Tricking malaria vectors to control infection

Tricking malaria vectors to

control infectionHenrique [email protected]

Plasmodium Life Cycle

HumansSchizogony – asexual stages

Anopheles Mosquito

HepaticErythrocytic

Plasmodium Life Cycle

Sporogony – sexual stages

gametócitos

gâmetas

zigoto

ookinete Glândulas salivares

Matriz peritrófica

Células epiteliais

Oocisto

Lâmina basal

Esporozoíto

Collins FH & James AA. 1996 Science & Medicine, Dec: 52-61

XMalaria transmission – Sporogonic cycle

Increase mosquito resistance to infection

Genetic modified organism

Anti-plasmodiumAnti-mosquito

molecules

How can we block transmission ?

New delivery systems

Salivary glands

Hemolinph

~~oocyst

sporozoites

ookinetezigote

gamets

P.M.

mid gut

Epithelium Fat body

Immunity as a way to control mosquito infection

Salivary glands

Hemolinph

~~oocyst

sporozoites

ookinetezigote

gamets

P.M.

mid gut

Epithelium Fat body

Immunity as a way to control mosquito infection

Is it enough?

Is there a way to boost mosquito response to infection?

Immunomodulatory nucleic acid sequences

DNAPAMPs PRM

Therapeutic prospective of CpG ODN

- Stimulation of protective immune response

- Modulation of the immune response to alergens

- Vaccine adjuvant

immune response

Working hypothesis

CpG oligos can modulate mosquito immune

response

increase resistance to Plasmodium infection

Methodology

24hp.i.

P. berghei

P. yoelii

- mid gut - fat body

Anopheles stephensiAnopheles gambiae

18hp.t

CpG ODN0604 TCCATGACGTTCCTGATGCT

Control - ODN

10dp.i.

Sampling - infection rate - infection intensity

Dissection

qRT-PCR

69nl of a 0.1mM ODN

Infection rate and intensity

Immune response?

Microarrays

Anopheles gambiae

GeneChip® Plasmodium/Anopheles Genome Array

Experimental design

- fat body

18hp.t

CpG ODN0604 TCCATGACGTTCCTGATGCT

Control

Sampling

MicroarraysResults

Total of 172 genes - 136 upregulated - 36 downregulated

Hypothesis

PPO

Serine proteases cascade

Coagulation Wounding

CpG-ODNrecognition

Transcript identifier Homology / gene name fold changePRM

ENSANGT00000021166 CTL4 1,3ENSANGT00000028469 Toll 1,3ENSANGT00000028101 LRR 1,3ENSANGT00000019522 TEP15 1,5ENSANGT00000013948 PGRP-LB 2,6

CLIP domain serine proteasesENSANGT00000020262 CLIPA1 1,3ENSANGT00000020252 CLIPA2 1,5ENSANGT00000025727 CLIPA4 1,4ENSANGT00000020259 CLIPA5 2,2ENSANGT00000021656 CLIPB7 1,4ENSANGT00000020324 CLIPB10 1,4ENSANGT00000018122 CLIPB14 -1,7ENSANGT00000021259 CLIPC7 2

*Other serine proteasesENSANGT00000018367 TRY1_ANOGA -1,6ENSANGT00000000070 1,4ENSANGT00000024108 1,7ENSANGT00000011374 1,4ENSANGT00000024591 1,5ENSANGT00000021418 1,3ENSANGT00000021354 1,7ENSANGT00000019333 1,4ENSANGT00000021336 Q7QAI0_ANOGA 1,5

Serine protease inhibitorsENSANGT00000007723 SRPN3 1,4ENSANGT00000016808 SRPN11 1,3ENSANGT00000021651 SRPN1 1,7ENSANGT00000021812 SRPN2 1,4ENSANGT00000003574 antistasin 1,5ENSANGT00000020094 Kazal 1,5ENSANGT00000018532 pacifastin 1,3ENSANGT00000013014 Proteinase inhibitor I4, serpin 1,6ENSANGT00000011846 PEBP 1,4ENSANGT00000018751 Allergen V5 1,6

PPOENSANGP00000002175 PPO5 -1,5

Wounding/CoagulationENSANGT00000016504 Transglutaminase 1,6ENSANGT00000022610 Fibrinogen, alpha/beta/gamma chain, C-terminal globular 1,5ENSANGT00000021200 CUB; EGF-like; Aspartic acid and asparagine hydroxylation -1,8ENSANGT00000012174 von Willebrand factor, type C; Fibronectin, type III 1,5ENSANGT00000028106 Lipocalin; Apolipoprotein D -1,4ENSANGT00000014823 CPR75 - cuticular protein 75 1,4ENSANGT00000022245 Extensin PRINT 3,5ENSANGT00000024865 Peritrophin-A domain 1,7ENSANGT00000020715 CPF3 - cuticular protein 3 2,2ENSANGT00000012817 Proline-rich region -1,4ENSANGT00000022326 Proline-rich region 1,6

Melenization ENSANGT00000022461 Q9GT44_ANOGA - Phenylalanine hydroxylase 1,7ENSANGT00000014813 Glutamate decarboxylase EC_4.1.1.15 1,6

Transcription factors/regulationENSANGT00000020147 REL2 1,4ENSANGT00000020341 EDRF1 (GATA) -1,5ENSANGT00000019204 TSC22 domain family 1 1,4

DiverseENSANGT00000021546 Hemolymph precursor 1,5ENSANGT00000010929 Placental 11 precursor, EC3.4.21-Serine endopeptidase 1,6*Some of these serine proteases might not be involved in the immune response.

