Plasmodium Lifecycle
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Transcript of Plasmodium Lifecycle
Dr. Alexis N. LaCrue and Dr. Dennis E. KyleSeptember 8, 2011
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
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
Reasons for Malaria Resurgence
• Insecticide resistant vectors
• Parasite drug resistance• Demographics • Economics and politics
• Vector control
• Vaccines
• Drugs
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].)
PERITROPHIC MATRIX
EPITHELIAL CELLS
OOCYST
BASAL LAMINA
SPOROZOITES
OOKINETE
ZYGOTEGAMETES
GAMETOCYTES
SALIVARY GLANDS
24hr PI
8-10 days
PI
14-16 days
PI
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
Eggs are Deposited Singly on Water Surface
Lateral floats function to keepeggs on water surface
Chorion of egg is sculpted
Drawing of Anopheles eggs
Larval embryogenesis (72 hr) and hatching must occur in 4 days
•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
The pupa is non-feeding, surface breathing, and is the stage of transition from aquatic larva to winged adult (24 hr)
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
1. Temperature2. Rainfall3. Relative Humidity4. Topography5. Soil Type
Factors that influence presence, abundance and longevity of mosquitoes.
Land cover(vegetation)
Entomologic Inoculation Rate (EIR) EIR = mosquito biting
rate times the proportion of infected mosquitoes
Sporozoite rates usually 1-20%
Transgenic mosquitoes to block transmission of malaria
Currently, there is no vaccine.
1. 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• Would be used in combination
with other vaccines and/or drug therapy
Protective mechanisms of immunity are shown for each stage.
CREDIT: KATHARINE SUTLIFF/SCIENCE
•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.
Prophylaxis Causal prophylaxis Suppressive prophylaxis Post-exposure “prophylaxis”
Treatment of acute, uncomplicated malaria Treatment of severe malaria Radical cure Presumptive Intermittent Therapy (IPT)
Malaria: progress, perils, and prospects for eradicationBrian 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
1. Cinchona alkaloidsquininequinidine
2. 4-aminoquinoloneschloroquineamodiaquine
3. 4-quinolinemethanolmefloquine
4. 8-aminoquinolinesprimaquine
5. Phenanthrenemethanols
halofantrine6. Biguanides
proguanil
7. Sulfonamidessulfadoxine
(with pyrimethamine)8. Sulfones
dapsone (with
chlorproguanil)9. Antifols
pyrimethamineproguanil
10. Artemisinins11. Antibiotics
DoxycyclineTetracyclineAzithromycin
WHO/UNICEF, 2005
Malaria-free areasSulfodoxine-pyrimethamine resistance
Chloroquine resistance Mefloquine resistance
ACT resistance-Thai cambodia border
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
Antimalarial drugYear of
introduction1st case ofresistance
Treatment Halofantrine Quinine (iv) plus
halofantrine Quinine (iv)
followed by Quinine (po) and Doxycycline
Halofantrine (po) with whipping cream!
Result Recrudesced at 4
wk MFQ prophylaxis,
Recrudesced ~3 wk
Cleared yet recrudesced 7d after Rx , but asymptomatic
Recrudesced at 12 days
Treatment Mefloquine plus
Doxycycline (7 days) Artesunate (po)
followed by Mefloquine
Result Recrudesced at day
27
Success at last!
“This case of imported multi-drug resistant falciparum malaria shows that artemisinin and derivatives will soonbe needed, in fact are already needed, in the westernworld.” Lancet 1994
Common name: Qinghaosu Isolated from: Chinese herb Artemisia annua Characteristics:
1. Rapidly kills asexual stages2. Short half-life3. 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
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
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
Survival of erythrocytic forms that leads to renewed manifestation
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).
Problem: Very few in peripheral blood
Difficult to identify in blood smearsWant 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 recrudescence3.Predict optimal dosing regimen of artemisinin combinations
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
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
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
MMV- R and D
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.
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.
NH
O
R
R'NH
O
R'
4Q PEQ
R
1
2
3456
78
12
345
6
78 9
10 NH
O
1
2
3456
78
OR
O
OO
THA
NH
O
O
endochin (RMMC103)
NH
O
O
O
ICI56,780(RMMC128)
O
O
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
Interested in exploring the anti-malarial activity of quinolones and tetrahydroacridones
Endochin,- an experimental anti-malarial quinolone from the 1940s
Recently shown to have poor activity in mammalian systems
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
Hit Validation
Early Lead Candidate
Hit to LeadKyle lab
Team: USFKyle lab
Team: USF and PortlandManetsch labRiscoe lab
Team: USF and PortlandManetsch labRiscoe lab
Charman lab
Kyle lab Manetsch and Riscoe
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
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
Compounds with low nM in vitro activity Schmatz Scouting protocol to screen for
compounds to use in the Thompson test
Overview
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% parasitemia
Compounds with ≥90% reduction in parasitemia go to
GSK
Methods- Thompson Test
Compound 4
Drug Dose per day (3 days) Vehicle Route
Activity (day 30PE)
UNTREATED None None None N/AAMODIAQUINE 30 mg/kg PEG400 Oral CATOVAQUONE 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
C= CURATIVE (100% INHIBITION)A= ACTIVE (>80% INHIBITION)S= SUPPRESSIVE (20-80% INHIBITION)N= NOT ACTIVE (<20% INHIBITION)
Group Drug Dosages per day
(3 days): -1,0, +1
Vehicle Route Activity
Control Untreated None None N/AExperiment
al1 10 or 50
mg/kgPEG 400 PO Suppressive at
50 mg/kgExperiment
al2 10 or 50
mg/kgPEG400 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.
