FIDIC - Cátedrasfci.uib.es/digitalAssets/177/177085_6.pdf · fidic. the search for ... patarroyo...

163
FUNDACION INSTITUTO DE INMUNOLOGIA DE COLOMBIA FIDIC

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FUNDACION INSTITUTO DE INMUNOLOGIA DE

COLOMBIAFIDIC

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THE SEARCH FOR

π

Or

A MATHEMATICAL METHOD FOR DEVELOPING

VACCINES

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Whooping coug355 thousand

Helminthiasis165 thousand

Diarrhoea3.1 millions

Tuberculosis3.1 millions

Malaria2.1 millions

Acute Respiratory Infections4.4 millions

Measles1 millionHIV/AIDS

1 million

Tetanus500 thousand

Hepatitis1.1 millions

TEN LEADING CAUSES OF MORTALITY IN THE WORLD

INDUCED BY TRANSMITTABLE DISEASES

WHO-1995

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BIOLOGICAL VACCINES

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VACCINES IN PROGRESS

SMALL POXPOLIO

MEASLESPAROTIDITIS

GERMAN MEASLESCHICKEN POX

ACTUAL VACCINES

HEPATITIS A & BYELLOW FEVER

RABIESBCGDPT

SCHISTOCARIESLYME

STREPTOCOCCUSRICKETS

INFLUENZAROTAVIRUS

RSVPAPILOMA, ETC

MALARIAAIDS

TYPHUSCHOLERA

MENINGITISDENGUEHERPES

S.T.D.LEISHMANIA

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PURPOSE :

To find a rational approach

for thedevelopment of

chemicallysynthetizedvaccines.

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Plasmodium falciparumPARASITE�S LIFE CYCLE

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Why malaria?• 300 million people are

infected and 3 million dieannually

• Acute disease• Easy diagnosis• Curable with treatment• Experimental model: the

Aotus monkey• Heavy economic burden

for the developing world

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0

2

4

6

8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15D ays

859876932994942978

01234567

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15D ays

643847983980981

01234567

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15D ays

883977874952

01234567

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15D ays

680872574515631860

TITRATION OF THE FVO P. falciparum STRAIN IN AOTUS

MONKEYS

106

105

104

107

Rodriguez R. et al. 1990. Am. J. Trop. Med. Hyg 43:339

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Accepted in IMMUNOGENETICSUnusual Variability in Aotus monkey MHC-E sequences

Paula P. Cárdenas1, Carlos F. Suárez1, Consuelo P. Martínez, Eugenio J. Llanos, Mateo Obregón, Manuel E.

Patarroyo, Manuel A. Patarroyo *

Aotus monkey MHC-E

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9 7 9 7

69

¤6 6

¤6

¤67

4 4 4¤46

6¤67

¤ ¤9

97¤

4 7¤ ¤

9¤9

¤

97 7

¤ ¤7

¤4¤ ¤

46

4 4¤

4 1¤

1 1¤

1¤ ¤

1 1

Ao-D

RB*W

104

HLA-

DRB1

*04

Ao-D

RB*W

304

HLA-

DRB1

*03

9 7 9 7

69

¤6 6

¤6

¤67

4 4 4¤46

6¤67

¤ ¤9

97¤

4 7¤ ¤

9¤9

¤

97 7

¤ ¤7

¤4¤ ¤

46

4 4¤

4 1¤

1 1¤

1¤ ¤

1 1

Logos obtenidos a partir de las secuencias de los Aotus (Ao-DRB*W) y de las secuencias de

humanos (HLA-DRB)