Transglutaminases

If oligo CpG modulates the immune response to Plasmodium through coagulation

Coagulation Wound healingfibrin

Transglutaminase

fibrinogencroslink

Transglutaminase

Increased susceptibility to infection

X

Transglutaminase inhibitor

1,3-Dimethyl-2-[(2-oxopropyl)thio]imidazolium chlorideD003

TGM

Transglutaminase inhibitorExperimental design

P. berghei

18hp.t

D003Control

10dp.i.

- infection rate - infection intensity

Dissection

Transglutaminase inhibitorInfection rate and intensity

MicroarraysExperimental design

P. berghei10-20%EP + exflagelação

Anopheles gambiae

4 daysp.tAGAP009098 dsRNA

Control dsRNA

10dp.i.

69nl of a 150uM

- infection rate - infection intensity

Dissection

fat body

qRT-PCR

Efficiency of silencing

AGAP009098 knockdown Infection rate and intensity

TransglutaminaseTGM AGAP009098

24h post-infection1.54 ± 0.42-fold in the fat bodies 2.05 ± 0.77-fold in the midgut.

Midgut invasion

Increase transcription

CpG Oligo

X Decrease invasionImpaired oocyst formation

ConclusionsInfection rate and intensity

1- Oligo CpG treatment leads to mosquito immunostimulation activating wound healing/coagulation effector mechanisms

2- Oligo CpG Immunostimulation results in decrease of infection rates and infection intensities

3- Transglutaminase inhibition results in susceptibility to infection

IMMUNOSTIME

What does IMUNOSTIM propose?

To identify other molecules able to stimulate mosquito

immunity to Plasmodium

To characterize immune pathways that are triggered by

immunostimulatory molecules and confer protection

against Plasmodium.

Immunostimulatory molecules

ISM Type Receptor Concentration (µg/ml)

M-TriDAP Peptidoglican-like molecule

NOD1/NOD2 94.5

Pam2CSK4 Synthetic bacterial lipoprotein

TLR2/6 0.945

Zym Zymosan from S.cerevisiae cell wall

TLR2 9.45 and 94.5

CL097 Imidazoquinoline compound

TLR7/8 9.45

Rec-Fla-ST Recombinant flagellin from Salmonella typhimurium

TLR5 9.45 and 945

sHz Synthetic hemozoin TLR9 100 and 200

Immunostimulatory moleculesExperimental design

Microinjectionwith different

concentrations of ISM

Microinjectionwith control

TREATMENT

Blood fed on P . berghei GFP

infected mice

INFECTION

(24h p-treatmentday 0 p-infection)

DATA COLLECTION

Midgut dissection and oocyst count

8-10th day p-infection

DATA ANALYSIS

Determination of infection rates and intensities

0

20

40

60

80

Test group100-150

A. gambiae females

Control group100-150

A. gambiae females

Immunostimulatory moleculesResults

Mean Infection Rate for the three experiments of each immunostimulant treatment

0

20

40

60

80Control Treated

Infe

ctio

n ra

te (%

)

Immunostimulatory moleculesResults

MtriDAP (9

4.5ug

/mL)

CL097

(9.45

ug/m

L)

REC-Flag (9

.45ug

/mL)

REC-Flag (9

45ug

/mL)

Zym (9

.45ug

/mL)

Zym (9

4.5ug

/mL)

Pam (0

.945u

g/mL)

sHz (

100u

g/mL)

sHz (

200u

g/mL)

0

10

20

30

40

50

60

70 Control Treated

Med

ian

oocy

st/m

idgu

tInfection intensity for the three experiments of each immunostimulant treatment

Immunostimulatory moleculesconclusion

Treatment with different concentrations of zymosan and hemozoin showed a reduction

in infection rates and intensities

Treatment with Pam2CSK4 and CL097 did have no effect on the infection outcome

Treatment with M-TriDAP had no effect in either the parameters analyzed which might

be consistent with the fact that they are ligands of NOD1/2, receptors only described in

vertebrates

HemozoinResults

HemozoinResults

HemozoinResults – Rel2-F silencing

Immunostimulatory moleculesconclusion

Hemozoin reduces Plasmodium berghei infection by REL2-mediated activation of the immune system

Zymozan acts through the Toll pathway

We can stimulate mosquito immune response

Resistance to infection

XX

Conclusion – Future

anti-PlasmodiumImmune response

Malaria elimination!

modulation

To Think !To think !

Thank you

[email protected]

Rua da Junqueira Nº 1001349-008 LisboaPortugal

Tel.: +351 213652678

Obrigado

Thank you

INSTITUTO DE HIGIENE E MEDICINA TROPICAL

Research area: Parasite Mosquito interactionsIP: Henrique Silveira ([email protected]) Team: Ana Custódio, Isa Pires, Joana Gomes, Luisa Simões, Ana Rhodes, Catarina Alves

- Mosquito immune response and immunomodulation to the different stages of the sporogonic cycle- Mechanisms of resistance to infection: recognition, signalling and effector

Ala35Ser

Ser37Asn

Ala39Ser

Ser58Ala

Ile123MetAla123Gly/Val

Thr137Asn

Thr137AsnThr144Ser

Arg140Gln

Asn141Gln

Ala35Ser

Ser37Asn

Ala39Ser

Ser58Ala

Ile123MetAla123Gly/Val

Thr137Asn

Thr137AsnThr144Ser

Arg140Gln

Asn141Gln Molecular evolution of immune associated genes of African Anopheles mosquitos

Detoxification and mosquito response to infection.