Plasmodium Lifecycle


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Presentation by Dr. Dennis E. Kyle and Dr. Alexis LaCrue from the University of South Florida on the Plasmodium Lifecycle for Stomping Out Malaria in Africa's Boot Camp trainings.

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  • There has been a resurgence of malaria and some of the reasons include: Drug Resistance In parts of South East Asia strains of malaria have developed resistance to anti-malarial drugs such as mefloquine and chloroquine. Insecticide resistance Mosquitoes are becoming resistant to commonly used insecticides such as DDT. Demographics can lead to a resurgence because, People traveling to and from places where malaria is endemic can be potential carriers of the disease. Politics and Economics Malaria-endemic countries are among the world ’ s most impoverished. A malaria-stricken family spends an average of over one quarter of its income on malaria treatment, as well as paying prevention costs and suffering loss of income because they cannot work when they are ill . The cost of malaria control and treatment can drain an economy. According to the WHO, it costs Arica $10-12 billion every year in gross domestic product.
  • With the increase in parasite resistance to drug treatments and insecticide resistant mosquitoes, novel control methods, are being looked at as a means of controlling malaria. Currently there is no vaccine against malaria. Drug resistance new drugs need to be developed………….. Insecticide treated bed nets to control the vector. Note: Nets can cut malaria transmission by at least 50% and child deaths by 20%; however, very few people have them due to the cost.
  • The transmission characteristics of regionally dominant malaria vector species were compared in the recent publication cited on this slide and used to explain regional differences in the force of transmission. This world map shows the tremendous diversity of vector species, both current and historic. Some colors represent only the potential for transmission, because malaria has been eradicated from most temperate zones of former endemicity, such as Europe and North America since ca WW II. Although this map differentiates between some members of species complexes (e.g. gambiae and arabiensis of the Gambiae Complex) others are not broken into their constituent species. I will be showing habitats of some of the South American vector shortly.
  • Box 1 Parasite stages under attack Current approaches to malaria vaccine development can be classified according to the parasite stages that are targeted: 1. Vaccines directed against sporozoites and/or liver stages (collectively termed pre-erythrocytic stages) are designed to prevent blood-stage infection and thereby avoid all manifestations of disease (anti-infection vaccines). 2. Vaccines directed against asexual blood stages are designed to reduce clinical severity (anti-morbidity/mortality vaccines). 3. Vaccines directed against mosquito stages are designed to halt development in the mosquito (transmission-blocking vaccines). Protective mechanisms of immunity are shown for each stage. In reality, the effects that can be anticipated for each type of vaccine overlap broadly: a pre-erythrocytic-stage vaccine, even if not 100% effective, could reduce transmission and morbidity — the latter is predicted by the reductions in morbidity and mortality associated with the use of insecticide-impregnated bednets; a highly effective blood-stage vaccine could eliminate blood stages as soon as they emerge from the liver, thereby curtailing both infection and transmission; and a transmission-blocking vaccine could reduce population-wide malaria infection rates and malaria-associated morbidity.
  • The fate of normal and attenuated malaria sporozoites in the host liver. A malaria infection is initiated by injection into the host blood of sporozoites by a female anopheline mosquito as she takes a blood meal. The sporozoites must migrate to the liver and colonize hepatocytes in order for the infection to progress. This involves traversal of resident macrophages (Kupffer cells) lining the liver's blood vessels and passage through a number of hepatocytes before invading a hepatocyte and beginning to develop. The sporozoite differentiates, grows, and multiplies within a vacuole in the host hepatocyte, giving rise to thousands of merozoites. These are released into the bloodstream where they invade red blood cells, initiating the erythrocytic stage of the disease. Sporozoites attenuated by irradiation (RAS) or by genetic manipulation (GAS) also transit through Kupffer cells and hepatocytes before invading liver cells. RAS undergo growth arrest but the infected hepatocytes remain intact, leading to the generation of a protective immune response involving B cells that attack free sporozoites and T cells that recognize infected hepatocytes. The intracellular development of GAS is different from that of RAS, as GAS-infected hepatocytes in culture disappear 24 hours after infection; the GAS-induced immune response may also be different. CREDIT: KATHARINE SUTLIFF/SCIENCE
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  • Artemisinin is effective against multi-drug resistant p. fal Qinghaosu used for the treatment of fever in Chinese traditional medicine for more than 2,000 years Front line treatment With ACTs, the duration of treatment is only 3 days (Maude et al) If used alone, the artemisinins will cure falciparum malaria in 7 days, but studies have shown that in combination with certain synthetic drugs they produce high cure rates in 3 days with higher adherence to treatment. (WHO Facts on ACTs January 2006 update)
  • Teuscher et al, tested different P. falciparum strains and found that only 0.04-1.313% of DHA treated parasites recover to resume growth. In an in vivo study, LaCrue et al found that recovery was based on the number of dormant parasites present in the host. So we know that dormancy occurs in vivo; however…
  • Following treatment with artemisinin, there are very few dormant parasites circulating in the peripheral blood possibly due to clearance by the spleen. This makes identification of dormant parasites difficult. Our goal is to..
