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The neglected research on Plasmodium vivax vaccine …G1 G2 G3 G4 G5 G6 G1: AdHu gal / Adjuvant G2:...
Transcript of The neglected research on Plasmodium vivax vaccine …G1 G2 G3 G4 G5 G6 G1: AdHu gal / Adjuvant G2:...
Ciclo de vida
Plasmodium sp.
Malária no mundo
2x108
108
107
P. falciparum
> 1 milhão de mortes/ano
2011
Proteína CS é o antígeno mais abundante na superfície dos esporozoítos
Alvo de Anticorpos protetores
SP RI Tandem repeats RIII TSR GPI
RII Plus
CS
Alvo de linfócitos T
Dr. Ruth S. Nussenzweig Dr. Victor Nussenzweig NYU
RTS,S
NH 2
HBsAg
COOH
CS Proteína (P. falciparum) (aa 207 -395)
HBsAg (NANP)3
Vacina recombinante de malária P. falciparum
Partícula RTS,S
RTS
S
Levedura
October 2011
Eficácia 50.5%
GSK &
US Army
P. falciparum
Eficácia 50.5%
Brown dots Infected
Blue dots protected
2011
Eficácia 17.4% Eficácia 64.3%
PfAMA-1 50 mg/dose X3
ASO2A
Crianças 1-6 anos
2011
Pesquisa de vacinas contra
malária no Brasil ?
2.85 billions at risk
132-391 M cases per
year
Americas ~80%
Asia ~50%
2674
18,218 174 (0.95%)
Publications
P. falciparum
World Brazil
2,623 35 (1.33%)
422 (13.8%)
Vaccine Plasmodium
P. vivax
Vaccine P. vivax 24 (5.68%)
204 (7.6%)
National Institute of Science and Technology in Vaccines
(INCTV)
FIOCRUZ (RJ, MG e Ba)
Universidades Federais (RJ , SP, MG)
Universidade Estadual (SP)
Dengue Malaria Chagas´ Disease Leishmaniasis Leptospiroses
Vectors Recombinant Proteins Plasmidial DNA Recombinant Viruses Chimeric virus
Experimental Models Mice Dogs Monkeys (Callitrix) Monkeys (Rhesus)
WWW.cpqrr.fiocruz.br/imtev/BR
Antigen discover: Dr. Irene S. Soares (FCF – USP) Dr. Mauricio M. Rodrigues (UNIFESP-EPM) Dr. Fabio T. Costa (Unicamp)
Adjuvants: Dr. Luis Carlos S. Ferreira (ICB – USP) Dr. Ricardo T. Gazzinelli (FIOCRUZ)
Malaria Vaccine
Viral Vectors: Dr. Oscar Bruna-Romero
Recombinant antigen expression Dr. Irene S. Soares Dr. Mauricio M. Rodrigues Dr. Luis Carlos S. Ferreira Dr. Oscar Bruna-Romero (IM – UFSC)
Immunogenicity
Dr. Mauricio M. Rodrigues Dr. Irene S. Soares
Dr. Oscar Bruna-Romero Dr. Fabio T. Costa
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
UNIFESP – EPM Lais H. Teixeira Dr. Cibele Tararam Ariane G. Camacho
Wistar Institute
Dr. Hildeglund Ertl Dr. Marcio Lasaro
New York University School of Medicine
Dr. Victor Nussenzweig Dr. Ruth S. Nussenzweig
USP ( Ciências Farmaceuticas ) Dr. Irene S. Soares
UFMG ( Departamento de Microbiologia ) Dr. Oscar Bruna - Romero
Vander J. Oliveira
Kátia Françoso
2014
CS Protein of Plasmodium vivax
NH2 COOH
Repeat region
VK210 = (DRADGQPAG)2 (DRAAGQPAG)2 DRADGQPAGD
VK247 = (ANGAGNQPG)4
P. vivax-like = (APGANQEGGAA)3 ?
77 249 1 331-3
Universal vaccine = immunity to the all three allelic forms.