Ao-D

RB*W

11HL

A-DR

B1*0

8-14

9 7 9 769¤

6 6¤

6¤67

4 4 4¤46

6¤67

¤ ¤9

97¤

4 7¤ ¤

9¤9

¤97 7

¤ ¤7

¤4¤ ¤

46

4 4¤

4 1¤

1 1¤

1¤ ¤

1 1

9 7 9 7

69¤

6 6¤

6¤67

4 4 4¤46

6¤67

¤ ¤9

97¤

4 7¤ ¤

9¤9

¤97 7

¤ ¤7

¤4¤ ¤

46

4 4¤

4 1¤

1 1¤

1¤ ¤

1 1

Ao-D

RB*W

101

HLA-

DRB1

*01

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% m

ean

% m

in

% m

ax

% m

ean

% m

in

% m

ax

Ao-DRB*W104 vs. HLA-DRB1*04 95 91 98 93 80 100Ao-DRB*W304 vs. HLA-DRB1*03 92 88 95 85 77 91Ao-DRB*W302 vs. HLA-DRB3 92 89 94 84 77 91Ao-DRB*W11 vs. HLA-DRB1*08 92 88 96 83 74 91Ao-DRB*W303 vs. HLA-DRB3 91 86 94 83 71 91Ao-DRB*W301 vs. HLA-DRB3 90 86 93 84 74 91Ao-DRB*W11 vs. HLA-DRB1*11 90 85 95 83 71 91Ao-DRB*W11 vs. HLA-DRB1*13 90 85 96 81 71 91Ao-DRB*W701/02 vs. HLA-DRB1*07 90 87 93 80 74 88Ao-DRB*W101 vs. HLA-DRB1*01 90 84 94 80 74 88Ao-DRB*W11 vs. HLA-DRB1*14 89 85 96 80 68 91Ao-DRB*W4 vs. HLA DRB1*10 88 84 89 74 68 80Ao-DRB*W4 vs. HLA DRB4 88 83 91 72 65 80

β domain Pockets

HumanOwl Monkey

Promedio y Rango de homología entre Ao-DRβ y HLA-DRβ en el dominio β y

posiciones de los Pockets

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IMMUNOBLOT OFP. falciparum ISOLATED

PROTEINSPatarroyo M. E. et. al. 1987. Vaccines. 117-124

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PARASITAEMIA IN MONKEYS IMMUNISED WITH MEROZOITE

PURIFIED PROTEINS

Immunizing Monkey Percentage of parasitemia on days (after challenge)molecule number 6 7 8 9 10 11 12 13 14 15 16 17 18 19

155K 087 0 0 0 0 0.88 0.30 2.0 3.0 5.35 8.8 Q 090 0 0 0 0 0 0 0.8 0.2 0.45 0.9 1.5 1.6 2.0 3.4

115K 085 0 0.46 0.84 3.48 3.0 9.93 Q150 0 0.05 0.20 1.12 1.67 nd 6.57 Q

105K 165 0 0.33 1.92 3.72 10.9 Q168 0 0.81 1.64 3.33 11.5 Q

90K 160 0 0.13 1.2 3.39 4.14 13.65 Q170 0 0.10 1.1 1.20 5.09 5.40 14.7 Q

83K 125 0 0 0 0 0 0 0 0 0 0 0 0 0 0130 0 0 0 0.6 0.2 0.3 3.63 1.6 1.1 0.6 nd 0 0 0144 0 0 0 0 0 0 0 0.1 0.05 0.5 0.3 0.07 0.03 0.01

60K 119 0 0 0 0.1 0.80 0.15 4.5 1.25 15.3 Q122 0 0.13 0.25 0.15 0.80 2.5 17.5 Q

55K 0.81 0 0 0 0 0 0.18 0.24 0.8 1.57 3.0 3.5 4.4 7.4 Q102 0 0 0 0 0 0.05 0.05 0.5 0 0.1 0.6 0.9 5.4 7.3

50K 0.29 0 0.40 1.50 3.6 4.67 10.0 Q 0.56 0 0.66 5.6 4.43 6.42 4.20 11.3 Q131 0 0.31 1.50 0.50 3.93 2.0 16.5 Q

40K 111 0 0 0 0 1.02 0.20 2.7 3.2 14.3 Q114 0 0 0 0 0.8 0.5 2.0 2.0 2.0 7.6 Q

35K 135 0 0 0 0 0 0 0.01 0.03 0.03 0.1 0.012 0.01 0 0159 0 0.25 0.25 1.3 2.23 1.03 2.5 2.1 4.0 2.1 1.6 0.7 0.08 0.01

30K 093 0 0.05 1.52 2.5 4.75 3.45 2.54 4.8 9.4 Q171 0 0.1 2.5 1.6 2.63 2.05 13.0 Q

23K 112 0 0.06 0 1.0 0 3.60 6.37 6.60 12.0 Q 137 0 0.05 0.05 0.20 0 1.1 3.2 nd +

Controls 501 0.40 0.76 3.0 2.0 7.82 9.07 Q 502 0.03 0.06 0.43 0.47 3.6 3.4 5.9 8.92 Q199 1.0 4.3 3.11 9.8 26.0 Q

