The neglected research on Plasmodium vivax vaccine development Financial Support: mrodrigues@unifesp.br
Ciclo de vida Plasmodium sp.
Malária no mundo 10 8 10 7 2x10 8 P. falciparum > 1 milhão de mortes/ano
2011
Proteína CS é o antígeno mais abundante na superfície dos esporozoítos CS SP RI Tandem repeats RIII TSR GPI RII Plus Alvo de Anticorpos protetores Alvo de linfócitos T Dr. Ruth S. Nussenzweig Dr. Victor Nussenzweig NYU
Vacina recombinante de malária P. falciparum RTS,S NH 2 HBsAg CS Proteína (P. falciparum) (aa 207-395) COOH HBsAg (NANP) 3
Partícula RTS,S RTS Levedura S
GSK & US Army P. falciparum October 2011 Eficácia 50.5%
Blue dots protected Eficácia 50.5% Brown dots Infected
2011 Crianças 1-6 anos PfAMA-1 50 mg/dose X3 ASO2A Eficácia 17.4% Eficácia 64.3%
2011
Pesquisa de vacinas contra malária no Brasil?
Americas ~80% Asia ~50% 2.85 billions at risk 132-391 M cases per year
Publications P. falciparum World Brazil 18,218 174 (0.95%) 2674 204 (7.6%) P. vivax Vaccine Plasmodium 2,623 35 (1.33%) Vaccine P. vivax 422 24 (5.68%) (13.8%)
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 Recombinant antigen expression Dr. Irene S. Soares Dr. Mauricio M. Rodrigues Dr. Luis Carlos S. Ferreira Dr. Oscar Bruna-Romero (IM UFSC) Viral Vectors: Dr. Oscar Bruna-Romero Immunogenicity Dr. Mauricio M. Rodrigues Dr. Irene S. Soares Dr. Oscar Bruna-Romero Dr. Fabio T. Costa
Sporozoite major surface antigen: CS protein CS SP RI Tandem repeats RIII TSR GPI RII Plus Target of protective antibodies Target of protective T cells
2014 UNIFESP EPM Lais H. Teixeira Dr. Cibele Tararam Ariane G. Camacho Vander J. Oliveira USP ( Ciências Farmaceuticas ) Dr. Irene S. Soares Kátia Françoso UFMG ( Departamento de Microbiologia ) Dr. Oscar Bruna - Romero New York University School of Medicine Dr. Victor Nussenzweig Dr. Ruth S. Nussenzweig Wistar Institute Dr. Hildeglund Ertl Dr. Marcio Lasaro
CS Protein of Plasmodium vivax Repeat region 1 77 249 331-3 NH 2 COOH VK210 = (DRADGQPAG) 2 (DRAAGQPAG) 2 DRADGQPAGD VK247 = (ANGAGNQPG) 4 P. vivax-like = (APGANQEGGAA) 3? Universal vaccine = immunity to the all three allelic forms.