Summary- Thompson Test
Group Drug Dosages per day
(3 days): -1,0, +1
Vehicle Route Activity
Experimental 4 0.3-10 mg/kg PEG 400 PO CURATIVE(all)
Experimental(October
2010)
5 0.3-10mg/kg PEG 400 PO CURATIVE(all)
Experimental(October
2010)
6 0.3-10mg/kg PEG 400 PO CURATIVE(3 and 10mg/kg)
Experimental(November
2010)
7 0.3-10mg/kg PEG 400 PO CURATIVE (0.3-10mg/kg)
Experimental(November
2010)
8 0.3-10mg/kg PEG400 PO CURATIVE(1-10mg/kg)
Experimental(December
2010)
9 0.3-10mg/kg PEG400 PO CURATIVE(1-10mg/kg)
Experimental(December
2010)
10 0.3-10mg/kg PEG400 PO CURATIVE(all)
Experimental(January
2011)
11 0.3-10mg/kg PEG 400 PO CURATIVE(all)
Experimental(January
2011)
12 0.3-10mg/kg PEG 400 PO CURATIVE(1-10mg/kg)
Thompson Test-Results
Compound 4 and 11 are now pre-clinical lead candidates.
Most of the drugs currently in use:
CQ, QN, AMO, MQ, AS, ATOV
Artemisinin derivatives
Primaquine
20-24 days
Plasmodium berghei-infected
mouse
Naïve female Anopheles
stephensi takes a blood meal
Dissect infected Sgs and purify
sporozoites
Inject 10,000 spz/mouse +
Dose(Day 0)
Dose mice
(Day -1)
Dose mice
(Day +1)
Follow for 30 days
6-7 days
Group Drug Dosages per day
(3 days): -1,0, +1
Vehicle Route Activity
Infection control
Untreated None None N/A
Drug control
1 50 mg/kg 10% DMSO, 0.5% Tween
SC CURATIVE
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
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!!
Non-invasive study of on-going biological processes No surgery needed
Captures light emitted by the reactions of luciferase and its substrate D-luciferan Firefly – attract a mate Sea pansy- repel predators Bacteria- repel predators
Common applications of BLI: Studies of infection using
bioluminescent pathogens Studies of cancer progression using
bioluminescent cell line Stem cell research
Sea pansy produces GFP protein
Squid with bioluminescent bacteria
Bioluminescent marine bacteria
Firefly
“production and emission of light by a living organism as a result of a chemical reaction”
20-24 days
Plasmodium berghei-infect
donor mice
Naïve female Anopheles
stephensi takes a blood meal
Dissect infected Sgs and purify
sporozoites
Seed into 96 well plate, 1,500
sporozoites per well & HepG2 cells
6-7 days
Drug concentrations decrease from right to left
Mice infected with parasite containing luciferase gene
Injected with D-luciferin which is oxidized by luciferase + ATP
ATP only present in living cells so the reaction allows for measurement of energy or life
Mice anesthetized and placed into Xenogen IVIS Spectrum(Imaged for 5-60 seconds)
Imaged for 5-60 seconds
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
20-24 days
Plasmodium berghei-infected
mouse
Naïve female Anopheles
stephensi takes a blood meal
Dissect infected Sgs and purify
sporozoites
Inject 10,000 spz/mouse +
Dose(Day 0)
Dose mice
(Day -1)
Dose mice
(Day +1)
BLI (44hr and Days
6,9,13 PE)
6-7 days
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
Blood-stage group: Follow days 6, 9, 13, 21, and 30PE
Liver collection group: Euthanize and image livers
Untreated SQ (50 mg/kg) PQ-50(50 mg/kg) (10 mg/kg) (50 mg/kg)
BLI-LIVER STAGE ACTIVITY
Drug 1 Drug 2
1 21 21 21 21 21 2
1
2
1
2
Untreated SQ (50 mg/kg)
PQ-50ORAL (50 mg/kg)
Drug 1
44HR PE
DAY 6PE
DAY 9PE
DAY 13P
E
Compound Dose (mg/kg) Route Activity (n=5)
Untreated N/A N/A N/A
1 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)
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
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)
Acknowledgments