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Development of a universal vaccine against malaria caused by Plasmodium vivax.
UNIFESP–EPMLais H. TeixeiraDr. Cibele TararamAriane G. Camacho
Wistar Institute
Dr. Hildeglund ErtlDr. Marcio Lasaro
New York University School of Medicine
Dr. Victor NussenzweigDr. Ruth S. Nussenzweig
USP (Ciências Farmaceuticas)Dr. Irene S. Soares
Unicamp (Departamento de Imunologia)Dr. Fabio T. M. Costa
UFMG (Departamento de Microbiologia)Dr. Oscar Bruna-Romero
Financial
Support:
Financial Financial
Support:Support:
Sporozoite major surface antigen: CS protein
Target of protective antibodies
SP RI Tandem repeats RIII TSR GPI
RII Plus
CS
Target of protective T cells
2.85 billions at risk
P. vivaxP. falciparum132-391 M
cases per year
P. vivaxAmericas
80%
P. vivaxAsia 50%
Duffy negative ind.
CS Protein of Plasmodium vivax
NH2 COOH
Repeat region
VK210 = (DRADGQPAG)2 (DRAAGQPAG)2 DRADGQPAGD
VK247 = (ANGAGNQPG)4
P. vivax-like = (APGANQEGGAA)3 ?
77 2491 331-3
Universal vaccine = immunity to the all three allelic forms.
His6-PvCS-VK210
His6-PvCS-Vivax-like
His6-PvCS-VK247
His6-PvCS-All-CS-epitopes
VK210- (DRADGQPAG)2 (DRAAGQPAG)2 DRADGQPAGD,
1- MGSSHHHHHH SSGLVPRGSH MTHCGHNVDL SKAINLNGVN FNNVDASSLG AAHVGQSASR
61- GRGLGENPDD EEGDAKKKKD GKKAEPKNPR ENKLKQPGDR ADGQPAGDRA DGQPAGDRAD
121- GQPAGDRADG QPAGDRAAGQ PAGDRADGQP AGDRADGQPA GDRADGQPAG DRADGQPAGD
181- RAAGQPAGDR AAGQPAGDRA DGQPAGDRAA GQPAGDRADG QPAGDRAAGQ PAGDRADGQP
241- AGDRAAGQPA GDRAAGQPAG DRAAGQAAGD RAAGQAAGGN AGGQGQNNEG ANAPNEKSVK
301- EYLDKVRATV GTEWTPCSVT CGVGVRVRRR VNAANKKPED LTLNDLETDV CT
VK-247- (ANGAGNQPG)4
1- MGSSHHHHHH SSGLVPRGSH MTHCGHNVDL SKAINLNGVN FNNVDASSLG AAHVGQSASR
61- GRGLGENPDD EEGDAKKKKD GKKAEPKNPR ENKLKQPGAN GAGNQPGANG AGNQPGANGA
121- GNQPGANGAG NQPGANGAGN QPGANGAGNQ PGANGAGNQP GANGAGNQPG ANGAGNQPGA
181- NGAGNQPGAN GAGNQPGANG AGNQPGANGA GNQPGANGAG NQPGANGAGN QPGANGAGNQ
241- PGANGAGNQP GANGAGNQPG ANGAGNQPGA NGAGNQPGGN AGGQGQNNEG ANAPNEKSVK
300- EYLDKVRATV GTEWTPCSVT CGVGVRVRRR VNAANKKPED LTLNDLETDV CT
(Vivax-like- APGANQEGGAA)3
1- MGSSHHHHHH SSGLVPRGSH MTHCGHNVDL SKAINLNGVN FNNVDASSLG AAHVGQSASR
61- GRGLGENPDD EEGDAKKKKD GKKAEPKNPR ENKLKQPGAP GANQEGGAAA PGANQEGGAA
121- APGANQEGGA AAPGANQEGG AAAPGANQEG GAAAPGANQE GGAAAPGANQ EGGAAAPGAN
181- QEGGAAAPGA NQEGGAAAPG ANQEGGAAAP GANQEGGAAA PGANQEGGAA APGANQEGGA
241- AAPGANQEGG AAAPGANQEG GAAAPGANQE GGAADRAAGQ AAGGNAGGQG QNNEGANAPN
300- EKSVKEYLDK VRATVGTEWT PCSVTCGVGV RVRRRVNAAN KKPEDLTLND LETDVCT
All-CS-epitopes
1- MGSSHHHHHH SSGLVPRGSH MTHCGHNVDL SKAINLNGVN FNNVDASSLG AAHVGQSASR
61- GRGLGENPDD EEGDAKKKKD GKKAEPKNPR ENKLKQPGPG DRADGQPAGD RADGQPAGDR
121- AAGQPAGDRA AGQPAGDRAD GQPAGDRADG QPAGDRADAP GANQEGGAAA PGANQEGGAA
181- APGANQEGGA AAAPGANQEG GAAAPGANQE GGAAAPGANQ EGGAAAANGA GNQPGANGAG
241- NQPGANGAGN QPGANGAGNQ PGANGAGNQP GDRAAGQAAG GNAGGQGQNN EGANAPNEKS
300- VKEYLDKVRA TVGTEWTPCS VTCGVGVRVR RRVNAANKKP EDLTLNDLET DVCT
Synthetic genes:
codon optmized
Cloning pET28b
Expression BL-21
Purification
(affinity and FPLC)
35
50
75105
30
160
A B C D kDa
A - His6-PvCS-VK210
B - His6-PvCS-VK247
C - His6-PvCS-Vivax-like
D - His6-PvCS-All-CS-epitopes
Bacterial recombinant CS proteins of Plasmodium vivax
116.0
66.2
45.0
35.0
25.0
A- yPvCS-VK210-His6
B- yPvCS-VK247-His6
C- yPvCS-VL-His6
D- yPvCS-All-CS-epitopes His6
A STD B C D
18.4
Yeast recombinant CS proteins of P. vivax
Cloning pPIC9K
Expression P. pastoris
Purification
(affinity and FPLC)
Synthetic genes:
codon optmized
Secretion
A- yPvCS-VK210-His6
B- yPvCS-VK247-His6
C- yPvCS-VL-His6
D- yPvCS-All-CS-epitopes His6
HPLC analysis
Titers and specificity
Three rec. proteins His6PvCS-All-CS-epitopes X
Poly (I:C) (TLR-3 agonist)
Universal vaccine = immunity to the all three allelic forms.