  • Plasmodium Lifecycle

    1. 1. Dr. Alexis N. LaCrue and Dr. Dennis E. KyleSeptember 8, 2011
    2. 2.  2.1 billion live in malarious areas Affects 300-500 million people worldwide; one million deaths annually Transmitted by the bite of a female Anopheles mosquito Five species that affect humans: Plasmodium falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi
    3. 3. Principle clinical sign is periodic fever Paroxysm: relapsing or periodic fever Periodicity corresponds to erythrocytic development (48 or 72 hr)  benign tertian: P. vivax  malignant tertian : P. falciparum  quartan : P. malariae The pattern of intermittent chills/fever mirrors the synchronized parasite development in an infected person’s blood This response is primarily due to toxins released with schizont rupture
    4. 4. Reasons for MalariaResurgence • Insecticide resistant vectors • Parasite drug resistance • Demographics • Economics and politics
    5. 5. • Vector control• Vaccines• Drugs
    6. 6. Global distribution of dominant or potentially important malaria vectors. This map does not include all regionallyimportant vectors or species complexes. (Ex Kiszewski et al. [2004] Am. J. Trop. Med. Hyg. 70[5].)
    8. 8.  Distribution  Over 420 species, most in tropics and subtropics  Temperate and summer arctic distribution  ~70 species capable of transmission (40 important) Feeding habits  Female requires blood meals for egg broods  Males feed on nectar Life cycle – 7 to 20 days (egg to adult)  egg > larva > pupa > adult  Females mate once and lay 200-1000 eggs in 3-12 batches over a lifetime  Find their host by chemical and physical stimuli  Average life span of mosquito < 3 weeks Malaria development – 7 to 12 days  Each male & female gametocyte produce >10,000 sporozoites
    9. 9. Eggs are Deposited Singly on Water Surface Lateral floats function to keep eggs on water surface Drawing of Anopheles eggs Chorion of egg is sculpted Larval embryogenesis (72 hr) and hatching must occur in 4 days
    10. 10. •Four larval stages (instars)•Feed on microbes•Breath at the water surface•Lie horizontally at the water surface•One week to months, temperaturedependent Non-wetable spiracle opens at water surface for respiration
    11. 11. The pupa is non-feeding, surface breathing, and is the stage oftransition from aquatic larva to winged adult (24 hr)
    12. 12.  Fully formed adult mosquito emerges from pupal stage at water surface. Males often emerge first and form swarms, can’t copulate until genitalia rotate 180° Females emerge, enter swarm, copulate in the air Females may mate more than once Sperm is stored in the spermatheca for lifetime Males feed on nectar, females primarily on blood Aestivation in adult females  cessation or slowing of activity in winter; especially slowing of metabolism
    13. 13. Factors that influence presence, abundance and longevity of mosquitoes. 1. Temperature 2. Rainfall 3. Relative Humidity Land cover 4. Topography (vegetation) 5. Soil Type
    14. 14.  Entomologic Inoculation Rate (EIR)  EIR = mosquito biting rate times the proportion of infected mosquitoes  Sporozoite rates usually 1-20%
    15. 15. Transgenic mosquitoes toblock transmission ofmalaria
    16. 16. Currently, there is no vaccine.3. Pre-erythrocytic/anti-infection vaccines • Directed against sporozoites and/or liver stages • Abundant surface proteins (CSP, TRAP) • Attenuated sporozoites • Designed to prevent blood-stage infection and thereby avoid all manifestations of disease.2. Anti-morbidity/mortality vaccines • Vaccines directed against asexual blood stages • Designed to reduce clinical severity.3. Transmission blocking vaccines • Directed against mosquito stages • Designed to halt development in the mosquito Protective mechanisms of immunity are shown for each stage. • Would be used in combination with other vaccines and/or drug therapy
    18. 18. •Does not treat the human but prevents infection•Better to have a vaccine or drug that does both.•We have new drug candidates that may do both.