E. coli rec. proteins His 6 -PvCS-VK210 His 6 -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 His 6 -VK247 = (ANGAGNQPG) 4 His 6 -PvCS-VK247 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 His 6 -Vivax-like = (APGANQEGGAA) 3 His 6 -PvCS-Vivax-like 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 His 6 -PvCS-All-CS-epitopes His 6 -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
PIC-PVCS-All epitopes TTCHHHHHHTHCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDE EGDAKKKKDGKKAEPKNPRENKLKQPGPGDRADGQPAGDRADGQPAGDRAAGQPAGDRA GQPAGDRADGQPAGDRADGQPAGDRADAPGANQEGGAAAPGANQEGGAAAPGANQEGGA AAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAANGAGNQPGANGAGNQPGANGAGN QPGANGAGNQPGANGAGNQPGDRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLDKVR ATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCTHHHHHH* P. pastoris rec. proteins Vander de Oliveira Jampaulo ypvcs-vk210-his 6 ypvcs-vk247-his 6 PvCS-Vivax-like-His 6 ypvcs-all-cs-epitopes-his 6 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*
Bacterial Recombinant CS proteins of P. vivax Synthetic genes: codon optmized Cloning pet28b Expression BL-21 kda 160 105 75 50 35 30 A B C D Purification (affinity and FPLC) A - His 6 -PvCS-VK210 B - His 6 -PvCS-VK247 C - His 6 -PvCS-Vivax-like D - His 6 -PvCS-All-CS-epitopes
Yeast recombinant CS proteins of P. vivax Synthetic genes: codon optmized Cloning ppic9k Expression P. pastoris 116.0 66.2 45.0 35.0 25.0 A STD B C D Secretion 18.4 A- ypvcs-vk210-his 6 B- ypvcs-vk247-his 6 Purification (affinity and FPLC) C- ypvcs-vl-his 6 D- ypvcs-all-cs-epitopes His 6 Vander O. Jampaulo
Yeast recombinant CS proteins of P. vivax B A- ypvcs-vk210-his 6 B- ypvcs-vk247-his 6 C D C- ypvcs-vl-his 6 E D- ypvcs-all-cs-epitopes His 6
Universal vaccine = immunity to the all three allelic forms. Three rec. proteins X His 6 PvCS-All-CS-epitopes Poly (I:C) (TLR-3 agonist) Titers and specificity
Why Poly (I:C)? TLR-3 agonist November 2011
C57Bl/6 mice immunization protocol with the recombinant proteins Rec. protein Rec. protein Rec. protein Antibody titers to the different allelic forms until day 180 Day 0 Day 21 Day 42 Gr. 1 Adjuvant alone Gr. 2 Gr. 3 ypvcs-vk210 ypvcs-vk247 1 mg/mouse/dose 1 mg/mouse/dose Adjuvant Gr. 4 Gr. 5 ypvcs-vivax-like Three rec. proteins 1 mg/mouse/dose 3 mg/mouse/dose Poly (I:C) (TLR-3 agonist) Gr. 6 ypvcs-all-cs-epitopes 3 mg/mouse/dose
Serum IgG titers Serum IgG titers Serum IgG titers Serum IgG titers B Antibodies to His 6 PvCSP - VK210 10 6 C 10 6 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 Antibodies to His 6 PvCSP-VK247 10 5 10 4 10 3 10 2 D 20 40 60 80 100 120 140 160 180 200 10 6 Days Antibodies to His 6 PvCSP-Vivax-like 10 5 10 5 10 4 10 4 10 3 10 3 10 2 10 2 20 40 60 80 100 120 140 160 180 200 Days 20 40 60 80 100 120 140 160 180 200 Days
Replication deficient adenovirus Human type 5 Simian C68 NH 2 1 SP aa repeats 100 253 23 355 COOH 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
C57Bl/6 mice immunization protocol with the recombinant proteins Rec. Adenovirus Rec. protein Antibody titers Day 0 Day 42
Serum IgG titers Serum IgG titers C 10 6 10 5 Prime Heterologous prime-boost vaccination After priming 10 7 His6 -PvCSP-Vk210 His 6 -PvCSP-Vk247 His 6 -PvCSP-Vivax-like NS D Boost 10 7 ** P<0.01 * P<0.05 10 6 10 5 After boosting NS ** * ** * NS 10 4 10 4 10 3 10 3 10 2 10 2 1 2 3 4 5 6 G1 G2 G3 G4 G5 G6 G1: AdHu5-bgal G2: AdC68-PvCSP G3: AdHu5-PvCSP G4: AdHu gal G5: AdC68-PvCSP G6: AdHu5-PvCSP 1 2 3 4 5 6 G1 G2 G3 G4 G5 G6 G1: AdHu5-bgal / Adjuvant G2: AdC68-PvCSP / Adjuvant G3: AdHu5-PvCSP / Adjuvant G4: AdHu gal / Protein Mix G5: AdC68-PvCSP / Protein Mix G6: AdHu5-PvCSP / Protein Mix
1:10,000 1:1,000 1:10,000 1:100 1:1,000 After priming Parasite recognition After boosting A Protein mix poly (I:C) AdC68-PvCSP / Protein mix poly (I:C) B poly (I:C) AdHußgal/ Poly (I:C) VK247 VK247 C Protein mix poly (I:C) D poly (I:C) VK210 VK210
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
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 VK210