plasmodium lifecycle

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Dr. Alexis N. LaCrue and Dr. Dennis E. Kyle September 8, 2011

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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.

TRANSCRIPT

Page 1: Plasmodium Lifecycle

Dr. Alexis N. LaCrue and Dr. Dennis E. KyleSeptember 8, 2011

Page 2: Plasmodium Lifecycle

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

Page 3: Plasmodium Lifecycle
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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

Page 5: Plasmodium Lifecycle
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Reasons for Malaria Resurgence

• Insecticide resistant vectors

• Parasite drug resistance• Demographics • Economics and politics

Page 7: Plasmodium Lifecycle

• Vector control

• Vaccines

• Drugs

Page 8: Plasmodium Lifecycle
Page 9: Plasmodium Lifecycle

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].)

Page 10: Plasmodium Lifecycle

PERITROPHIC MATRIX

EPITHELIAL CELLS

OOCYST

BASAL LAMINA

SPOROZOITES

OOKINETE

ZYGOTEGAMETES

GAMETOCYTES

SALIVARY GLANDS

24hr PI

8-10 days

PI

14-16 days

PI

Page 11: Plasmodium Lifecycle

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

Page 12: Plasmodium Lifecycle

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

Page 13: Plasmodium Lifecycle

•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

Page 14: Plasmodium Lifecycle

The pupa is non-feeding, surface breathing, and is the stage of transition from aquatic larva to winged adult (24 hr)

Page 15: Plasmodium Lifecycle

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

Page 16: Plasmodium Lifecycle

1. Temperature2. Rainfall3. Relative Humidity4. Topography5. Soil Type

Factors that influence presence, abundance and longevity of mosquitoes.

Land cover(vegetation)

Page 17: Plasmodium Lifecycle
Page 18: Plasmodium Lifecycle

Entomologic Inoculation Rate (EIR) EIR = mosquito biting

rate times the proportion of infected mosquitoes

Sporozoite rates usually 1-20%

Page 19: Plasmodium Lifecycle
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Transgenic mosquitoes to block transmission of malaria

Page 24: Plasmodium Lifecycle
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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.

Page 27: Plasmodium Lifecycle

CREDIT: KATHARINE SUTLIFF/SCIENCE

Page 28: Plasmodium Lifecycle

•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.

Page 29: Plasmodium Lifecycle
Page 30: Plasmodium Lifecycle

Prophylaxis Causal prophylaxis Suppressive prophylaxis Post-exposure “prophylaxis”

Treatment of acute, uncomplicated malaria Treatment of severe malaria Radical cure Presumptive Intermittent Therapy (IPT)

Page 31: Plasmodium Lifecycle

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

Page 32: Plasmodium Lifecycle

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

Page 33: Plasmodium Lifecycle

WHO/UNICEF, 2005

Malaria-free areasSulfodoxine-pyrimethamine resistance

Chloroquine resistance Mefloquine resistance

ACT resistance-Thai cambodia border

Page 34: Plasmodium Lifecycle

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

Page 35: Plasmodium Lifecycle

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

Page 36: Plasmodium Lifecycle

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

Page 37: Plasmodium Lifecycle

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

Page 38: Plasmodium Lifecycle

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

Page 39: Plasmodium Lifecycle

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

Page 40: Plasmodium Lifecycle
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Survival of erythrocytic forms that leads to renewed manifestation

Page 42: Plasmodium Lifecycle
Page 43: Plasmodium Lifecycle

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).

Page 44: Plasmodium Lifecycle

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

Page 45: Plasmodium Lifecycle

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

Page 46: Plasmodium Lifecycle

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

Page 47: Plasmodium Lifecycle

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

Page 48: Plasmodium Lifecycle

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.

Page 49: Plasmodium Lifecycle

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

Page 50: Plasmodium Lifecycle

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

Page 51: Plasmodium Lifecycle

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

Page 52: Plasmodium Lifecycle

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

Page 53: Plasmodium Lifecycle
Page 54: Plasmodium Lifecycle

Compounds with low nM in vitro activity Schmatz Scouting protocol to screen for

compounds to use in the Thompson test

Overview

Page 55: Plasmodium Lifecycle

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

Page 56: Plasmodium Lifecycle

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)

Page 57: Plasmodium Lifecycle

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

Page 58: Plasmodium Lifecycle

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.

Page 59: Plasmodium Lifecycle
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Most of the drugs currently in use:

CQ, QN, AMO, MQ, AS, ATOV

Artemisinin derivatives

Primaquine

Page 61: Plasmodium Lifecycle

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

Page 62: Plasmodium Lifecycle

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

Page 63: Plasmodium Lifecycle

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!!

Page 64: Plasmodium Lifecycle

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”

Page 65: Plasmodium Lifecycle

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

Page 66: Plasmodium Lifecycle

Drug concentrations decrease from right to left

Page 67: Plasmodium Lifecycle

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

Page 68: Plasmodium Lifecycle

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

Page 69: Plasmodium Lifecycle

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

Page 70: Plasmodium Lifecycle

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

Page 71: Plasmodium Lifecycle

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

Page 72: Plasmodium Lifecycle

Untreated SQ (50 mg/kg)

PQ-50ORAL (50 mg/kg)

Drug 1

44HR PE

DAY 6PE

DAY 9PE

DAY 13P

E

Page 73: Plasmodium Lifecycle

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)

Page 74: Plasmodium Lifecycle

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

Page 75: Plasmodium Lifecycle

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