    19. 19.  Prophylaxis  Causal prophylaxis  Suppressive prophylaxis  Post-exposure “prophylaxis” Treatment of acute, uncomplicated malaria Treatment of severe malaria Radical cure Presumptive Intermittent Therapy (IPT)
    20. 20. Malaria: progress, perils, and prospects for eradicationBrian M. Greenwood, David A. Fidock, Dennis E. Kyle, Stefan H.I. Kappe, Pedro L. Alonso, FrankH. Collins, Patrick E. Duffy. J Clin Invest. 2008;118(4):1266–1276 doi:10.1172/JCI33996
    21. 21. 1. Cinchona alkaloids 7. Sulfonamides quinine sulfadoxine (with quinidine pyrimethamine)2. 4-aminoquinolones 8. Sulfones chloroquine dapsone (with chlorproguanil) amodiaquine 9. Antifols3. 4-quinolinemethanol pyrimethamine mefloquine proguanil4. 8-aminoquinolines 10. Artemisinins primaquine 11. Antibiotics5. Phenanthrenemethanols Doxycycline halofantrine Tetracycline6. Biguanides Azithromycin proguanil
    22. 22. ACT resistance-Thai cambodia border Chloroquine resistance Mefloquine resistance Sulfodoxine-pyrimethamine resistance Malaria-free areasWHO/UNICEF, 2005
    23. 23. Year of 1st case of Antimalarial drug introduction resistance Quinine 1632 1910 Chloroquine 1945 1957 12 years Proguanil 1948 1949 1 year Sulfadoxine- pyrimethamine 1967 1967 <1 year Mefloquine 1977 1982 5 years Atovaquone 1996 1996 <1 year
    24. 24.  Treatment  Result  Halofantrine  Recrudesced at 4 wk  Quinine (iv) plus  MFQ prophylaxis, halofantrine Recrudesced ~3 wk  Quinine (iv) followed  Cleared yet by Quinine (po) and recrudesced 7d after Rx Doxycycline , but asymptomatic  Halofantrine (po) with  Recrudesced at 12 days whipping cream!
    25. 25.  Treatment  Result  Mefloquine plus Doxycycline  Recrudesced at day 27 (7 days)  Artesunate (po) followed by  Success at last! Mefloquine “This case of imported multi-drug resistant falciparum malaria shows that artemisinin and derivatives will soon be needed, in fact are already needed, in the western world.” Lancet 1994
    26. 26.  Common name: Qinghaosu Isolated from: Chinese herb Artemisia annua Characteristics: 1. Rapidly kills asexual stages 2. Short half-life 3. Frequent recrudescence when used as monotherapy WHO recommendation: Use in combination w/ other antimalarials which have a longer half-life  Artemisinin Combination Therapy (ACT)  High cure rate in 3 days
    27. 27.  Severe malaria disease High levels of parasites in the blood Inability to take oral medications Lack of timely access to intravenous quinidine Quinidine intolerance or contraindications Quinidine failure
    28. 28.  Recrudescence rates  5 days of treatment < 10%  3 days of treatment 40% - 70%  1 day of treatment > 90% Recrudescent parasites remain susceptible to drug in in vitro drug susceptibility tests
    29. 29. Survival of erythrocytic forms that leads to renewed manifestation
    30. 30. In vitro Evidence In vivo Evidence Recovery rates ranging from 0.044% to 1.313% Recovery based on number of dormant parasites present in host Teuscher et al. (2010). JID 202: 1362-1368 LaCrue et al. (2011).
