perturbation of plasmodium vivax hypnozoite formation ... · workflow between cidr and mvru m"...

Perturbation of Plasmodium vivax hypnozoite formation, growth and reactivation in vivo in a human-liver chimeric mouse

Erika L. FlanneryDecember 7, 2017

JITMM: Bangkok, Thailand

GOAL: predict radical cure activity using FRG huHep modelP. vivax radical cure in vivo model development

• Infection with P. vivax can result in a secondary, blood-stage infection (relapse)

• Hypnozoites emerge from the liver and infect RBCs

• Relapse rates vary with geographic region• Latent hypnozoite infection is a significant barrier toglobal malaria eradication efforts

acuteinfection relapse

para

sitem

iatime since mosquito bite



para

sitem

iapa

rasit

emia

December 7, 2017 | JITMM 2017 | 2

treatment

para

sitem

ia

time since mosquito bite

+ primaquine

• P. vivax infections are treated upon clinical detection (blood-stage)• First-line treatment, in most countries

• 3d of chloroquine (CQ) + 14d of primaquine (PQ) is used

• This prevents subsequent relapse (in most cases)• Primaquine is the only anti-relapse drug and it has many liabilities

GOAL: predict radical cure activity using FRG huHep modelP. vivax radical cure in vivo model development

December 7, 2017 | JITMM 2017 | 3

cell engraftment: 0.5-1 million hepatocytes

4 weeks: 1-5 million 8 weeks: 5-10 million

12+ weeks: 50-150 million

FRG huHep mouse

FAH (fumaryl acetoacetate hydrolase) knockout: mouse hepatocyte death which can be controlled with drug

Rag2 (recombination activating gene 2) knockout: T cell and B cell deficiency

Il2rg (interleukin 2 subunit γ-chain) knockout: NK and NK(T) cell deficiency

Vaughan, Mikolajczak et al., Mol Biochem Parsitology 2012 Vaughan, Mikolajczak et al., J Clinical Investigation 2012

Mikolajczak, Vaughan et al., Cell Host Microbe 2015

A liver-chimeric mouse to study human malaria infectionFRG huHep model

December 7, 2017 | JITMM 2017 | 4

Workflow between CIDR and MVRU

M"F"

K"

R"

T"

Liver samples(formalin fixed or trizol)

December 7, 2017 | JITMM 2017 | 5

ContributorsCenter for Infectious Disease Research

Sebastian MikolajczakStefan KappeEve CheunchobCarola SchaeferAshley VaughanLander FoquetMatthew FishbaugherMary Jane NavarroWilliam Betz

Mahidol Vivax Research UnitJetsumon SattabongkotNiwat KangwanrangsanNarathatai YimamnuavchokWanlapa RoobsoongRapatbhorn Patrapuvich

University of WashingtonSean MurphyZachary Billman

December 7, 2017 | JITMM 2017 | 6

Blood stage infection> 7 days post-infection

Primary liver stage infectionSchizonts mature approx. 7 days post-infection

Hypnozoite persistence (latency)Hypnozoites remain for months to years after infection

Hypnozoite formation0-3 days post-infection

Hypnozoite activation (relapse)Replication and blood-stage infection

Liver stage efficacy testing against P. vivaxFRG huHep model

December 7, 2017 | JITMM 2017 | 7

Size and UIS4 staining differentiate hypnozoitesHypnozoite formation

• Liver tissue sectioning and antibody staining in FRG huHep mice allowsquantification of P. vivax infections

Parasite size

nd not determined

trophozoiteschizonthypnozoite

Day 8Day 8

Hypn

ozoi

te

Schi

zont

Scale bar: 15 m

Morphological differentiation

UIS4 parasitophorous vacuole membrane markerACP apicoplast markerDAPI DNA

1 2 3 4 5 6 7 8 9 101

10

100

1000

10000

days post infection

para

site

siz

e (

m2 )

ndndndndndnd

December 7, 2017 | JITMM 2017 | 8

Size and UIS4 staining differentiate hypnozoitesHypnozoite formation

• Liver tissue sectioning and antibody staining in FRG huHep mice allowsquantification of P. vivax infections

