Immunity to malaria: more questions than answers Jean Langhorne, Francis M Ndungu, Anne-Marit Sponaas & Kevin Marsh Nature Immunology, July th January 2009

Overview  Introduction to malaria  Immunity to malaria mechanisms of immunity immune response Immune memory 2

Malaria Vector-borne infectious disease Caused by protozoan parasites of the genus plasmodium Human malaria  P. falciparum  P. vivax  P. ovale  P. malariae all persons become ill on the first exposure  Causes flu-like illness anemia, fever, chills  followed by control of parasitemia  in severe cases coma and death 3 Center of disease control and prevention, Atlanta, USA. common & severe rare & mild

Malaria Wide spread in tropical and subtropical regions 400 million cases per year 3 million death Majority 2-5 years 4 © DPDx: CDC's web site for laboratory identification of parasitesDPDx

Life cycle of Plasmodium falcipare 5 Involves two hosts - Anopheles mosquito - Human host three distinct cycles - Sporogonic cycle (mosquito) - Exo-erythrocytic cycle - Erytrocytic cycle © DPDx: CDC's web site for laboratory identification of parasitesDPDx

6 Life cycle of Plasmodium falcipare Exo-erythrocytic cycle infected (female anopheles) mosquito bites through the skin sporozoites travel from dermis to the liver/hepatocytes undergo an amplification phase lasting between 2 and 9 days (asymptomatic stage) parasite reenters the bloodstream by rupture of hepatocytes (Merozoites) © DPDx: CDC's web site for laboratory identification of parasitesDPDx

Life cycle of Plasmodium falcipare 7 Erythrocytic cycle merozoites invade red blood cells (RBC) and initiate the asexual cycle in RBCs exponential expansion of parasite populations leads to febrile illness sexual stages (male and female gametocytes) are formed during the erythrocytic cycle this stage continues the life cycle in the mosquito after a blood meal © DPDx: CDC's web site for laboratory identification of parasitesDPDx

Life cycle of Plasmodium falcipare 8 © DPDx: CDC's web site for laboratory identification of parasitesDPDx Sporogonic cycle While in the mosquito's stomach, the microgametes penetrate the macrogametes generating zygotes zygotes become motile and elongated (ookinetes) invade the midgut wall of the mosquito to develop into oocysts oocysts grow, rupture, and release sporozoites which make their way to the mosquito's salivary glands.

2. Immunity to malaria mechanisms of immunity immune response Immune memory 9

Immunity to malaria naturally acquired immunity (NAI) to malaria Immunity to malaria is seen as asymptomatic infection  more resistant to new infections  limits parasitemia  reduces the frequency and severity of illness human studies showed that immunity to malaria is  relatively slow to develop and incomplete  immunity to death is acquired more quickly Langhorne et al., 2008

Mechanisms of Immunity Picture of human immunity to malaria has been provided by two main sources:  Deliberately induced malaria in non immune persons  Natural history studies in endemic populations Immune attack could be directed at any point in the life cycle of plasmodium Pre-erythrocytic cycle (asymptomatic stage)  Probably has limited involvement Erythrocytic cycle

Mechanisms of Immunity Pre-erythrocytic cycle targets are free sporocytes and infected hepatocytes Requires mainly CD8 + effector cells producing interferon-γ Need the help of CD4+ T cells Antibodies to sporozoites are thought to have a lesser function Kill parasites in infected hepatocytes 1. Antibodies to sporocyte neutralize sprozoites and/or block invasion of hepatocytes 2. IFN-γ and CD8+ T, NK, NKT, and γδT cells kill intrahepatic parasites 1 2 Langhorne et al., 2008

Mechanisms of Immunity Erythrocytic cycle targets are free merozoites or intraerythrocytic parasites (Schizont) Humoral responses are the key to blood stage immunity (protective antibody response) 3. Antibodies to merozoites opsonize merozites for uptake and/or inhibit invasion of RBCs 4. Antibodies to block infection of RBCs by merozoites Antibody-dependent cellular killing mediated by cytophilic antibodies Block adhesion of infected RBCs to endothelium Neutralize parasite toxins and prevent the induction of excessive inflammation Langhorne et al.,

