Development of a universal vaccine against malaria Unicamp ... · [email protected] Development of a universal vaccine against malaria caused by Plasmodium vivax. UNIFESP² EPM

[email protected]

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

Plasmodium sp. life cycle

Immune mechanisms

Antibodies to CS

CD4 and CD8 T cells

August 2014

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

Antibody

titers Parasitemia

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