Parasite size

nd not determined

trophozoiteschizonthypnozoite

1 2 3 4 5 6 7 8 9 101

10

100

1000

10000

days post infection

para

site

siz

e (

m2 )

ndndndndndnd

Frequency of hypnozoite formation

0

20

40

60

frequencies from several infections(each point represents one mouse)

freq

uenc

y of

hyp

nozo

itefo

rmat

ion

(%)

December 7, 2017 | JITMM 2017 | 9

• Volunteers were bitten by 10 P. falciparum infected mosquitoes• 30 mg single dose treatment with primaquine (PQ)

Timing of PQ treatment and efficacy in humans: a comparisonHypnozoite functional experimentation

Adapted from: Arnold et al.,1955 J Lab and Clin Med

1 3 50

50

100

days post infectionPQ treatment

perc

ent o

f vol

unte

ers

pate

nt

December 7, 2017 | JITMM 2017 | 10

Control PQ (3,4)

• Livers harvested 8 days after infection to allow surviving parasites to mature

• Primaquine prevents hypnozoite development• Primaquine kills early stage hypnozoites and schizonts• Primaquine is active in the FRG huHep mice

Mature hypnozoites respond differently to PQHypnozoite functional experimentation

0

5

10

15

2050

100150

No.

par

asite

spe

r 100

mm

2 liv

er ti

ssue Control

PQ (0,1)PQ (1,2)PQ (3,4)

schizonts hypnozoites

ACP UIS4

UIS4 parasitophorous vacuole membrane markerACP apicoplast markerDAPI DNA

December 7, 2017 | JITMM 2017 | 11

The complexity and size of hypnozoites change over timeHypnozoite persistence

Day 24

Day 8

Day 49

UIS4 parasitophorous vacuole membranemarker

ACP apicoplast markerDAPI DNA

• Persistent hypnozoite infection present for at least 49 days

Parasite size

235 10 14 21 30 36 491

10

100

1000

10000

Days post infection*Only labelled days were assayed

3 5 10 14 21 30 36 490

20

40

60

80

100

para

site

siz

e (μ

M)

Days post infection

December 7, 2017 | JITMM 2017 | 12

Control PQ Control PQ-0.5

0.0

0.5

1.0

1.5

No.

of p

aras

ites

per 1

00 m

m2


Unexpected results in FRG huHep mice following PQ treatmentHypnozoite persistence

liver harvest for IFA

Infection by 1M spz i.v.(VUNL-23)

30 mg/kg primaquine (PQ) p.o.

6 8 100 2 4days post-infection

12 14 16

Day 16 post-infection liver

December 7, 2017 | JITMM 2017 | 13

PQ treatment

• 60 mg/kg PQ treatment days 3-8

• Liver harvest at day 8 or day 16

• Primaquine arrests parasite growth, but parasites are not immediately clearedfrom the cell

(Day 3)

(Day 5)

(Day 10)

(Day 8)

Long-duration PQ treatment arrests schizont growthHypnozoite persistence

December 7, 2017 | JITMM 2017 | 14

• Single dose treatment prevents transmission

• 24 hours post treatment gametocytes there is no reduction in the presence of gametocytes by blood smear

• At the same time, there is a large reduction in transmission

White, 2012 Lancet Infectious Diseases

Single-dose PQ is effective, but does not clear gametocytes from the bloodHypnozoite persistence

December 7, 2017 | JITMM 2017 | 15

relapsepossible

6 8 10 12 14 16 180 2 4 20 22 24 26

****primary infection

Same treatment as in the clinic• 14d PQ treatment + 3d CQ• We start at day 4 because we know these

are true hypnozoites. • Follow with blood sampling for weeks

Treatment with PQ alone at day 4 would arrest schizonts confounding day 24 readouts

liver harvest for IFA and rtPCR

infection with 1.0M spz i.v.(Vxxx-xxx:VK2xx)

30 mg/kg primaquine p.o.

