ Transitory inflammatory responses (tissue or systemic) _ Tolerance


Infectious Process (2)Parasite-host interaction:

a complex and durable equilibrium during which the parasite agression by the immune response is limited (no parasite elimination).

Chronic infection and non sterile immunity.

Protective immunity to reinfection with the same parasite strain.

AdaptationChronicity


Effector mechanisms to protozoan parasite infection:the example of Plasmodium

• Natural immunity• Immunity to exoerythrocytic

stages• Immunity to blood stages• Innate immunity



Clinical features of malaria and possible mechanisms of disease

Syndromes Clinical features Disease mechanisms

Severe anaemia

Shock; impaired consciousness; respiratory distress

Reduced RBC production (reduced erythropoietin activity, proinflammatory cytokines); increased RBC destruction (parasite-mediated, eythrophagocytosis, antibody and complement-mediated lysis)

Cerebral complications (cerebral malaria)

Impaired consciousness; convulsions; long-term neurological deficits

Microvascular obstruction (parasites, platelets, rosettes, microparticles); proinflammatory cytokines; parasite toxins(e.g. GPI)

Metabolicacidosis

Respiratory distress, hypoxia, tachypnea; acidaemia; reduced central venouspressure

Reduced tissue perfusion (hypovolaemia, reduced cardiacoutput, anaemia); parasite products; parasite products; proinflammatory cytokines; pulmonary pathology (airwayobstruction, reduced diffusion)

Other Hypoglycaemia; disseminated intravascular coagulation

Parasite products and/or toxins; proinflammatorycytokines; cytoadherence

Malaria in pregnancy

Placental infection; low birth weight and fetal loss; maternal anaemia

Premature delivery and fetal growth restriction; placental mononuclear cell infiltrates and inflammation; proinflammatory cytokines


Innate Immunity Adaptive ImmunityPre-erythrocytic stages

Erythrocytic stagesSexual cycle

Asexualcycle

?

?

Homeostasis?Infection?


I. Natural immunity• Elicited after several years of continuous

exposure to infection by malaria parasite andillness.

• Occurs in children older than 10 years, in area of high transmission.

• Principally mediated by antibodies directedagainst antigens or toxins of the erythrocyticstages.

• Antibodies against liver stage and sporozoite are also observed in preimmune individuals.


• Declines very rapidly once exposure to theparasite cease.

• Absence of complete protection in individualsliving in endemic areas:

- could be a mean by which the parasite ensure its survival by evading full engagment of the host immune system.



Comparative proteomics throughout the life cycle

1147103683910492415

---X513

--X-204

-X--286

X---376

--XX65

-X-X80

-XX-84

X--X120

X-X-73

XX--148

-XXX36

X-XX28

XX-X53

XXX-197

XXXX152

GametocytesTrophozoitesMerozoitesSporozoitesProtein count

Florens L., Nature 2002, 419:520


II. Immunity to Exoerythrocyticstages• Liver stage has been discovered by Garnham, in 1947, in

an african monkey infected by P. Kochi and, in 1948, for P. falciparum (Short and Garnham).

• Liver stage of the plasmodium life cycle is to completethe transition from mosquito vector to human host.

• Process of amplification and molecular changes for theparasite: - few sporozoites injected (10-100)

- a dozen of liver schizont (104 to 4.104

merozoites) whereas blood stage schizont contains 8 to 24 merozoites)



• A Large range of immune effectors are able to block theliver stage compared to the limited number of mechanisms involved in immunity against blood stages

• MHC class I-restricted parasite antigens presentationby infected hepatocytes (major difference withinfected red blood cells)

• High level of immunogenicity of pre-erythrocytic stage antigens compared to the blood stage


• 1941, Mulligan et al., protection could be conferred by the bite of infected mosquitoes exposed to UV.

• 1950s, Foundation of research aiming to developa sporozoite-induced vaccine

• 1967, Confirmation by Vanderberg and Nussenzweigusing X-irradiation.

• To achieve protection, irradiation dose should allowinvasion of hepatocytes by the sporozoite andtransformation into trophozoite and abort the shizontdevelopment

1. Attenuated sporozoite model of protective immunity


• Protection is induced by sporozoite and aborted liverform antigens that are either retained in hepatocytesand /or Kupffer cells.

• Promote a local inflammation which will favour thepriming of parasite antigen specific T cells by DC or Kupffer cells.

• Several protective mechanisms: – Prevent invasion of hepatocytes by sporozoites– Inhibit the intrahepatocytic development of the

parasite


• Protection induces by immunisation with γ-irradiatedsporozoites is directed against pre-erythrocytic and not blood stages.