    31. 31. Problem: Very few in peripheral blood Difficult to identify in blood smears Want to be able to easily identify in the fieldGoal:Develop a method that will enhance the detection of dormantparasites in patient samples and thick smears 1.Faster identification and quantification of dormant parasites (i.e. distinguish dormant rings from rings, merozoites, Howell Jolly bodies) 2.Determine if there is a correlation between the number of dormant rings in the first 72hr of treatment and recrudescence 3.Predict optimal dosing regimen of artemisinin combinations
    32. 32.  Not for profit partnership established in Switzerland in 1999 Mission: To reduce burden of malaria in endemic countries through the development of novel and effective anti-malarials Vision: To have a malaria-free
    33. 33. MMV- R and D Lead identification (in vitro high throughput screens and in vivo studies) Lead optimization (in vitro and in vivo studies to identify absorption, distribution, metabolism, and excretion characteristics) Preclinical development and candidate selection (in vitro and in vivo studies to assess safety in humans) Clinical Phase I (volunteers administered increasing doses of drug; adverse effects assessed) Clinical Phase II (Proof of concept- small group of patients) Clinical Phase III (Large group of patients) Registration and Launch
    34. 34. MMV- R and D Lead identification (in vitro high throughput screens and in vivo studies) Lead optimization (in vitro and in vivo studies to identify absorption, distribution, metabolism, and excretion characteristics) Preclinical development and candidate selection (in vitro and in vivo studies to assess safety in humans) Clinical Phase I (volunteers administered increasing doses of drug; adverse effects assessed) Clinical Phase II (Proof of concept- small group of patients) Clinical Phase III (Large group of patients) Registration and Launch
    35. 35.  To determine if compounds from the Roman Manetsch lab at USF and the Michael Riscoe lab at the Portland Veterans Affairs Medical Center have anti-malarial activity in vivo and in vitro.
    36. 36. O O O O 5 8 9 1 5 4 3 R 4 Interested in exploring the anti-malarial activity of 3 6 7 2 6 OR R 2 R R O 2 7 N1 6 N10 3 O 7 N1 quinolones and tetrahydroacridones 8 H 5 H 4 8 H 4Q THA PEQ Endochin,- an experimental anti-malarial quinolone from O O O the 1940s O O O N H  Recently shown to have poor activity in mammalian O N H endochin ICI56,780 systems (RMMC103) (RMMC128) ICI 56, 780- Screened by Walter Reed and shown to have activity against liver stages in P. cynomolgi in 1970s Tetrahydroacridones (THAs)- anti-malarial activity known since 1940s Name Type Activity Endochin 4(1H)-quinolone (4Q) EE and erythrocytic activity in avians not mammals ICI 56,780 (RMMC128) Phenoxy-ethoxy-4(1H)-quinolone (PEQ) EE stages in monkeys RMMC93 Tetrahydroacridone (THA) In vitro erythrocytic activity and gametocyte activity  Potent blood stage activity and demonstrated potential to kill hypnozoites makes the 4Qs, THAs and PEQs ideal for novel drug development.  Novel antimalarials were subjected to an in vitro high throughput screen and those with low nanomolar activity were selected for in vivo studies.