*30 mg/kg MMV048 p.o.10 mg/kg chloroquine p.o.

An in vivo relapse model of infectionHypnozoite activation

December 7, 2017 | JITMM 2017 | 16

Primary infection

Latent/relapse infection

*30 mg/kg MMV048 p.o.bleed to detect merosome release


infection with 0.6M spz i.v.(VTTY-111)

6 8 10 12 14 16 180 2 4 20

6 8 10 12 14 16 180 2 4 20****

6 8 10 12 14 16 180 2 4 20

6 8 10 12 14 16 180 2 4 20****

0

20

40

60

80

num

ber o

f par

asite

s pe

r 10

0 m

2 tis

sue

sect

ion

n.s.

MMV048Control MMV048Control


primary infection(day 8)

Absence of schizonts in the liver (primary infection)

Absence of merozoites in the blood (primary infection)

Demonstration that late timepoint schizonts are relapsesHypnozoite activation

December 7, 2017 | JITMM 2017 | 17

Primary infection


*30 mg/kg MMV048 p.o.bleed to detect merosome release


infection with 0.6M spz i.v.(VTTY-111)

6 8 10 12 14 16 180 2 4 20

6 8 10 12 14 16 180 2 4 20****

6 8 10 12 14 16 180 2 4 20

6 8 10 12 14 16 180 2 4 20****

0

1

2

3

4

5

num

ber o

f lpa

rasi

tes

per

100 m

2 tis

sue

sect

ion

n.s.

n.s.



latent infection(day 18)

0

20

40

60

80

num

ber o

f par

asite

s pe

r 10

0 m

2 tis

sue

sect

ion

n.s.



primary infection(day 8)

Presence of schizonts in the liver (latent infection)

Presence of merozoites in the blood (latent infection)

Demonstration that late timepoint schizonts are relapsesHypnozoite activation

December 7, 2017 | JITMM 2017 | 18

Additional tools to advance P. vivax studiesP. vivax radical cure in vivo model development

RNA probes result in 506x enrichment

0.00.51.040

50

60

70

80

perc

ent P

. viv

ax re

ads

bulk liver RNAPvivax enriched RNA

Control MMV048 treated

500x enrichment

30x enrichment

Molecular methods• Contamination: interspecies and

intraspecies (hypnozoites v. schizonts)• Tools for enrichment: P. vivax specific

RNA probes• Exosome enriched serum (in

collaboration with Hernando del Portillo, ISB global)

December 7, 2017 | JITMM 2017 | 19

Completion of the full P. vivax transmission cycle in FRG huHep mice• Inject human reticulocytes• Egress of liver merosomes into the blood stream• Feed mosquitoes directly on mice• This would allow repeated experiments with the same P. vivax isolate

Summary

• Complete P. vivax liver stage development and blood stage transition can be recapitulated in the FRG huHep mouse

• P. vivax hypnozoites appear metabolically active because they grow and mature over time

• Relapse propensity of hypnozoites can be assessed using the FRG huHep model

• Genomics tools can inform drug discovery• Assay design• Biomarkers of infection

• Potential to complete the transmission cycle in FRG huHep mice

• Source of P. vivax sporozoites

December 7, 2017 | JITMM 2017 | 20

relapsepossible

6 8 10 12 14 16 180 2 4 20 22 24 26

****primary infection

Same treatment as in the clinic• 14d PQ treatment + 3d CQ• We start at day 4 because we know these are

true hypnozoites. • Begin at day first symptomatic? (10)• Earlier shortens the overall study period

Treatment with PQ alone at day 4 would arrest schizonts confounding day 24 readouts





November 28, 2017 | BMGF update TC | 6

0

5

10

15

2050

100150

No.

par

asite

spe

r 100

mm

2 liv

er ti

ssue Control

PQ (0,1)PQ (1,2)PQ (3,4)


no difference!

