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Parasitology 2017 Introduction to malaria – chemotherapy and vaccines Susan Wyllie Malaria Disease Burden One third of world population at risk ~200 million infections annually 0.6 million deaths (90% in Africa) 3,000 children under 5 die every day $12 billion lost GDP Consumes 40 % of public health spending Malaria Parasites of Humans Plasmodium falciparum Plasmodium vivax Plasmodium ovale Plasmodium malariae (Plasmodium knowlesi)* Merozoites escaping from an infected blood cell *Primarily infects macaques Mosquito Vectors of Human Malaria 50 out of 500 Anopheles spp e.g. Anopheles gambiae (Africa) e.g. Anopheles atroparvus (Europe) Anopheles gambiae Life Cycle of Plasmodium falciparum Sporozoites (5-100) MOSQUITO SALIVARY GLANDS Oocyst Ookinete LIVER Zygote Gametes Merozoites (105 -106) Schizont Ring BLOOD STAGES (pathology - fevers) MOSQUITO GUT Schizont Gametocyte Trophozoite haemoglobin digestion Malaria Control Strategies Vaccines Drugs Vector control methods: Vaccines Barrier Insecticides Biological Environmental Drugs & Vaccines Drugs Infected erythrocytes Insecticide treated bed nets Drugs for Malaria Quinoline and aminoalcohols Quinolines, etc. Chloroquine Amodiaquine Quinine Mefloquine Halofantrine HN ACT – combinations Lumefantrine – artemether (Coartem) H H3CO Cl Chloroquine N HO N Cl OH N H N CH3 Cl CH3 N CF3 Cl Cl Cl H 3C Halofantrine Mefloquine Lumefantrine Artemesinin analogues Compliance / safety / availability / cost of raw material CH3 H3C Dihydroartemsinin Artemether Arteether Artesunate O O O O CH3 RO Resistance potential / compliance / cost ($2.40) / availability O O N H N N N H3C NH2 N OCH3 OCH3 NH2 Cl Sulphadoxine Pyrimethamine Cl O H N O Atovaquone Resistance potential / cost R=H R=Methyl R=Ethyl R=Succinyl Other antimalarials S Resistance (25¢) OH N H 3C OH Atovaquone proguanil Quinine Amodiaquine F3C H2N Antifolates Sulphadoxine – pyrimethamine N HO N Cl CF3 Artemesinins Artemether Arteether Artesunate N HN Resistance (20¢) Safety / resistance Compliance / safety / resistance Resistance / safety / cost Safety / resistance / cost H H2C OH N Cl H N NH H N NH Proguanil CH3 CH3 Current Malaria Drug Targets CH3 N HN Cl O O H3C O Haem polymerisation / detoxification (Haemozoin) Chloroquine 4-Aminoquinolines and sesquiterpene endoperoxides O N H3C N Cl Artemisinins NH2 N NH2 Pyrimethamine Folate metabolism (DHFR / DHPS) Pyrimethamine / Sulphadoxine O O S H2N N H N CH3 RO N OCH3 OCH3 Sulphadoxine Haemoglobin Degradation Pathway PV Limited capacity to synthesise amino acids Trophozoite Need to scavenge from the host cell 60-80 % of haemoglobin digested in 48 h erythrocytic life cycle Cys, Glu, Gln, Ile, Met, Pro, Tyr are required Toxic by-products produced by this process must be dealt with FV Haemozoin (malaria pigment) N Pinocytosis (haemoglobin from the erythrocyte by pinocytosis) Erythrocyte N=nucleus; FV=food vacuole; PVM=parasitophorous vacuole Haemoglobin degradation pathway Food vacuole Cytoplasm 4 Aspartate protease (Plasmepsins I, II, IV and HAP) 3 Cysteine proteases (Falcipains 1-3) 1 Zinc metallopeptidase (Falcilysin) Haemoglobin globin Large peptides Aminopeptidases Small peptides Amino acids -Free haem is extremely toxic Free haem Fe2+ Superoxide dismutase O2 H2O2 H2O Peroxidases O2- -Can generate ROS -Is lipophilic and can intercalate into membranes causing cell lysis Haematin Fe3+ -Haematin Free haem metabolised to an inert chemical Haemozoin form called haemozoin by a process known as biomineralisation Kumar et al., Life Sciences 80 (2007) 813-828 Detoxification of Haematin into Inert Haemozoin Haematin -Haematin Haemozoin Free Fe2+ haem oxidation His-rich proteins and Phospholipids Fe3+ 1-4 linkages of haematin Membrane lysis Biomineralisation Dimers of haematin – 1-4 linkages are formed Dimers then begin to crystallise in a process known as biomineralisation to generate haemozoin Process not fully understood but is thought to be promoted by several factors including – the low pH of the food vacuole, association of haematin with histidine-rich proteins and phospholipids Ultimately haemozoin crystals are formed which are chemically inert and a safe storage mechanism for the parasite Artemisinin Combination Therapy (ACT) – current frontline therapy CH3 H3C O O O • Artemisinins reduce parasite burden rapidly • Used in combination with other drugs to protect emergence of resistance to partner drug (ACT) O CH3 RO Artemisia annua – sweet wormwood Youyou Tu Nobel Prize – Medicine 2015 Haem and Mode of Action of Artemisinins Haematin Artemesinin accumulates in the FV -Haematin Haemozoin Endoperoxide bridge CH3 H3 C O Cleavage