nd indicates not determined

Patarroyo M. E.. et al. 1987. Vaccines 87:117

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AMINOACID SEQUENCE OF THE SYNTHETIC MALARIA

VACCINESPf66

Sl 2 3 4 5 6 7 8 9 10 1 2 3 4 5

- Gly- Asp- Glu- Leu- Glu- Ala - Glu- Thr - Gln- Asn- Val - Tyr - Ala - Ala

Pro16 7 8 9 20 1 2 3 4 5 6 7 8 9 30

- Asn- Ala - Asn- Pro- Tyr - Ser - Leu- Phe- Gln - Lys- Glu- Lys- Met- Val

1 2 3 4 5 6 7 8 9 40 1 2 3 4 5- Pro- Asn- Ala - Asn- Pro- Pro- Ala - Asn- Lys- Lys- Asn- Ala - Gly- Cys

lS

Cys

Leu

Patarroyo M. et al. 1988. Nature. 332:158

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PROYECTO MACRO-TUMACOPROYECTO LA TOLAPROYECTO RONDONIAPROYECTO �LA TE�PROYECTO ARIPAOPROYECTO LAS MAJADASPROYECTO RIO ROSARIO

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We conclude that SPf66 does not protectagainst clinical falciparum malaria and thatfurther efficacy trials are not warranted.

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COLFAVACThe COLombian FAlciparum VACcine

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0.7

0.6

0.5

0.4

0.3

0.2

0.1

00 15 30 45 60 90 120 180

MINUTES

BOUND PICOMOLESHIGH BINDING ACTIVITY PEPTIDES

0.2

0.15

0.05

0.1

00 15 30 45 60 90 120 180

MINUTES

BOUND PICOMOLESHIGH BINDING ACTIVITY PEPTIDES

MINUTES

MEDIUM BINDING ACTIVITY PEPTIDES

0 15 30 45 60 90 120 1800

BOUND PICOMOLES

0.05

0.1

0.15

0.2

0 15 30 45 60 90 120 180MINUTES

0

0.05

0.1

0.15

0.2BOUND PICOMOLES

LOW BINDING ACTIVITY PEPTIDES

Calvo M. et al. Peptide Research, 1991, 4, 324-333

SPECIFIC UPTAKE OF THE DIFFERENT PEPTIDES BY

HUMAN R.B.C.

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PRINCIPLES FOR VACCINE DEVELOPMENT