In vivo P. vivax radical cure model – modeling relapsePv radical cure model

6 8 10 12 14 16 180 2 4

****6 8 10 12 14 16 180 2 4

Primary infection


MMV048

MMV048 +

PQ PQ

PQ + CQ

-2

0

2

4

6

8

log 10

Pla

smod

ium

18S

rRN

Ape

r ug

of li

ver

day 24 post-infection

contro

l

MMV048

MMV048 +

PQ

6

7

8

9

10

11

log 10

Pla

smod

ium

18S

rRN

Ape

r ug

of li

ver

day 8 post-infection


6 8 10 12 14 16 180 2 4 20 22 24 26****

6 8 10 12 14 16 180 2 4 20 22 24 26

****

6 8 10 12 14 16 180 2 4 20 22 24 26

6 8 10 12 14 16 180 2 4 20 22 24 26


infection with 0.85M spz i.v.(VYBN-14:VK210)



May 2017 experiment used to inform November experimentPv radical cure model

Objective: reproduce relapse model using same dosing regimen as used in the clinic• start primaquine treatment on day 4 because we know these are true hypnozoites• 5 mice per group• Treatment with primaquine alone would arrest schizonts confounding results

• MMV048 is used to clear out schizonts



bleed to detect merosome release, i.e. relapse

6 8 10 12 14 16 180 2 4 20 22 24 26

Control no drug treatment

1.

6 8 10 12 14 16 180 2 4 20 22 24 26

30 mg/kg primaquine p.o.10 mg/kg chloroquine p.o.

3.

6 8 10 12 14 16 180 2 4 20 22 24 26

****

30 mg/kg primaquine p.o.*30 mg/kg MMV048 p.o.

10 mg/kg chloroquine p.o.

2.

6 8 10 12 14 16 180 2 4 20 22 24 26****

*30 mg/kg MMV048 p.o.

4.

November – full relapse model experimentPv radical cure model


Results:• Mouse death 4/22• All treatments and bleeding performed as scheduled• Will ship to CIDR next week



bleed to detect merosome release, i.e. relapse

6 8 10 12 14 16 180 2 4 20 22 24 26

Control no drug treatment

1.

6 8 10 12 14 16 180 2 4 20 22 24 26

30 mg/kg primaquine p.o.10 mg/kg chloroquine p.o.

3.

6 8 10 12 14 16 180 2 4 20 22 24 26

****

30 mg/kg primaquine p.o.*30 mg/kg MMV048 p.o.

10 mg/kg chloroquine p.o.

2.

6 8 10 12 14 16 180 2 4 20 22 24 26****

*30 mg/kg MMV048 p.o.

4.

November – full relapse model experimentPv radical cure model


• Repeat relapse model – add PK study

• Novel drug testing• Monensin

• Formulation can be a problem (MMV assessment)• Days of dosing

• Timing of relapse• Repopulation with reticulocytes to model viability• Activation of relapse

• HDAC inhibitors• Pf co-infection• RBC lysis

Future experimentsPv radical cure model


Thank-you

CIDRSebastian MikolajczakEve ChuenchobCarola SchaeferAshley VaughanLander FoquetMatt FishbaugherMary Jane NavarroWill Betz

MVRUJetsumon PrachumsriNiwat KangwanrangsanNarathatai YimamnuavchokWanlapa Roobsoong

UWSean MurphyZach Billman

Funding

Bill and Melinda Gates Foundation


• in silico probe design• tiled across full genome

• 85,000 120 bp probes• tiled every 100bp

• Shear size of 350-400bp• BLAT’d against human genome• 30% mismatch is allowed

probe

spliced transcript

exon exon

Towards a molecular marker of hypnozoitesP. vivax radical cure in vivo model development

date | place | slide#

perturbation of plasmodium vivax hypnozoite formation ... · workflow between cidr and mvru m"...

Documents