• Sera from mice immunized with γ-SPZ do not passivelyprotect naive mice to sporozoite challenge, whereasadoptive transfer of spleenic cells does confer protection: protective immunity is mediated by cellular mechanisms

• However, Mabs to CSP repeat region inhibit sporozoiteinfectivity in vitro: antisporozoite antibodies could alsocontribute to protection



2. Antibody dependant effectormechanisms


• Inhibition of sporozoite penetration intohepatocytes

• Inhibition of schizont growth


• Antibody dependant cytoctoxicity mechanisms

Enhancement of sporozoite penetration intohepatocytes by low doses of anti CS antibody


3. CD8+ T cells mediated-protection

• A Critical role for CD8+ T cells was demonstrated in rodent malaria system.

• Depletion of CD8+ T cells abolished protective immunityinduced by immunization with γ-SPZ .

• Depletion of CD4+ T cells had no effect.

• Effector CD8T cells are active against the infectedhepatocyte able to present in a MHC class I contextparasite antigen derived peptides.




Représentation relative des différentes sous-populations lymphocytaires CD3+ dans le foie d’une

souris C57BL/6 normale

35% TCRαβ+ NK1.1+

60% TCRαβ+ NK1.1-

5% TCRγδ+

70% CD4+

25% CD4-CD8- (DN)

5% CD8+

Dont 85% expriment un

TCRαβ “semi-invariant”

(Vα14-Jα281+Vβ8, Vβ7 ou

Vβ2) reconnaissant des

antigènes de nature

glycolipidique présentés

par CD1d.


Liver αβT cells

• Conventional CD4 CD8 CD4-CD8-

• Unconventional

CD4+NK1.1 CD4-CD8- NK1.1 CD8+


TNK cells

• T cells HSAlo

C44hi

CD45RBhi

LECAM-1lo

Ly6Chi

Thy1hi

CD5hi

B220neg

CD3int

DN CD4

• NK cells

NK1.1int

IL-2Rβint (CD122) CD 69int

CD 16 Ly49A

Ly49C


Specificity of NKT cells

• TCR repertoire Vβ8 (8.2, 8.3, 8.1)

Vβ7 Vβ2

Vα14-Jα281 associated

• Restriction CD1• Cytokines γ−IFN+++ IL-4+++


Days after infectionMea

n nu

mbe

r of p

ositi

ve c

ells

(x 1

06 )

0

1

2

3

4

5

6

30 10 30

DN

CD3CD4CD8

Liver T cell populations



0,0E+00

2,0E+03

4,0E+03

6,0E+03

8,0E+03

1,0E+04

1,2E+04

1,4E+04

1,6E+04

0 2 3 6 10day p.i.

IL4IL10TNFaIFNgIC

Cytokines-secretion profile of hepatic NK TCRαβ Tet+During P. yoelii infection


Inhibitory activity of CD3+NK1.1+ T cells onP. yoelii hepatic stages

0 400 800 1200

Mean number of Schizonts

control

NK1.1+

CD3int

NK1.1+

CD3int

+ anti-IFN-γ

anti-IFN-γ

NK1.1+CD3

int+ anti-CD3

anti-CD3


• Secondary effector mechanisms

• CD8 CTL do not kill malaria infected hepatocytes via the perforin/Fas pathways as perforin deficient miceas well as CD95L mutant mice were protected as well as wild-type mice after immunization with γ-SPZ.

• Protection is mediated via IFN-γ because it could beabolished by a treatment with anti-IFN-γ antibodies.

• Other cytokines are also involved….



LADEM

NONO3-

NO2-L-arginine

iNO synthase

L-citrulline

Monoxyde d’azote

Nitrites

Nitrates


CD8

CD4

Mø, DC

NK

Nucleus

ER

Malaria protein

Proteosome

MHC class I-peptide complex

TAP 1and 2

Infected HepatocyteParasitophorous

vacuole

GolgiPeptides

iNOS

IFN-γ

IL-12

IL-12

STAT

IFN-γ

IFN-γ


Oxygen radicals

– NADP NADPH+ +H+

NADPH Oxydase

O2O2

.-

H2O2

OH.

SOD

CatalaseGlutathioneperoxydase


Production of acute phase proteins

PIED S., NUSSLER A., PONTET M., MILTGEN F., MATILE H., LAMBERT P.H. & MAZIER D. C-reactive protein protects against pre-erythrocytic stages of malaria. Infection and Immunity.1989:57, 278-282.



III. Immunity to blood stage malaria parasites




• Passive transfer of immune sera resulted in a temporary(10-15 days) but drastic (500 fold) reduction in peripheralparasitemia with improvement in the clinical condition.

• Antibody do not block red blood cell invasion by parasites in the volunteers.