Antibody titers
to the different
allelic forms
until day 180
Rec.
protein
Day 0 Day 21 Day 42
Rec.
protein
Rec.
protein
Gr. 1 Adjuvant alone
Gr. 2 His6PvCS-VK210
Gr. 3 His6PvCS-VK247
Gr. 4 His6PvCS-Vivax-like
Gr. 5 Three rec. proteins
Gr. 6 His6PvCS-All-CS-epitopes
Adjuvants
Poly (I:C)
(TLR-3 agonist)
C57Bl/6 mice immunization protocol with the recombinant proteins
1 mg/mouse/dose
1 mg/mouse/dose
1 mg/mouse/dose
3 mg/mouse/dose
3 mg/mouse/dose
G1: Adjuvant
G2: yPvCSP-VK210 / Adjuvant
G3: yPvCSP-VK247 / Adjuvant
G4: yPvCSP-Vivax-like / Adjuvant
G5: yPvCSP-All-CS-epitopes / Adjuvant
G6: Protein Mix /Adjuvant
Days
20 40 60 80 100 120 140 160 180 200
102
103
104
105
106Antibodies to
His6PvCSP-VK210
Days
20 40 60 80 100 120 140 160 180 200
102
103
104
105
106Antibodies to
His6PvCSP-VK247
Days
20 40 60 80 100 120 140 160 180 200
102
103
104
105
106Antibodies to
His6PvCSP-Vivax-like
Amtibod
ytite
rs
Amtibod
ytite
rsAmtibod
ytite
rs
Antibody immune response to the three different allelic forms of the P. vivax CS protein
Replication deficient adenovirusHuman type 5Simian C68
NH2 COOH
aa repeats100 25323 3551
SP
1- mgmqvqsiql fllllwvpgs rgthcghnvd lskainlngv nfnnvdassl gaahvgqsas
61- rgrglgenpd deegdakkkk dgkkaepknp renklkqpgp gdradgqpag dradgqpagd
121-raagqpagdr aagqpagdra dgqpagdrad gqpagdrada pganqeggaa apganqegga
181-aapganqegg aaaapganqe ggaaapganq eggaaapgan qeggaaaang agnqpganga
241-gnqpgangag nqpgangagn qpgangagnq pgdraagqaa ggnaggqgqn neganapnek
301-svkeyldkvr atvgtewtpc svtcgvgvrv rrrvnaankk pedltlndle tdvct
Antibody
titers
Rec. Adenovirus
Day 0 Day 42
Rec. protein
C57Bl/6 mice immunization protocol with the recombinant proteins
Heterologous prime-boost vaccination
After priming After boosting
Seru
m Ig
Gtite
rs
G1: AdHu gal
G2: AdC68-PvCSP
G3: AdHu5-PvCSP
G4: AdHu gal
G5: AdC68-PvCSP
G6: AdHu5-PvCSP
C D
1 2 3 4 5 6
102
103
104
105
106
107
His6-PvCSP-Vk210
His6-PvCSP-Vk247
His6-PvCSP-Vivax-like
Prime
NS
G1 G2 G3 G4 G5 G6
G1: AdHu gal / Adjuvant
G2: AdC68-PvCSP / Adjuvant
G3: AdHu5-PvCSP / Adjuvant
G4: AdHu gal / Protein Mix
G5: AdC68-PvCSP / Protein Mix
G6: AdHu5-PvCSP / Protein Mix
Seru
m Ig
Gtite
rs
1 2 3 4 5 6
102
103
104
105
106
107
Boost
*
**
NS
NS
*
**
** P<0.01
P<0.05*
G1 G2 G3 G4 G5 G6
AdHu5-bgal AdHu5-bgal
Parasite recognition
poly (I:C)Protein mix
poly (I:C)
1:1
,00
01
:10
,000
AdC68-PvCSP /Protein mix poly (I:C)
A B
C Protein mix
poly (I:C)poly (I:C)
VK247
1:1
00
1:1
0,0
00
VK247
VK210VK210
1:1
,00
0
D
AdHußgal/
Poly (I:C)
Experimental challenge
Antibody
titers
Rec. protein
Day 0 Day 14
Rec. protein
Day 28
Challenge P. bergheitransfected with PvCS
Parasitemia
Dr. Rogerio Amino Institut Pasteur
Conclusions
Three rec. proteinsPoly (I:C)
His6PvCS-All-CS-epitopesPoly (I:C)
=Heterologous prime-boost vaccination rec. adenovirus
followed by protein works well in the mouse model using the P. vivax CS protein.
Plasmodium vivax vaccine compositions
USPTO 275220201WO1
PatentGenetically modified sequences encoding
Plasmodium vivax antigens WO2010 127420
2014