Tricking malaria vectors to control infection - Henrique Silvira

1. Tricking malaria vectors to
control infection
Henrique Silveira
hsilveira@ihmt.unl.pt

2. Plasmodium Life Cycle

3. Plasmodium Life Cycle
Humans
Schizogony – asexual stages
Hepatic
Erythrocytic
Anopheles Mosquito
Sporogony – sexual stages

4. gametócitos
Malaria transmission – Sporogonic cycle
gâmetas
zigoto
ookinete Glândulas
salivares
Matriz peritrófica
Células
epiteliais
Oocisto
Lâmina basal
Esporozoíto
Collins FH & James AA. 1996 Science & Medicine, Dec: 52-61

5. How can we block transmission ?
Increase mosquito resistance to infection
Genetic modified
organism
Anti-plasmodium
Anti-mosquito
molecules
New delivery
systems

6. Immunity as a way to control mosquito infection
Hemolinph
ooc~~yst
sporozoites
Salivary glands
zigote ookinete
gamets
P.M.
mid gut
Epithelium Fat body

7. Immunity as a way to control mosquito infection
Hemolinph
ooc~~yst
sporozoites
Salivary glands
zigote ookinete
gamets
P.M.
mid gut
Epithelium Fat body
Is it enough?

8. Is there a way to boost mosquito response to
infection?

9. Immunomodulatory nucleic acid sequences
DNA
PAMPs PRM
Therapeutic prospective of CpG ODN
- Stimulation of protective immune response
- Modulation of the immune response to alergens
- Vaccine adjuvant
immune
response

10. Working hypothesis
CpG oligos can modulate
mosquito immune response
increase resistance to Plasmodium infection

11. Methodology
24h
p.i.
P. berghei
P. yoelii
- mid gut
- fat body
Anopheles stephensi
Anopheles gambiae
18h
p.t
CpG ODN
0604
TCCATGACGTTCCTGATGCT
Control - ODN
10d
p.i.
Dissection
Sampling - infection rate
- infection intensity
qRT-PCR
69nl of a 0.1mM ODN

12. Infection rate and intensity
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Anopheles stephensi / Plasmodium yoelii Anopheles gambiae / Plasmodium berghei
N=231
n=3
PI=51.1
N=207
n=3
PI=63.8
N=225
n=3
PI=25.8
N=265
n=3
PI=49.8
N=265
n=3
PI=49.8
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13. Immune response?

14. Microarrays
Anopheles gambiae
18h
p.t
GeneChip®
Plasmodium/Anopheles Genome Array
Experimental design
- fat body
CpG ODN
0604
TCCATGACGTTCCTGATGCT
Control
Sampling

15. Microarrays
Results
Total of 172 genes
- 136 upregulated
- 36 downregulated
Cytoskeleton
2% Egg
production/
Hormone
4%
Metabolism/
Cell function
26%
Oxidative
Stress
12%
Transport
6%
Unknown
Function
12%
Unknown
Gene
8%
Immunity
31%
21%
17%
17%
15%
23% 4% 2%
2%
Recognition
CLIP serine proteases
Other serine proteases
Serine proteases inhibitors
Coagulation/wounding
AMP
PPO
Transcription factor

16. Hypothesis
CpG-ODN recognition
PPO
Serine proteases
cascade
Coagulation
Wounding
Transcript identifier Homology / gene name fold change
PRM
ENSANGT00000021166 CTL4 1,3
ENSANGT00000028469 Toll 1,3
ENSANGT00000028101 LRR 1,3
ENSANGT00000019522 TEP15 1,5
ENSANGT00000013948 PGRP-LB 2,6
CLIP domain serine proteases
ENSANGT00000020262 CLIPA1 1,3
ENSANGT00000020252 CLIPA2 1,5
ENSANGT00000025727 CLIPA4 1,4
ENSANGT00000020259 CLIPA5 2,2
ENSANGT00000021656 CLIPB7 1,4
ENSANGT00000020324 CLIPB10 1,4
ENSANGT00000018122 CLIPB14 -1,7
ENSANGT00000021259 CLIPC7 2
*Other serine proteases
ENSANGT00000018367 TRY1_ANOGA -1,6
ENSANGT00000000070 1,4
ENSANGT00000024108 1,7
ENSANGT00000011374 1,4
ENSANGT00000024591 1,5
ENSANGT00000021418 1,3
ENSANGT00000021354 1,7
ENSANGT00000019333 1,4
ENSANGT00000021336 Q7QAI0_ANOGA 1,5
Serine protease inhibitors
ENSANGT00000007723 SRPN3 1,4
ENSANGT00000016808 SRPN11 1,3
ENSANGT00000021651 SRPN1 1,7
ENSANGT00000021812 SRPN2 1,4
ENSANGT00000003574 antistasin 1,5
ENSANGT00000020094 Kazal 1,5
ENSANGT00000018532 pacifastin 1,3
ENSANGT00000013014 Proteinase inhibitor I4, serpin 1,6
ENSANGT00000011846 PEBP 1,4
ENSANGT00000018751 Allergen V5 1,6
PPO
ENSANGP00000002175 PPO5 -1,5
Wounding/Coagulation
ENSANGT00000016504 Transglutaminase 1,6
ENSANGT00000022610 Fibrinogen, alpha/beta/gamma chain, C-terminal globular 1,5
ENSANGT00000021200 CUB; EGF-like; Aspartic acid and asparagine hydroxylation -1,8
ENSANGT00000012174 von Willebrand factor, type C; Fibronectin, type III 1,5
ENSANGT00000028106 Lipocalin; Apolipoprotein D -1,4
ENSANGT00000014823 CPR75 - cuticular protein 75 1,4
ENSANGT00000022245 Extensin PRINT 3,5
ENSANGT00000024865 Peritrophin-A domain 1,7
ENSANGT00000020715 CPF3 - cuticular protein 3 2,2
ENSANGT00000012817 Proline-rich region -1,4
ENSANGT00000022326 Proline-rich region 1,6
Melenization
ENSANGT00000022461 Q9GT44_ANOGA - Phenylalanine hydroxylase 1,7
ENSANGT00000014813 Glutamate decarboxylase EC_4.1.1.15 1,6
Transcription factors/regulation
ENSANGT00000020147 REL2 1,4
ENSANGT00000020341 EDRF1 (GATA) -1,5
ENSANGT00000019204 TSC22 domain family 1 1,4
Diverse
ENSANGT00000021546 Hemolymph precursor 1,5
ENSANGT00000010929 Placental 11 precursor, EC3.4.21-Serine endopeptidase 1,6
*Some of these serine proteases might not be involved in the immune response.