of endoperoxide bridge by haem O O O CH3 O haem-artemesinin adducts (“haemarts”) Carbon-centred free radicals generated Possible targets of artemisinin free radicals: TCTP (translationally controlled tumour protein homolog) SERCA (sarco/endoplasmic reticulum Ca2+‡ -ATPase) Cysteine proteases Food Vacuole Mode of Action of Quinolines Haematin Induces oxidative stress -Haematin Haemozoin CQ adduct formation Membrane lysis Chloroquine CQ CQH+ H+ CQH2++ pH FV ~5.5 Accumulates following protonisation HN N pK = 10.41 Cl N pK = 8.11 Basic CQ V-type ATPase ATP H+ ADP pH cytosol ~7.2 Disruption of Folate Metabolism Aspartate + CO2 + PRPP Ser SHMT NADP+ H4F Pyrimethamine DHFR Gly NADPH Reduce Methylene and -H4F methylate folate Dihydropteroate synthase GTP H2F Sulphadoxine TS Uridine UMP TK dTMP dUMP O O HN Deoxyuridine monophosphateO HN N O Deoxyribose-P CH3 Deoxythymidine N monophosphate Deoxyribose-P RNA SHMT – serine hydroxymethyltransferase Thymidine DNA DHFR – dihydrofolate reductase Cell death TS- thymidylate synthase Antimalarials – mechanisms of resistance Emerging resistance to artemisinin in Plasmodium falciparum malaria - Recent studies have discovered emerging resistance to artesunate (artemisinin monotherapy) on the Thai-Cambodian border* - Average time taken to kill off parasites in the body following treatment increased from 48 hours to 84 hours in this area - Rates of infection recurrence following treatment had risen from 10% to 30% - Should this resistance spread from this geographical area – artemisinin could become completely useless in the treatment of this infection (disastrous!) *Dondrop et al., New England Journal of Medicine, 361, 455-467 Why is resistance developing? - In this area of Asia – public health system is weak and the use of anti-malarial drugs is uncontrolled - Non-compliance – sub-lethal drug concentrations in the body (antibiotics) - Ideal conditions for drug resistance to develop - In this area artemisinin is available as a monotherapy – Far easier for resistance to develop against a single drug (single mutation) than against a combination (chances of two advantageous mutations happening in one parasite exponentially higher) - Artesunate (oral artemisinin) should always be given in combination (mefloquine and amodiaquine often used) Drug Resistance Mechanisms – molecular basis Altered Drug Level Altered Target Level By exclusion Decreased import Increased export Modified Decreased affinity By sequestration Drug-binding molecule Compartmentalization By Metabolism Pro-drug not activated Drug inactivation Amplified Increased sequestration Increased enzyme activity Missing By-passed via salvage pathway Repaired / protected Increased damage repair Protected by metabolite Molecular basis of artemisinin resistance Artemisinin-resistant Plasmodium spp. have enhanced cell stress responses - survive environmental stressors and repair damage Resistance also associated with mutations in the Kelch 13 (K13) gene K13 proteins facilitate poly-ubiquitinylation of specific proteins – ubiquitinylated proteins then targeted for degradation by the proteasome A transcriptional regulator (Nrf2) which regulates the parasites response to oxidative stress is degraded in this way Mutation of K13 believed to reduce degradation of Nrf2 leading to enhanced anti-oxidant defences allowing the parasite to protect/repair artemisinin-induced oxidative damage Trends in Parasitology 2016; 32(9):682-96. Malaria drug resistance - molecular mechanisms Drug Gene Major mutations Mechanism Sulfadoxine DHPS A437G (K540E, A581G) Decreased affinity (higher Ki) for target Pyrimethamine DHFR S108N (N51I, C59R, I164L) Decreased affinity (higher Ki) for target Chloroquine MDR1 D86Y Increased efflux from FV Artemisinin K13 Multiple Increased repair and protection from damage Insecticides LLIN/ITN There is also growing resistance to the insecticide used on nets 45 countries have identified resistance to at lease one of the four classes of insecticides used Insecticide treated bed nets Antimalarials – the future The Ideal Antimalarial Drug (Target Product Profile) • • • • • • • • Active against resistant strains Inexpensive (< $2 / treatment; once daily; 3 days max) Long half-life (no recrudescence for at least 28 days post-treatment) Safe in pregnancy Safe in children Option of oral formulation Gametocytocidal (prevent transmission) Active against exo-erythrocytic (liver) stage of plasmodia where P. vivax is endemic Life Cycle Stages for Drug Intervention Prevent relapse (hypnozoite stage in P.vivax) Hypnozoite LIVER Reduce Transmission e.g. artemesinins Merozoites Schizont Ring BLOOD STAGES Schizont Gametocyte Trophozoite