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.
1. Dr. Alexis N. LaCrue and Dr. Dennis E. Kyle
September 8, 2011
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
4. 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
6. Reasons for Malaria
Resurgence
• Insecticide resistant vectors
• Parasite drug resistance
• Demographics
• Economics and politics
9. Global distribution of dominant or potentially important malaria vectors. This map does not include all regionally
important vectors or species complexes. (Ex Kiszewski et al. [2004] Am. J. Trop. Med. Hyg. 70[5].)
10. GAMETOCYTES
GAMETES
14-16 ZYGOTE 24hr
days PI
PI
OOKINETE
SALIVARY 8-10
GLANDS days PI
PERITROPHIC MATRIX
SPOROZOITES
OOCYST EPITHELIAL
CELLS
BASAL LAMINA
11. 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
12. 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
13. •Four larval stages (instars)
•Feed on microbes
•Breath at the water surface
•Lie horizontally at the water surface
•One week to months, temperature
dependent
Non-wetable spiracle opens at water surface for respiration
14. The pupa is non-feeding, surface breathing, and is the stage of
transition from aquatic larva to winged adult (24 hr)
15. 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
16. Factors that influence presence, abundance and longevity of mosquitoes.
1. Temperature
2. Rainfall
3. Relative Humidity Land cover
4. Topography (vegetation)
5. Soil Type
18. Entomologic Inoculation
Rate (EIR)
EIR = mosquito biting rate
times the proportion of
infected mosquitoes
Sporozoite rates usually 1-20%
26. 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
31. Malaria: progress, perils, and prospects for eradication
Brian M. Greenwood, David A. Fidock, Dennis E. Kyle, Stefan H.I. Kappe, Pedro L. Alonso, Frank
H. Collins, Patrick E. Duffy. J Clin Invest. 2008;118(4):1266–1276 doi:10.1172/JCI33996
34. 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
35. 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!
36. 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
37. 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
38. 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
39. 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
43. 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).
44. Problem:
Very few in peripheral blood
Difficult to identify in blood smears
Want to be able to easily identify in the field
Goal:
Develop a method that will enhance the detection of dormant
parasites 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
45. 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 world
www.mmv.org
46. 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
47. 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
48. 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.
49. 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.
50. Team: USF and
Portland
Manetsch lab
Riscoe lab
Team: USF and
Portland
Manetsch lab
Riscoe lab
Team: USF
Kyle lab
Hit Validation
Kyle lab Manetsch and Riscoe
Hit to Lead
Kyle lab Charman lab
Early Lead
Candidate
51. 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
52. Microsoft Sharepoint (paid)
http://sharepoint.microsoft.com/en-us/Pages/default.aspx
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
54. Overview
Compounds with low nM in vitro activity
Schmatz Scouting protocol to screen for
compounds to use in the Thompson test
55. 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
56. 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
57. 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.
58. 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.
60. Primaquine
Most of the drugs
currently in use:
CQ, QN, AMO, MQ,
AS, ATOV
Artemisinin
derivatives
61. Plasmodium berghei-
infected mouse
6-7 days
Naïve female
Anopheles stephensi
takes a blood meal
20-24 days
Dissect infected Sgs
and purify
sporozoites
Dose mice Inject 10,000 Dose mice Follow for
(Day -1) spz/mouse + Dose (Day +1) 30 days
(Day 0)
62. Group Drug Dosages per day Vehicle Route Activity
(3 days):
-1,0, +1
Infection Untreated None None N/A
control
Drug control 1 50 mg/kg 10% DMSO, 0.5% SC CURATIVE
Tween
Drug 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
63. 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!!
64. “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 needed
Squid 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 predators
Bioluminescent marine bacteria
Common applications of BLI:
Studies of infection using bioluminescent
pathogens
Studies of cancer progression using
bioluminescent cell line
Stem cell research
65. Plasmodium berghei-
infect donor mice
6-7 days
Naïve female
Anopheles stephensi
takes a blood meal
20-24 days
Dissect infected Sgs
and purify
sporozoites
Seed into 96 well plate,
1,500 sporozoites per
well & HepG2 cells
67. Mice infected with parasite Injected with D-luciferin Mice anesthetized and placed
containing 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
68. 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
69. Plasmodium berghei-
infected mouse
6-7 days
Naïve female
Anopheles stephensi
takes a blood meal
20-24 days
Dissect infected Sgs
and purify
sporozoites
Dose mice Inject 10,000 Dose mice BLI
(Day -1) spz/mouse + Dose (Day +1) (44hr and Days 6,9,13 PE)
(Day 0)
70. 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
74. 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
75. 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)
Editor's Notes
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..