Ligand

Host Cell Parasite

Blocking Receptor- Ligand

Vaccine

Receptor

INDUCE IMMUNITY

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AMINO ACID SEQUENCE OF THE EBA-175 SYNTHETIC PEPTIDES

Peptide

number

1755 20 KARNEYDIKENEKFLDVYKE 39

1756 40 KFNELDKKKYGNVQKTDKKI 59

1757 60 FTFIENKLDILNNSKFNKRW 79

1758 80 KSYGTPDNIDKNMSLINKHN 99

1759 100 NEEMFNNNYQSFLSTSSLIK 119

1760 120 QNKYVPINAVRVSRILSFLD 139

1761 140 SRINNGRNTSSNNEVLSNCR 159

1762 160 EKRKGMKWDCKKKNDRSNYV 179

1763 180 CIPDRRIQLCIVNLSIIKTY 199

1764 200 TKETMKDHFIEASKKESQLL 219

1765 220 LKKNDNKYNSKFCNDLKNSF 239

1766 240 LDYGHLAMGNDMDFGGYSTK 259

1767 260 AENKIQEVFKGAHGEISEHK 279

1768 280 IKNFRKEWWNEFREKLWEAM 299

1769 300 LSEHKNNINNCKNIPQEELQ 319

1770 320 ITQWIKEWHGEFLLERDNRS 339

1771 340 KLPKSKCKNNTLYEACEKEC 359

1772 360 IDPCMKYRDWIIRSKFEWHT 379

1773 380 LSKEYETQKVPKENAENYLI 399

1774 400 KISENKNDAKVSLLLNNCDA 419

1775 420 EYSKYCDCKHTTTLVKSVLN 439

1776 440 GNDNTIKEKREHIDLDDFSK 459

1777 460 FGCDKNSVDTNTKVWECKNP 479

1778 480 YILSTKDVCVPPRRQELCLG 499

1779 500 NIDRIYDKNLLMIKEHILAI 519

1780 520 AIYESRILKRKYKNKDDKEV 539

1781 540 CKIINKTFADIRDIIGGTDY 559

1782 560 WNDLSNRKLVGKINTNSKYV 579

1783 580 HRNKKNDKLFRDEWWKVIKK 599

1784 600 DVWNVISWVFKDKTVCKEDD 619

1785 620 IENIPQFFRWFSEWGDDYCQ 639

1786 640 DKTKMIETLKVECKEKPCED 659

1787 660 DNCKSKCNSYKEWISKKKEE 679

1788 680 YNKQAKQYQEYQKGNNYKMY 699

1789 700 SEFKSIKPEVYLKKYSEKCS 719

1790 720 NLNFEDEFKEELHSDYKNKC 739

Peptidenumber

1791 740 TMCPEVKDVPISIIRNNEQT 759

1792 760 SQEAVPEENTEIAHRTETPS 779

1793 780 ISEGPKGNEQKERDDDSLSK 799

1794 800 ISVSPENSRPETDAKDTSNL 819

1795 820 LKLKGDVDISMPKAVIGSSP 839

1796 840 NDNINVTEQGDNISGVNSKP 859

1797 860 LSDDVRPDKKELEDQNSDES 879

1798 880 EETVVNHISKSPSINNGDDS 899

1799 900 GSGSATVSESSSSNTGLSID 919

1800 920 DDRNGDTFVRTQDTANTEDV 939

1801 940 IRKENADKDEDEKGADEERH 959

1802 960 STSESLSSPEEKMLTDNEGG 979

1803 980 NSLNHEEVKEHTSNSDNVQQ 999

1804 1000 SGGIVNMNVEKELKDTLENP 1019

1805 1020 SSSLDEGKAHEELSEPNLSS 1039

1806 1040 DQDMSNTPGPLDNTSEETTE 1059

1807 1060 RISNNEYKVNEREDERTLTK 1079

1808 1080 EYEDIVLKSHMNRESDDGEL 1099

1809 1100 YDENSDLSTVNDESEDAEAK 1119

1810 1120 MKGNDTSEMSHNSSQHIESD 1139

1811 1140 QQKNDMKTVGDLGTTHVQNE 1159

1812 1160 ISVPVTGEIDEKLRESKESK 1179

1813 1180 IHKAEEERLSHTDIHKINPE 1199

1814 1200 DRNSNTLHLKDIRNEENERH 1219

1815 1220 LTNQNINISQERDLQKHGFH 1239

1816 1240 TMNNLHGDGVSERSQINHSH 1259

1817 1260 HGNRQDRGGNSGNVLNMRSN 1279

1818 1280 NNNFNNIPSRYNLYDKKLDL 1299

1819 1300 DLYENRNDSTTKELIKKLAE 1319

1820 1320 INKCENEISVKYCDHMIHEE 1339

1821 1340 IPLKTCTKEKTRNLCCAVSD 1359

1822 1360 YCMSYFTYDSEEYYNCTKRE 1379

1823 1380 FDDPSYTCFRKEAFSSMIFK 1399

1824 1400 FLITNKIYYYFYTYKTAKVT 1419

1825 1420 IKKINFSLIFFFFFSF 1435

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PEPTIDE RBC BINDING BEHAVIOURPEPTIDE RBC BINDING BEHAVIOUR

AddedAdded PeptidePeptide

Peptide Binding activity is the curve’s specific slope

0

0.055

0.115

0 0.5 1 0 0.5 1

1585 EBA P. 4

0 0.5 1

1795

Total Binding Non-specific Binding Specífic Binding

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Peptide 15 30 45 6015111512151315141516151715181519152015211522152315241525152683K15271528152915401541154215431544154515461547154815491550155115521553155415551556155715581559

