Download Introduction to malaria and chemotherapy

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.
.