Curative treatment All drugs Drug Treatment Strategies • Curative treatment (erythrocytic stages) • Prevent relapse (P.vivax hypnozoite stage) • Reduce transmission (gametocytocidal agents) • Slow emergence of resistance (ACT policy) • Reduce pathology in pregnancy (IPTp) • Stimulate partial immunity in infants; reduce anaemia (IPTi) • Prophylactic treatment (travellers) DDD107498: New potent antimalarial in development P. falciparum blood stage EC50 = <1 nM, including resistant lines Nature 522, 315–320 (2015) University of Dundee with Monash University, Columbia University, Universities of South Florida, California, Washington & Imperial College, Swiss Tropical and Public Health Institute and Sanger Institute Potent against multiple life-cycle stages EC50 membrane feeding assay = 1.8 nM EC50 Liver = 1.8 nM EC50 Gametocytes ♂1.8 nM; ♀1.2 nM EC50 Blood = 1 nM Nature 522, 315–320 (2015) Inhibits protein synthesis in the parasites Potential single dose treatment University of Dundee with Monash University, Columbia University, Universities of South Florida, California, Washington & Imperial College, Swiss Tropical and Public Health Institute and Sanger Institute Malaria vaccines Ideal malarial vaccine • prevent infection in the first instance • reduce the clinical disease severity • reduce the rate of transmission • Low cost Minimum requirement - protect children (ages 0 - 5 years) - protect during pregnancy Problems in developing a vaccine • Four antigenically distinct malaria species – Each has ~6,000 genes • Immunity in malaria is complex and immunological responses/requirements for protection are incompletely understood • Identifying and assessing vaccine candidates takes time and is expensive • There is no clear ‘best approach’ for designing a malaria vaccine RTS,S vaccine development Sporozoites Surface of sporozoites covered with an antigen known as circumsporozoite protein (CSP) LIVER CSP is involved in hepatocyte binding Antibodies to CSP shown to protect against infection Original hybrid vaccine was created combining an independent T-cell epitope alongside the P. falciparum CS protein and hepatitis B surface antigen Included 16 tandem repeats of the epitope from the CS protein (RTS) RTS,S vaccine development - Structure/function of CSP highly conserved across the various strains of malaria Immunodominant region RTS later redesigned to include T- and B-cell epitopes from the C-terminus of the CS and was renamed to RTS,S RTS,S: ‘R’ for the CS “repeats” circumsporozoite protein structure ‘T’ for T-cell epitope ‘S’ for Hepatitis B antigen ‘S’ for genetically transformed yeast (Saccharomyces cerevisiae) used to express the vaccine Moorthy, V., & Ballou, R. (2009). Immunological Mechanisms Underlying Protection Mediated by RTS,S: a review of the available data. Malaria Journal, 8(312). RTS,S vaccine trials Children aged 5-17 months and babies 6-12 weeks recruited into trial Vaccinated +/- booster 18 month follow-up Results published 2012 and updated in 2015 N Engl J Med. (2012) 367:2284-95. A phase 3 trial of RTS,S/AS01 malaria vaccine in African infants. Lancet. (2015) 386:31-45. Efficacy and safety of RTS,S/AS01 malaria vaccine with or without a booster dose in infants and children in Africa: final results of a phase 3, individually randomised, controlled trial. RTS,S vaccine trial outcome Efficacy ranges from 26 to 50% in infants and young children Duration of protection – reduces significantly over time (18 months max) RTS,S vaccine approved in July 2015 for use in Africa for babies at risk from malaria RTS,S - the world's first approved malaria vaccine Caveats “Apparent protection …is modest both in extent and duration” in 5-17 month age group. Requires booster dose of vaccine to reduce severe malaria by 32.2% “After 20 months, vaccinated children who were not boosted showed an increased risk of severe malaria over the next 27 months compared with non-vaccinated controls.” No significant efficacy against severe malaria in 6-12 week age group Logistical and cost implications. Funding must not be directed from access to drugs (ACTs), rapid diagnostic tests, bed-nets (ITNs) and other control measures Reading list (SW) A novel multiple-stage antimalarial agent that inhibits protein synthesis. Baragaña et al. Nature. (2015) 522, 315-320 Efficacy and safety of RTS,S/AS01 malaria vaccine with or without a booster dose in infants and children in Africa: final results of a phase 3, individually randomised, controlled trial. RTS,S Clinical Trials Partnership. Lancet. (2015) 386, 31-45. Artemisinin Action and Resistance in Plasmodium falciparum. Tilley et al. Trends in Parasitology (2016) 32, 682-96. .