    37. 37. Team: USF and Portland Manetsch lab Riscoe lab Team: USF and Portland Manetsch lab Riscoe lab Team: USF Kyle labHit Validation Kyle lab Manetsch and RiscoeHit to Lead Kyle lab Charman labEarly LeadCandidate
    38. 38.  SKYPE Google documents (free)  Everyone must have a google account to access  Share folders and calendars  Share excel, word, powerpoint, and pdf-like documents  export to microsoft excel, microsoft word, or adobe acrobat  work on documents at the same time
    39. 39.  Microsoft Sharepoint (paid)  Must be given access to a server  Share folders and calendars  Share microsoft excel, word, powerpoint and adobe acrobat documents  Great for working from the same files  Create team sites within a main site  Give specific permissions to documents, folders, and sites
    40. 40. Overview Compounds with low nM in vitro activity Schmatz Scouting protocol to screen for compounds to use in the Thompson test
    41. 41. Methods- Thompson Test Infect Mice with 1x106 Plasmodium berghei-GFP parasites Day 3-5 PE-Treat mice with 10 mg/kg and 50 mg/kg of compound diluted in PEG400 Day 3,6,9,13,21 and 30PE-Check parasitemia via flow cytometry Euthanize when animals reach >40% Compounds with ≥90% reduction in parasitemia parasitemia go to GSK
    42. 42. Compound 4 C= CURATIVE (100% INHIBITION) A= ACTIVE (>80% INHIBITION) S= SUPPRESSIVE (20-80% INHIBITION) N= NOT ACTIVE (<20% INHIBITION) Drug Dose per day (3 days) Vehicle Route Activity (day 30PE) UNTREATED None None None N/A AMODIAQUINE 30 mg/kg PEG400 Oral C ATOVAQUONE 50 mg/kg PEG400 Oral C (0.3MG/KG) 10 mg/kg PEG400 Oral N (1.0MG/KG) 50 mg/kg PEG400 Oral C (3.0MG/KG) 50 mg/kg PEG400 Oral C (10.0MG/KG) 50 mg/kg PEG400 Oral C
    43. 43. Summary- Thompson Test Group Drug Dosages per day Vehicle Route Activity (3 days): -1,0, +1 Control Untreated None None N/A Experimental 1 10 or 50 mg/kg PEG 400 PO Suppressive at 50 mg/kg Experimental 2 10 or 50 mg/kg PEG400 PO Suppressive at both concentrations Experimental 3 10 or 50 mg/kg PEG 400 PO CURATIVE Experimental 4 0.3-10 mg/kg PEG 400 PO CURATIVE Compound 4 was selected for further modifications to improve the bioavailability and produce a lead candidate.
    44. 44. Thompson Test-Results Group Drug Dosages per day Vehicle Route Activity (3 days): -1,0, +1 Experimental 4 0.3-10 mg/kg PEG 400 PO CURATIVE (all) Experimental 5 0.3-10mg/kg PEG 400 PO CURATIVE (October 2010) (all) Experimental 6 0.3-10mg/kg PEG 400 PO CURATIVE (October 2010) (3 and 10mg/kg) Experimental 7 0.3-10mg/kg PEG 400 PO CURATIVE (November 2010) (0.3-10mg/kg) Experimental 8 0.3-10mg/kg PEG400 PO CURATIVE (November 2010) (1-10mg/kg) Experimental 9 0.3-10mg/kg PEG400 PO CURATIVE (December 2010) (1-10mg/kg) Experimental 10 0.3-10mg/kg PEG400 PO CURATIVE (December 2010) (all) Experimental 11 0.3-10mg/kg PEG 400 PO CURATIVE (January 2011) (all) Experimental 12 0.3-10mg/kg PEG 400 PO CURATIVE (January 2011) (1-10mg/kg) Compound 4 and 11 are now pre-clinical lead candidates.
    45. 45. Primaquine Most of the drugs currently in use: CQ, QN, AMO, MQ, AS, ATOVArtemisininderivatives
    46. 46. Plasmodium berghei- infected mouse 6-7 days Naïve female Anopheles stephensi takes a blood meal 20-24 days Dissect infected Sgs and purify sporozoitesDose mice Inject 10,000 Dose mice Follow for (Day -1) spz/mouse + Dose (Day +1) 30 days (Day 0)
    47. 47. Group Drug Dosages per day Vehicle Route Activity (3 days): -1,0, +1 Infection Untreated None None N/A controlDrug control 1 50 mg/kg 10% DMSO, 0.5% SC CURATIVE TweenDrug control PRIMAQUINE 50 mg/kg PEG400 PO CURATIVE Test 2 10 and 50 mg/kg PEG 400 PO NONE Test 3 10 or 50 mg/kg PEG 400 PO CURATIVE Test 4 3-10 mg/kg PEG 400 PO CURATIVE
    48. 48.  In-direct method- Assessment of pre-patent period (i.e. time from injection of sporozoites to peripheral blood infection)  Pros:  All or none effect of drug or vaccine  Cons:  Complicated if drug effects liver and erythrocytic forms Direct methods- Study of parasites in vivo (i.e. examination of liver sections, QRT- PCR, flow cytometry, intra-vital imaging)  Pros:  Significant advances for detecting liver stages  Cons:  Mice must be sacrificed. Prevents long term study of infection.  Labor intensive (requires lots of mice to achieve statistical power)  Expensive LET’S TRY BIOLUMINESCENCE!!
