The neglected research on Plasmodium vivax vaccine …G1 G2 G3 G4 G5 G6 G1: AdHu gal / Adjuvant G2: AdC68-PvCSP / Adjuvant G3: AdHu5-PvCSP / Adjuvant G4: AdHu gal / Protein Mix G5:

The neglected research on Plasmodium vivax vaccine development

Financial

Support:

[email protected]

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