Mechanisms of Immunity Erythrocytic cycle targets are free gametocytes Humoral responses are the key to blood stage immunity (protective antibody response) 5. Antibodies prevent sequestration and maturation of gametocytes 6. Antibody and complement mediate lysis of gametocytes and prevent further development Langhorne et al.,

Immune response to plasmodium Many features are similar to those of sepsis, over-vigorous or disordered immune responses are central CD4+ and CD8+ T cells are chief participants in acute pathology Enhanced amounts of cytokine responses  TNF  IL-1 β, IL-6, IL-10  IFN-γ  CCL3 (MIP-1 α ), CCL4 (MIP-1 β )

Immune response to plasmodium Specific cytokine profiles associated with different syndromes  Relatively low IL-10 in severe malaria anaemia  Large amounts of IL-10 in respiratory distress  Low amounts of CCL5 in severe disease and mortality Polymorphism in host genes is associated with susceptibility  IFN-γ  Interferon regulatory factors  TNF  IL-10, IL-4 Balance in the regulation of pro- and anti-inflammatory cytokines may be critical in determining the extent of pathology

Trigger for cytokine production Depends on interaction of host cells and parasite via pattern-recognition receptors (PRR) Can be induced by infected RBCs or parasite products with several Toll-like receptors (erythrocytic cycle)  Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)  Glycosylphosphatidylinositol anchors (GPI)  Hemazoin  Ds DNA

Trigger for cytokine production

MyD88  Intracellular adaptor for several TLRs  Important for the induction of some but not all of the pathology  Also required for controlling acute-stage parasitemia Immune response via several PRR Quantity of inflammatory cytokines? Qualitiy of a person’s response to a particular clone? Difficult to distinguish pathological from protective responses

Immune memory in malaria Immunity to malaria develops Relatively slow Is not sterile Continued exposure to malarial antigens is needed for  Generation of memory and effector cells  Persistence or memory and effector cells  Maintenance of immunity It is still unclear what components of immunity is lost without exposure  Short lived immunity  Longlived immunity

Immune memory in malaria Rapid boosting of antibody responses indicate the presence of memory B cells Accumulation of memory B cell specific for malaria indicates the presence of memory B cells  anti-P. falciparum memory B cells present in adults for over 8 years without evident re-exposure  a subsequent study in children in contrast presence of anti-P. falciparum serum antibody only very low frequencies of malaria-specific memory B cells

Immune memory in malaria Pre-Erythrocytic cycle To be protective specific CD4+ and CD8+ T cells  must be maintained as effector cells to differentiate very rapidly into effector-killer cells  Population shift between mostly protective effector phenotype to the less protective memory phenotype This could explain why immunological memory is not always correlated with immunity

Immune memory in malaria Erythrocytic cycle  CD4+ T cells are induced by natural infection Frequencies of responding cells and the prevalence of exposed people with measurable malaria-specific CD4+ T cells are often low  lower concentrations of malaria-specific antibodies in malaria-infected people positive for HIV suggests that CD4+ T cell help is necessary for the induction and also for the maintenance of protective antibody Requirment for memory CD4+ T cells in immunity has still to be elucidated

Immune memory in malaria  Inhibition of DC maturation  Hemozoin can inhibit macrophage monoyte function  IL-10 from DCs and macrophages modulated by infect RBCs inhibit CD4+ T cell activation  CD4+ T cells produce IL-10 and transforming growth factor- β, which inhibts the generation of central and memory-effector cells

Immune memory in malaria 5.Infected RBCs induce apoptosis and/or depletion of memory B cells 6.The niche for plasma cells of many different specificities may be limiting 7.Acceleration of the catabolism of immunoglobulin molecules 8.Antigenic variation for immune escape 9.Circulating immune complexes and low-affinity immunoglobulin molecules can trigger apoptosis of long-lived plasma cells

Conclusions- more questions than answers Advanced understanding the host response to plasmodium But many questions remain: What to measure as a correlate for immunity? What mechanisms regulate immune pathology in semi- immune people? What defects contribute to the relatively ineffective immunity in children? Why immunity to plasmodium infection can be short- lived?

Thank you for your attention!

Complete life cycle

Trigger for cytokine production Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)  Adhesive molecules  sequestration in the post-capillary venules Glycosylphosphatidylinositol anchors (GPI)  Anchor on many plamodium membrane proteins  induction of proinflammatory responses Hemazoin  disposal product formed from the digestion of erythrocytes