30K 156015611562 nd15631564156515661567 nd15691570157115721573157438K1575157615771578157915801581158215831584 nd1585158615871588

33K1589159015911592159315941595 nd159615971598159919K 16001601

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15 30 454042

4043

4044

4045

4046

4047

4048

4049

4050

4051

4052

4053

4054

4055

4056

4057

4058

4059

4060

4061

4063

4064

4065

2.0

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Peptide Sequence26327 1 LINIETEMKHKQKQLINKMNY 2026328 21 DIEKDNITDQYMHDVQQNIFY 4026329 41 EPITLKMNEYNTLLNDNHNN 6026330 61 NINNEHQFNHLNSLHTKIFSY 8026331 81 HNYNKEQQQEYITNIMQRID 10026332 101 VFINDLDTYQYEYYFYEWNQ 12026333 121 EYKQIDKNKINQHINNIKNN 14026334 141 LIHVKKQFEHTLENIKNNENY 16026335 161 IFDNIQLKKKDIDDIIININY 18026336 181 NTKETYLKELNKKKMLQNKK 20026337 201 KVDEKSEINNHHTLQHDNQNY 22026338 221 VEQKNKIKDHNLITKPNNNSY 24026339 241 SEESHQNEQMKEQNKNILEKY 26026340 261 QTRNIKPHHVHNHNHNHNHNY 28026341 281 HNQNQNQKDSTKLQEQDISTY 30026342 301 HKLHNTIHEQQSKDNHQGNRY 32026343 321 EKKQKNGNHERMYFASGIVV 34026344 341 SILFLSSLGFVINSKNNKQEY 36026345 361 YDKEQEKQQQNDFVCDNNKM 38026346 381 DDKSTQKYGRNQEEVMEISF 40026347 386 QKYGRNQEEVMEISFDNDYI 405

Normocyte Binding Protein PfNBP-1 Binding Activity 1 % 2%

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Sequence

4313 134 DAEVAGTQYRLPSGKCPVFG 153

4314 154 KG I I I ENSNTTFLTPVATGNY173

4315 174 QYLKDGGFAFPPTEPLMSPM 193

4316 194 TLDEMRHFYKDNKYVKNLDE 113

4317 214 LTLCSRHAGNMI PDNDKNSNY233

4318 234 YKYPAVYDDKDKKCH I LY I A 253

4319 254 AQENNGPRYCNKDESKRNSM 273

4320 274 FCFRPAKD I SFQNYTYLSKN 293

4321 294 VVDNWEKVCPRKNLQNAKFGY313

4322 314 LWVDGNCED I PHVNEFSA I DY333

4323 334 LFECNKLVFELSASDQPKQY 353

4324 354 EQHLTDYEK I KEGFKNKNAS 373

4325 374 MI KSAFLPTGAFKADRYKSH 393

4326 394 GKGYNWGNYNTETQKCE I FN 413

4327 414 VKPTCL I NNSSY I ATTALSH 433

4328 434 P I EVEHNFPCSLYKNE I MKE 453

4329 454 I ERESKR I KLNDNDDEGNKK 473

4330 474 I I APR I F I SDDKDSLKCPCDY493

4331 494 PE I VSNSTCNFFVCKCVERRY513

4332 514 AEVTSNNEVVVKEEYKDEYA 533

4333 534 D I PEHKPTYDKMK I I I ASSA 553

4334 554 AVAVLAT I LMVYLYKRKGNA 573

4335 574 EKYDKMDEPQHYGKSNSRND 593

4336 594 EMLDPEASFWGEEKRASHTTY613

4337 603 WGEEKRASHTTPVLMEKPYY 622

4307 14 EFTYMI NFGRGQNYWEHPYQ 33

4308 34 KSDVYHP I NEHREHPKEYQY 53

4309 54 PLHQEHTYQQEDSGEDENTL 73

4310 74 QHAYP I DHEGAEPAPQEQNL 93

4311 94 FSS I E I VERSNYMGNPWTEY 113

114 MAKYD I EEVHGSG I RVDLGE 1334312

Peptide 2.0FIDICFIDIC

AMA-1

Peptide Kd (nM)

