tricking malaria vectors to control infection
TRANSCRIPT
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
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Tel.: +351 213652678
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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.