    49. 49. “production and emission of light by a living organism as a result of a chemical reaction”Sea pansy produces GFP protein Firefly  Non-invasive study of on-going biological processes  No surgery neededSquid with bioluminescent bacteria  Captures light emitted by the reactions of luciferase and its substrate D-luciferan  Firefly – attract a mate  Sea pansy- repel predators  Bacteria- repel predatorsBioluminescent marine bacteria  Common applications of BLI:  Studies of infection using bioluminescent pathogens  Studies of cancer progression using bioluminescent cell line  Stem cell research
    50. 50. Plasmodium berghei- infect donor mice 6-7 days Naïve female Anopheles stephensi takes a blood meal 20-24 days Dissect infected Sgs and purify sporozoitesSeed into 96 well plate, 1,500 sporozoites per well & HepG2 cells
    51. 51. Drug concentrations decrease from right to left
    52. 52. Mice infected with parasite Injected with D-luciferin Mice anesthetized and placedcontaining luciferase gene which is oxidized by into Xenogen IVIS Spectrum ATP only present in living cells so the luciferase + ATP (Imaged for 5-60 seconds) reaction allows for measurement of energy or life Imaged for 5-60 seconds
    53. 53.  Parasite used: P. berghei ANKA 1052 cl1: GFP and luciferase under the control of AMA-1 promoter (Leiden) Compound Dose (mg/kg) Route Untreated N/A N/A 1 50 Sub-q 1 50 Oral 2 10 Oral 2 50 Oral and Sub-q 2 100 Oral 3 3 Oral 3 10 Oral
    54. 54. Plasmodium berghei- infected mouse 6-7 days Naïve female Anopheles stephensi takes a blood meal 20-24 days Dissect infected Sgs and purify sporozoitesDose mice Inject 10,000 Dose mice BLI (Day -1) spz/mouse + Dose (Day +1) (44hr and Days 6,9,13 PE) (Day 0)
    55. 55. 44hr post-infection Inject sporozoite-infected mice with D-luciferin (100mg/kg) Anesthetize for 5 min with isofluorane Image mice using the IVIS spectrum system Liver collection group: Blood-stage group:Euthanize and image livers Follow days 6, 9, 13, 21, and 30PE
    56. 56. BLI-LIVER STAGE ACTIVITY Drug 1 Drug 2Untreated SQ (50 mg/kg) (50 mg/kg) (10 mg/kg) (50 mg/kg) PQ-50 1 2 1 2 1 2 1 2 1 2 1 21 12 2
    57. 57. Drug 1 Untreated SQ (50 mg/kg) ORAL (50 mg/kg) PQ-5044HR PEDAY6PEDAY9PEDAY13PE
    58. 58. Compound Dose (mg/kg) Route Activity (n=5)Untreated N/A N/A N/A1 50 Sub-q Curative (all)1 50 Oral Curative (all)2 10 Oral Curative (3)2 50 Oral Curative (4)2 50 Sub-q Curative (4)2 100 Oral Curative (4)3 3 Oral Curative (all)3 10 Oral Curative (all)
    59. 59.  We have screened more than 100 compounds since April 2009 Found compounds with blood, liver, gametocyte, and mosquito stage activity Best 2 analogs have moved forward as pre- clinical leads
    60. 60. Acknowledgments  Kyle lab  Dennis E. Kyle (PI)  Tina Mutka (research assistant)  Ken Udenze (graduate student)  Steven Stein (graduate student)  Manetsch lab  Roman Manetsch  Matt Cross  Andrii Monastyrskyi  Jordany Maignan  Riscoe Lab  Portland, Oregon  PK studies  Monash University, Sue Charman  Parasites  Leiden University  Funding  Medicines for Malaria Ventures (MMV)