4313 120 ± 124315 120 ± 104316 150 ± 144322 700 ± 214325 100 ± 094328 140 ± 11

Urquiza M., et al., 2001, Vaccine, 19: 508

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Peptide

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FIDICFIDIC

Bozedech Z et al August 18 2003. PLOS Biology 1, 001

PHASEOGRAM OF PUTATIVE VACCINE TARGETS

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C : CONSERVED

V: VARIABLE

High RBC binding activity peptides

FIDICFIDIC

PROTEIN C V Reference

MSP1 3 6 Urquiza M. et. al . 1996. Parasite Immunology . 18: 515-526

RESA 2 − Vera R. et al . 2000. Vaccine. 18: 1289-1293

SERA 6 1 Puentes A. et. al . 2000. Parasitology International. 49: 105-117

MSA2 1 2 Ocampo M. et. al . J. Peptide Research. 55: 216-233

EBA175 6 − Rodríguez L.E et. al. 2000. Parasitology. 120: 225-235

GBP130 1 − Suárez J. et. al . 2000. Mem. Inst. Oswaldo Cruz . 95: 495-501

HRP I, II, II 3 1 López R. et a l. 2000. Acta Tropica. 75: 349-359

ABRA 5 − Curtidor H. et. al . 2001. Vaccine. 19: 4496-4504

AMA1 4 3 Urquiza M. et. al . 2001. Vaccine. 19: 508-513

EBA140 6 − Rodríguez L.E.. et. al . 2003. J. Peptide Research. 62: 175-184

NBP1 2 − Valbuena J. et al . 2003. Peptides . 24: 1007-1014

MSP8 5 − Puentes A. et. al. 2003. Peptides .24: 1015-1023

MAEBL 9 − Ocampo M. et al . 2004. Biochem. Biophys. Res. Comm. 315: 319-329

RAP2 4 − López R. et al. 2004. Biochemie . 86: 1-6

RBP2 Ha/Hb 9 3 Ocampo M. et. al . 2004. Parasitol. Int. 53: 77-88

RAP1 4 − Curtidor H. et. al . 2004. Vaccine . 22: 1054-1062

EBA160 5 − Valbuena J. et al. 2004. Biochem. Biophys. Res. Comm . 321: 835-844

CLAG3 5 − Ocampo M. et. al. 2005. Proteins Science . 14: 504-513

EBL1 5 − Curtidor H. et. al. 2005. Protein Science . 14: 464-473

JESEBL 5 − Vera R. et al . 2004. Biochemie. In press

STEVOR − 3 García J. et al. 2004. Peptides. In press

MSP10 3 − Puentes A. et al . 2004. Biochemie. In press

MSP3 3 − Rodríguez L.E. et. al. 2004. Submitted to Protein Science

SORTILIN 6 − Vera R. et al. 2004. Submitted to Biochemistry

RESA-LIKE 3 − Rodríguez L.E. et. al. 2004. Submitted to J. Int. Parasitol.

TryThrA 4 − Curtidor H. et. al. 2004. Submitted to Chembiochem

HAP 2 − Valbuena J. et. al . 2004. Submitted to Biol. Chem.

MSP6 2 1 López R. et al. 2004. Submitted to Peptides

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1. Plasmodia (falciparum & vivax) use CONSERVED as well as VARIABLE sequences to bind to host cells.

Synthetic vaccines - Emergingprinciples

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ANTIGENICITYSTUDIES

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Aotus Monkeys develop resistance toreinfection after being infected severaltimes with 5 to 500 X 106 P. falciparum FVO infected erythrocytes and treated withchloroquine

HYPERIMMUNE MONKEYS

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HIPERIMMUNE FA CONTROL NAIVE