• Immunity was correlated with the presence of cytophilicantibodies (IgG1/G3) anti MSP3 that cooperate withmonocytes and malaria antigens via FC receptor to produce TNF = ADCI

• TNFa inhibits the ring stages parasite development.• Targetted antigens: MSP1, AMA-1

Asexual blood stages


Possible action of antibody-independantcell-mediated mechanisms


IV. Innate immunity in malaria





Malaria ligand that induces innate immuneresponses and their respective receptors


Effector mechanismsmetazoan parasite infection:

the example of Helminths


• Target are larvae (no multiplication in the vertebrate host)• Destruction by cell cytoxicicity mechanisms

- ADCC- NK- Antibody opsonisation inducing phagocytosis by macrophage- Agglutination- Complement activation

• Helminth infection are characterized by an elevated IgE responseand increase of percentage of eosinophil

• IgE Fixation on macrophages, eosinophils and/or platelets via FcεRII induces receptors aggregation and release of toxicmolecules:

ROI, NO, Proteases, IFNγ

• Eosinophils mediated cytotoxicity can also be induced by IgG2a• Anaphylatoxic Ab can induce intestinal worm expulsion via

mastocytes

Protective mechanisms



Th2 type dominant responseShistosomiasis induces a IL-4 dominant response


• Masking and MimicryS. Mansoni fixed fibronectin, C-reactive protein, glycolipids from the ABO group.Conserved peptidic sequence between parasite and host protein (cytokines, proteases, repeat sequences of thrombospondin, HSP)

• Blockingantigen-antibody complexes in serum bind to the parasite's surfacemechanically block the actions of cytotoxic antibodies or lymphocytesdirectly inhibiting the actions of lymphocytes (helminth, trypanosoma)

• Intracellular locationMany protozoan parasites grow and divide within host cells: Plasmodia in hepatocytes, red blood cells,Leishmania and Toxoplasma in macrophagesTheilera in lymphocytesT cruzi and Toxoplasma in several type of cells

IMMUNE ESCAPE (1)


• Antigenic VariationThree major groups of parasitic protozoa are known to be able to change the antigenic properties of their surface coat :

African trypanosoma :glycocalyx

Plasmodium, Babesia, and Giardia.Developmental stage, clone, strain and specie specificity of surface antigens

Use of commutation for gene rearangement as for Ab and TCR repertoirediversity.

existence of repeat in the antigenic sequence

No involvement of immunodominant epitopes in protective responses

• Immunosuppression

IMMUNE ESCAPE (2)




D. Immunopathology



What is cerebral malaria?



Causes of Cerebral Malaria?

Mechanical Immunological

Sequestration of mature formsof parasitised erythrocytes:

Interaction PfEMP1 withICAM or E selectin

Sequestration of leucocytes

T cells?Cytokines

TNFα, IFNγ




Événements immunophysiopathologiques associés au NP

INF-γ

TNF-α

TP

Rupture de l’intégrité de la barrière hémato-encéphalique

TNF-α

+ +

+

+

+

ϕPNN

A

µ


Lymphocytes T and cerebral malaria

T

Cyclosporine ABetamethasone

Treatment withmonoclonal antibodies

(anti-CD3, anti-CD4, anti-CD8)Anti-lymphocytes serum

SpleenectomyThymectomy

Mutations Nude and SCID

Cerebral malaria

KO miceTCRαβ

CD4/CD8


0,0E+00

2,0E+04

4,0E+04

6,0E+04

8,0E+04

1,0E+05

1,2E+05

1,4E+05

0,0E+00

2,0E+04

4,0E+04

6,0E+04

8,0E+04

1,0E+05

1,2E+05

1,4E+05

Blood stages Sporozoites

Recruitment of αβT cells in Brain of CM+ Mice

Day 0 Day 0CM - CM-CM + CM +

CD8+

CD4+

Cel

lnum

ber



Cytokines produced by Brain CD8+T cellsfrom CM+ Mice


CD8+ T cells in brain of CM+ mice

LT CD8+ LFA-1ICAM-1

IFN-γTNF-α

CD69CD25

Activation

CD44CD62L (-)

(re)localisation

Participation in the cerebral inflammation

APC

Kb,d

Db,d

TCRVβ4Vβ6Vβ8.1+++Vβ11


Implication potentielle du TGF-β2Cytokine importante dans la régulation négative de l’inflammation

INF-γ

TNF-α

T

TGFTGF--ββ22

endo

ϕ

PNN

µA

AdhérenceSéquestration

T8

INF-γ

TNF-α


D.Difficulties for a malaria vaccine development

• Size and complexicity of parasite genome (6000 genes). Each infection presents thouzands of antigens to theimmune system.

• Parasite changes through several life cycle stages andlocation, presenting a different array of antigens duringeach stage.

• Involvement of a series of immune evasion strategies by the parasite that allows it to confuse, hide andmisdirect the human immune system.

• In endemic areas, a person can be infected not only by different strains but also by different species



The genome of Plasmodium falciparum




Malaria pathogenesisNeurological signsComa stade II or moreRepeated convulsions (<2per 24h)

ConvulsionsRespiratory distressPulmonary oedemaSevere anemiaHypoglycemiaRenal failureDiffuse haemorageHaemoglobinuriaMetabolic acidose

FeverHeadacheVomitingParasitemia (<5%)

Koko J. et al. Medecine tropicale, 1997

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Basis of Immunology and Immunophysiopathology of ...adrien.six.online.fr/IP-HCMC2005/Document/IP-HCMC2005_IAIa_SP... · Basis of Immunology and Immunophysiopathology of Infectious