17. Transglutaminases
If oligo CpG modulates the immune response to
Plasmodium through coagulation
Coagulation
Wound healing
fibrin
Transglutaminase
fibrinogen
croslink
X
Transglutaminase
Increased susceptibility to infection

18. Transglutaminase inhibitor
1,3-Dimethyl-D003 2-[(2-oxopropyl)thio]imidazolium chloride
TGM

19. Transglutaminase inhibitor
Experimental design
P. berghei
18h
p.t
D003
Control
10d
p.i.
Dissection
- infection rate
- infection intensity

20. Transglutaminase inhibitor
Infection rate and intensity
N=152
n=3
PI=51.1
N=130
n=3
PI=63.8
(***)
***

21. Microarrays
Experimental design
Anopheles gambiae
P. berghei
10-20%EP + exflagelação
4 days
p.t AGAP009098 dsRNA
Control dsRNA
10d
p.i.
69nl of a 150uM
Dissection
- infection rate
- infection intensity
fat body
qRT-PCR
Efficiency of silencing

22. AGAP009098 knockdown
Infection rate and intensity
N=132
n=3
PI=83.3
N=141
n=3
PI=69.5
N=216
n=3
PI=50.5
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23. Transglutaminase
TGM AGAP009098
24h post-infection
1.54 ± 0.42-fold in the fat bodies
2.05 ± 0.77-fold in the midgut.
Midgut invasion
Increase transcription
CpG Oligo
X Decrease invasion
Impaired oocyst formation

24. Conclusions
Infection 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

25. IMMUNOSTIME

26. 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.

27. 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

28. Immunostimulatory molecules
Experimental design
TREATMENT
Microinjection
with different
concentrations
of ISM
Microinjection
with control
INFECTION
(24h p-treatment
day 0 p-infection)
Blood fed on
P . berghei GFP
infected mice
DATA
COLLECTION
8-10th
day p-infection
Midgut
dissection and
oocyst count
DATA
ANALYSIS
100
50
0
Determination of
infection rates
and intensities
Test group
100-150
A. gambiae
females
Control group
100-150
A. gambiae
females

29. Immunostimulatory molecules
Results
Mean Infection Rate for the three experiments of each immunostimulant
treatment
80
70
60
50
40
30
20
10
0
Infection rate (%)
Control Treated

30. Immunostimulatory molecules
Results
Infection intensity for the three experiments of each immunostimulant
treatment
70
60
50
40
30
20
10
0
Median oocyst/midgut
Control Treated

31. Immunostimulatory molecules
conclusion
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

32. Hemozoin
Results

33. Hemozoin
Results

34. Hemozoin
Results – Rel2-F silencing

35. Immunostimulatory molecules
conclusion
Hemozoin reduces Plasmodium berghei infection by REL2-mediated activation of the immune system
Zymozan acts through the Toll pathway

36. Conclusion – Future
We can stimulate mosquito
immune response
Resistance to infection
X

37. anti-Plasmodium
Immune response
modulation
Malaria
elimination!
TToo Tthhiinnkk !!

38. Thank you
hsilveira@ihmt.unl.pt
Rua da Junqueira Nº 100
1349-008 Lisboa
Portugal
Tel.: +351 213652678

39. Obrigado
Thank you

40. INSTITUTO DE HIGIENE E MEDICINA TROPICAL
Research area: Parasite Mosquito interactions
IP: Henrique Silveira (hsilveira@ihmt.unl.pt)
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
GeneChip®
Ala35Ser
Ser37Asn
Ala39Ser
Ser58Ala
Ile123Met
Ala123Gly/Val
Thr137Asn
Thr137Asn
Arg140Gln
Asn141Gln
Thr144Ser
Molecular evolution of
immune associated
genes of African
Anopheles mosquitos
A B
A. arabiensis
C
A. gambiae
A. arabiensis
A. gambiae
Figure 1 - Median-joining network based
on thirteen haplotypes for PGRP-S1 gene,
twenty-five haplotypes for PGRP-S2 gene
and forty three haplotypes for PGRP-S3.
The area of circles is proportional to the
frequency of the haplotypes.
A . arabiensis – Tanzania
A. arabiensis – Mozambique
A. gambiae – Mozambique
A. gambiae - Tanzania
Detoxification and
mosquito response
to infection.
Imunostimulation
MtriDAP, CL097, Zymosan, alginato, CpG, hemozoin, Flagelina
(recFLA-ST), Pam2CSK4
infection STOP% Malaria
Resistance to
Transmission
Which molecules are involved?
Which pathways?
How to modulate them?
Methodologies:
- In vivo e in vitro models
- Funcional genomics
- Proteomics
- Gene silencing