Peptide1228 1522 1218 1220 1487 1464 1224 1228 1582 1599 1538 1564 15 30 45

404240434044404540464047

40484049

4050

4051405240534054

40554056405740584059

40604061406340644065

AOTUS ABS AGAINST MSP-2 PEPTIDES Vs. BINDING ACTIVITY

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ACUTE RECOVERED EXPOSED

MSA2 1X 2X 3X 1X 2X 3X BINDING

Peptide F03

F10

F18

F19

F47

F35

F51

F68

F82

F13

F34

F52

F08

F31

F60

F76

F84

F02

F77

F40

F61

F62

F45

F50

F01

15 30 45

4042

4043

4044

4045

4046

4047 ND>

4048

4049

4050

4051

4052

4053

4054

4055

4056

83K 4057

4058

4059

4060

4061

4063

4064

4065

HUMAN ABS AGAINST MSP-2 PEPTIDES Vs. BINDING ACTIVITY

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HUMAN Abs AGAINST EBA-175 PEPTIDES vs. BINDING

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Plasmodia (falciparum & vivax) use CONSERVED as wellas VARIABLE sequences to bind to host cells.

1.

Synthetic vaccines - Emergingprinciples

CONSERVED binding sequences are NOT antigenic, but VARIABLE sequences are

2.

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IMMUNOGENICITY AND

PROTECTION STUDIES

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PROTOCOL FOR IMMUNIZATION OFAOTUS MONKEYS

Monkeys were immunized with 125 µgr of peptidein FA on days 1, 20, and 40, bled on day 60 for

immunological studies, and challenge on day 65.

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EBA 175 Kd

GROUP H 1678 1692 1713 1730 17371758 0 0 0 200 01º779 0 0 0 0 01783 0 100 0 100 0

GROUP I 1679 1693 1714 1731 1738EBA 175 Kd 1814 100 200 12800 6400 6400VARIABLE 1815 12800 25600 51200 12800 1600

1783 0 0 0 800 0

GROUP J 1681 1694 1715 1732 17391758 0 0 0 400 100

EBA 175 Kd 1779 0 0 0 0 0C&V 1783 0 0 0 800 0

1814 200 100 100 100 01815 25600 204800 25600 3200 6400

AOTUS SERA REACTIVITY AGAINST ORIGINAL

EBA-175 IMMUNIZING PEPTIDES

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AOTUS MONKEYS IMMUNIZED WITH EBA UNMODIFIED PEPTIDES

GROUP H: IMMUNIZED WITH CONSERVED HBAP1758, 1779, 1783.

0

5

10

5 7 9

11 13 15

% P

AR

ASI

TEM

IA

1678

1692

1713

1730

1737

DAYS

GRUPO I: IMMUNIZED WITH VARIABLE HBAP1814, 1815.

0

5

10

5 7 9

11 13 15

% P

AR

ASI

TEM

IA

1679

1693

1714

1731

1738

DAYS

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AOTUS MONKEYS IMMUNIZED WITH MSP-1

UNMODIFIED PEPTIDE MIXTURES

1513, 1517, 1522

0

5

10

5 7 9 11 13 15

% P

AR

ASI

TAEM

IA

1656

1659

1666

1670

1682

DAYS

GROUP B: IMMUNIZED WITH PEPTIDES FROM 38 Kd : 1577, 1582.

0

5

10

5 7 9 11 13 15

% P

AR

ASI

TAEM

IA

1655

1657

1660

1667

1683

DAYS

GROUP D: IMMUNIZED WITH PEPTIDES FROM 19 Kd:

1598, 1599.

0

5

10

5 7 9 11 13 15

% P

AR

ASI

TAEM

IA

1662

1669

1673

1680

1685

DAYS

GROUP A: IMMUNIZED WITH PEPTIDES FROM 83 Kd :

1585, 1590, 4017.

0

5

10

5 7 9 11 13 15

% P

AR

ASI

TAEM

IA 1658

1661

1668

1672

1684

DAYS

GROUP C: IMMUNIZED WITH PEPTIDES FROM 33 Kd:

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MAPS USED IN THE MONKEY TRIALS