His6-Vivax-like = (APGANQEGGAA)3
1- thcghnvdls kainlngvnf nnvdasslga ahvgqsasrg rglgenpdde egdakkkkdg
61- kkaepknpre nklkqpgapg anqeggaaap ganqeggaaa pganqeggaa apganqegga
121-aapganqegg aaapganqeg gaaapganqe ggaaapganq eggaaapgan qeggaaapga
181-nqeggaaapg anqeggaaap ganqeggaaa pganqeggaa apganqegga aapganqegg
241-aaapganqeg gaadraagqa aggnaggqgq nneganapne ksvkeyldkv ratvgtewtp
301-csvtcgvgvr vrrrvnaank kpedltlndl etdvct
His6-VK210 = (DRADGQPAG)2 (DRAAGQPAG)2 DRADGQPAGD
1- thcghnvdls kainlngvnf nnvdasslga ahvgqsasrg rglgenpdde egdakkkkdg
61- kkaepknpre nklkqpgdra dgqpagdrad gqpagdradg qpagdradgq pagdraagqp
121-agdradgqpa gdradgqpag dradgqpagd radgqpagdr aagqpagdra agqpagdrad
181-gqpagdraag qpagdradgq pagdraagqp agdradgqpa gdraagqpag draagqpagd
241-raagqaagdr aagqaaggna ggqgqnnega napneksvke yldkvratvg tewtpcsvtc
301-gvgvrvrrrv naankkpedl tlndletdvc t
His6-VK247 = (ANGAGNQPG)4
1- thcghnvdls kainlngvnf nnvdasslga ahvgqsasrg rglgenpdde egdakkkkdg
61- kkaepknpre nklkqpgang agnqpganga gnqpgangag nqpgangagn qpgangagnq
121-pgangagnqp gangagnqpg angagnqpga ngagnqpgan gagnqpgang agnqpganga
181-gnqpgangag nqpgangagn qpgangagnq pgangagnqp gangagnqpg angagnqpga
241-ngagnqpgan gagnqpggna ggqgqnnega napneksvke yldkvratvg tewtpcsvtc
301-gvgvrvrrrv naankkpedl tlndletdvc t
His6-All-CS-Epitopes
(DRADGQPAG)2 (DRAAGQPAG)2 DRADGQPAGD(APGANQEGGAA)3
(ANGAGNQPG)4
1- thcghnvdls kainlngvnf nnvdasslga ahvgqsasrg rglgenpdde egdakkkkdg
61- kkaepknpre nklkqpgpgd radgqpagdr adgqpagdra agqpagdraa gqpagdradg
121-qpagdradgq pagdradapg anqeggaaap ganqeggaaa pganqeggaa aapganqegg
181-aaapganqeg gaaapganqe ggaaaangag nqpgangagn qpgangagnq pgangagnqp
241-gangagnqpg draagqaagg naggqgqnne ganapneksv keyldkvrat vgtewtpcsv
301-tcgvgvrvrr rvnaankkpe dltlndletd vct
His6-PvCS-VK210
His6-PvCS-Vivax-like
His6-PvCS-VK247
His6-PvCS-All-CS-epitopes
E. coli rec. proteins
yPvCS-VK210-His6
PvCS-Vivax-like-His6
yPvCS-VK247-His6
yPvCS-All-CS-epitopes-His6
PIC-PVCS-All epitopes TTCHHHHHHTHCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDE
EGDAKKKKDGKKAEPKNPRENKLKQPGPGDRADGQPAGDRADGQPAGDRAAGQPAGDRA
GQPAGDRADGQPAGDRADGQPAGDRADAPGANQEGGAAAPGANQEGGAAAPGANQEGGA
AAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAANGAGNQPGANGAGNQPGANGAGN
QPGANGAGNQPGANGAGNQPGDRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLDKVR
ATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCTHHHHHH*
PIC-PVCS-210 PRENKLKQPGDRADGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAAGQPAGDRAD
GQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRADGQPAG
DRAAGQPAGDRADGQPAGDRAAGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRAAG
QAAGDRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVG
VRVRRRVNAANKKPEDLTLNDLETDVCTHHHHHHH*
PIC-PVCS-247 PRENKLKQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGA
GNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPG
ANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAG
NQPGANGAGNQPGGNAGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVG
VRVRRRVNAANKKPEDLTLNDLETDVCTHHHHHH*
PIC-PVCS-Vivax-like PRENKLKQPGAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGAN
QEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGG
AAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAP
GANQEGGAADRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSV
TCGVGVRVRRRVNAANKKPEDLTLNDLETDVCTHHHHHH*
PIC-PVCS-Allepitopes PRENKLKQPGPGDRADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRADGQPAGDR
ADGQPAGDRADAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAAPGANQEGGAAAPG
ANQEGGAAAPGANQEGGAAAANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANG
AGNQPGDRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCG
VGVRVRRRVNAANKKPEDLTLNDLETDVCTHHHHHH*
P. pastoris rec. proteins Vander de Oliveira Jampaulo
Synthetic genes:
codon optmized
Cloning pET28b
Expression BL-21
Purification
(affinity and FPLC)
35
50
75
105
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 P. 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
Vander O. Jampaulo
Yeast recombinant CS proteins of P. vivax
B C
D E
A- yPvCS-VK210-His6 B- yPvCS-VK247-His6
C- yPvCS-VL-His6 D- yPvCS-All-CS-epitopes His6
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.
Why Poly (I:C)?
TLR-3 agonist
November 2011
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 yPvCS-VK210
Gr. 3 yPvCS-VK247
Gr. 4 yPvCS-Vivax-like
Gr. 5 Three rec. proteins
Gr. 6 yPvCS-All-CS-epitopes
Adjuvant
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
106
Antibodies to His6PvCSP
-VK210
Days
20 40 60 80 100 120 140 160 180 200
102
103
104
105
106
CAntibodies to
His6PvCSP-VK247
Days
20 40 60 80 100 120 140 160 180 200
102
103
104
105
106
Antibodies to His6PvCSP-Vivax-like
D
B
Seru
m Ig
G t
iters
Seru
m Ig
G t
iters
Seru
m Ig
G t
iters
Seru
m Ig
G t
iters
Replication deficient adenovirus Human type 5 Simian 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 After priming
After boosting
poly (I:C)
Protein mix
poly (I:C)
1:1
,00
0
1:1
0,0
00
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
VK210
VK210
1:1
,00
0
D
AdHußgal/
Poly (I:C)
Conclusions
High antibody titers
Three rec. proteins + Poly (I:C)
yPvCS-All-CS-epitopes + Poly (I:C)
Heterologous prime-boost vaccination with rec. adenovirus followed by protein.
Plasmodium vivax vaccine compositions 27522-0201WO1
VK210
Perspectives
Immunization of mice and challenge with P. berghei
transgenic expressing the P. vivax CS protein
University of Oxford (Dr. A. Reyes Sandoval)
Institut Pasteur (Dr. R. Amino)
cGMP protein production
Walter Reed Army Institute