1. Parasitology 2017 Susan Wyllie
Introduction to malaria – chemotherapy and vaccines
Susan Wyllie

2. 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

3. 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

4. Life Cycle of Plasmodium falciparum
Sporozoites (5-100)
MOSQUITO SALIVARY GLANDS
Oocyst
Ookinete
LIVER
Zygote
Merozoites
( )
Gametes
Schizont
Ring
MOSQUITO GUT
BLOOD STAGES
(pathology - fevers)
Schizont
Gametocyte
Trophozoite
haemoglobin digestion

5. Malaria Control Strategies
Vaccines
Drugs
Vector control methods:
Barrier
Insecticides
Biological
Environmental
Vaccines
Drugs & Vaccines
Drugs
Infected erythrocytes
Insecticide treated bed nets

6. Drugs for Malaria Quinoline and aminoalcohols Quinolines, etc.
Chloroquine
Amodiaquine
Quinine
Mefloquine
Halofantrine
Resistance (20¢)
Safety / resistance
Compliance / safety / resistance
Resistance / safety / cost
Safety / resistance / cost
Artemesinins
Artemether
Arteether
Artesunate
Compliance / safety /
availability / cost of raw material
ACT – combinations
Lumefantrine – artemether
(Coartem)
Resistance potential / compliance / cost ($2.40) / availability
Antifolates
Sulphadoxine – pyrimethamine
Resistance (25¢)
Atovaquone - proguanil
Resistance potential / cost
Chloroquine
Quinine
Halofantrine
Artemesinin analogues
Other antimalarials
Proguanil
Atovaquone

7. Current Malaria Drug Targets
Chloroquine
Haem polymerisation / detoxification
(Haemozoin)
4-Aminoquinolines and sesquiterpene endoperoxides
Folate metabolism
(DHFR / DHPS)
Pyrimethamine / Sulphadoxine
Artemisinins

8. (haemoglobin from the erythrocyte by pinocytosis)
Haemoglobin Degradation Pathway PV
Limited capacity to synthesise amino acids
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
Trophozoite FV
Haemozoin
(malaria pigment) N
Pinocytosis
(haemoglobin from the erythrocyte by pinocytosis)
Erythrocyte
N=nucleus; FV=food vacuole; PVM=parasitophorous vacuole

9. 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)
Amino-
peptidases
Large peptides
globin
Small peptides
Amino acids
Haemoglobin
-Free haem is extremely toxic
-Can generate ROS
-Is lipophilic and can intercalate into membranes causing cell lysis
Free haem
Fe2+ O2
Superoxide dismutase
Haematin
Fe3+
O2-
-Haematin
H2O2
Free haem metabolised to an inert chemical form called haemozoin by a process known as biomineralisation
H2O
Haemozoin
Peroxidases
Kumar et al., Life Sciences 80 (2007)

10. Detoxification of Haematin into Inert Haemozoin
Free haem
Fe2+
His-rich proteins
and
oxidation
Fe3+
Phospholipids
Membrane lysis
b1-4 linkages of haematin
Biomineralisation
Dimers of haematin – b1-4 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

11. Artemisinin Combination Therapy (ACT) – current frontline therapy
Artemisinins reduce parasite burden rapidly
Used in combination with other drugs to protect emergence of resistance to partner drug (ACT)
Artemisia annua – sweet wormwood
Youyou Tu
Nobel Prize – Medicine 2015

12. Haem and Mode of Action of Artemisinins
Haematin
-Haematin
Haemozoin
haem-artemesinin adducts
(“haemarts”)
Carbon-centred free radicals generated
Cleavage of endoperoxide bridge by haem
Endoperoxide bridge
Food Vacuole
Artemesinin accumulates in the FV
Possible targets of artemisinin free radicals:
TCTP (translationally controlled tumour protein homolog)
SERCA (sarco/endoplasmic reticulum Ca2+‡-ATPase)
Cysteine proteases

13. Mode of Action of Quinolines
Haematin
-Haematin
Haemozoin
Induces oxidative stress
CQ adduct formation
Membrane lysis
Chloroquine
pH cytosol ~7.2 H+
ATP
ADP
V-type
ATPase CQ
CQH+
CQH2++
pH FV ~5.5
Accumulates following protonisation CQ
Basic

14. Reduce and methylate folate
Disruption of Folate Metabolism
Ser
Aspartate + CO2 + PRPP
H4F
NADP+
Sulphadoxine
Pyrimethamine
Cell
death
SHMT
DHFR
Gly
NADPH
Reduce and methylate folate
Dihydropteroate synthase
Methylene
-H4F
H2F
GTP TS TK
Uridine
UMP
dUMP
dTMP
Thymidine
Deoxyuridine monophosphate
Deoxythymidine monophosphate
RNA
DNA
SHMT – serine hydroxymethyltransferase
DHFR – dihydrofolate reductase
TS- thymidylate synthase

15. Antimalarials – mechanisms of resistance

16. 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,

17. 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)

18. Drug Resistance Mechanisms – molecular basis
Altered Drug Level
By exclusion
Decreased import
Increased export
By sequestration
Drug-binding molecule
Compartmentalization
By Metabolism
Pro-drug not activated
Drug inactivation
Altered Target Level
Modified
Decreased affinity
Amplified
Increased sequestration
Increased enzyme activity
Missing
By-passed via salvage pathway
Repaired / protected
Increased damage repair
Protected by metabolite

19. 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):

20. Malaria drug resistance - molecular mechanisms
Gene
Major mutations
Mechanism
Sulfadoxine
DHPS
A437G (K540E, A581G)
Decreased affinity (higher Ki) for target
Pyrimethamine
DHFR
S108N (N51I, C59R, I164L)
Chloroquine
MDR1
D86Y
Increased efflux from FV
Artemisinin
K13
Multiple
Increased repair and protection from damage

21. Insecticide treated bed nets
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

22. Antimalarials – the future

23. 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

24. Life Cycle Stages for Drug Intervention
Prevent relapse
(hypnozoite stage in P.vivax)
Hypnozoite
LIVER
Reduce
Transmission
e.g. artemesinins
Merozoites
Schizont
Curative treatment
All drugs
Ring
BLOOD STAGES
Schizont
Gametocyte
Trophozoite

25. 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)

26. 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

27. 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

28. Malaria vaccines

29. 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 years)
- protect during pregnancy

30. 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

31. RTS,S vaccine development
Sporozoites
Surface of sporozoites covered with an antigen known as circumsporozoite protein (CSP)
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)
LIVER

32. RTS,S vaccine development
Structure/function of CSP highly conserved across the various strains of malaria
RTS later redesigned to include T- and B-cell epitopes from the C-terminus of the CS and was renamed to RTS,S
Immunodominant
region
RTS,S:
‘R’ for the CS “repeats”
‘T’ for T-cell epitope
‘S’ for Hepatitis B antigen
‘S’ for genetically transformed yeast (Saccharomyces cerevisiae) used to express the vaccine
circumsporozoite protein structure
Moorthy, V., & Ballou, R. (2009). Immunological Mechanisms Underlying Protection Mediated by RTS,S: a review of the available data. Malaria Journal, 8(312).

33. 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
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.
N Engl J Med. (2012) 367:
A phase 3 trial of RTS,S/AS01 malaria vaccine in African infants.

34. 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

35. Baragaña et al. Nature. (2015) 522, 315-320
Reading list (SW)
A novel multiple-stage antimalarial agent that inhibits protein synthesis.
Baragaña et al. Nature. (2015) 522,
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,
Artemisinin Action and Resistance in Plasmodium falciparum.
Tilley et al. Trends in Parasitology (2016) 32, .