MAP 5A 1512 ( H E S Y Q E L V K K L E A L E D A V L T ) 4 - P3C

MAP 5B 1513 ( G Y S L F QK E KM V L N E ) 4 - P3C

MAP 6A 1522 ( Q I P F N L K I R A N E L D V L K K L V ) 4 - P3C

MAP 6B 1523 ( F G Y R K P L D N I K D N V G KM E D Y ) 4 - P3C

MAP 8B 1584 ( L GQ V V T G E A V T P S V I D N I L S K I E N E Y ) 4 - P3C

MAP 9B 1585 ( D V I Y L K P L AG V Y R S L K KQ I E ) 4 - P3C

MAP 11A 1596 ( I A D L S T D Y N H N N L L T K F L S T G ) 4 - P3C

MAP 11B 1597x ( M V F E N L A K T V L S N L L D N L QGM L N I S QHQ ) 4 - P3C

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AOTUS MONKEYS IMMUNIZED WITH MAP MIXTURES

Days

11 14 17 20

%

1985

1994

2003

2005

2010

2019

2023

MAPS 3X

0

10

5 8

0

10

5 7 9 11 13 15 17 19Days

%1989

1995

1997

2013

2020

MAPS 5X

0

10

5 7 9 11 13 15 17 19

Days

%

1987

2004

2168

2169

2212

CONTROLS

Days

%

1991

2007

2035

2025

0

10

5 7 9 13 15 17 1911

CONTROLS

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Aminoacid Sequence of the MSP-1,EBA-175, GBP-130, and MSP-2 Expanded Peptides

HHII

JJKK

ML

ABCDEG

Patarroyo M. E. et al. Summitted to J. Structural Biology

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AOTUS SERA REACTIVITY AGAINSTEBA-175 EXPANDED PEPTIDES

1845 1874 1903 1919 19341757 25600 3200 0 12800 01758 0 100 0 100 01814 400 100 100 200 8001815 51200 6400 6400 25600 6400

GROUP F

18731843 1902 1912 19171779 0 0 0 0 0 01780 800 0 6400 0 100 16001782 12800 800 3200 12800 12800 400

0 0 100 100 200 100

GROUP E1918

1783

Patarroyo M. E. et al. Summitted to J. Structural Biology

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GROUP A

0

5

10

5 7 9 11 13 15 17 19

DAYS

%

18341838185318621901

GROUP B

0

5

10

5 7 9 11 13 15 17 19

DAYS

%

1837185618651904

GROUP D

0

5

105 7 9 11 13 15 17 19

DAYS

%

1857187219061916

GROUP C

0

5

10

5 7 9 11 13 15 17 19

DAYS

%

18411854187919051910

GROUP E

0

5

10

%

184318731902191219171918

5 7 9 11 13 15 17 19

DAYS

GROUP F

%

18451874190319191934

0

5

10

5 7 9 11 13 15 17 19

DAYS

GROUP G

0

5

10

5 7 9 11 13 15 17 19

DAYS

%

184718761907192019251944

AOTUS MONKEYS IMMUNIZED WITH MSP-1 EXPANDED PEPTIDES

Patarroyo M. E. et al. Summitted to J. Structural Biology

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IMMUNOGENICITY OF THEBINDING PEPTIDES IN RABBITS

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IMMUNOGENICITY OF THECONSERVED BINDING PEPTIDES

For the production of Mo Abs groups of4 Balb/c mice were immunized i.p. 5x with 15 µgr of the individual MSP1 (1513, 1522, 1585, 5501) EBA - 175 (1758, 1779, 1783, 1818) GBP - 130 (2220) or MSP2 (4044) conservedpeptides. After the 3rd immunizationNOT EVEN A SINGLE MOUSE, showedantibodies against the correspondingimmunizing peptide, as assessed byELISA western blots or IFA. The samewas also true for mice of the AKR andDBA strains.

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Synthetic vaccines - Emergingprinciples

Plasmodia (falciparum & vivax) use CONSERVED as wellas VARIABLE sequences to bind to host cells.

1.

CONSERVED binding sequences are NOT antigenic, butVARIABLE sequences are

2.

3. CONSERVED binding sequences are NOT immunogenic in ANY of the testedspecies when administered as monomers, polymerized, associatedwith T helper epitopes or presented as MAPs. While VARIABLE sequences can be immunogenic they are strain-specific

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AN EMPIRICAL APPROACH

Changing some residues.

To convert putative alpha helicesinto beta II or beta VII turns.

To modify putative aromaticcomposition.

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STRUCTURALLY REPLACED ANALOGUES OF THE 1513

PEPTIDE

TGYSLHQKEKMVLNE34887TGHSLFQKEKMVLNE24865TGMSLPQKEKMVLNE14863

TGYSLFQKEKMVLNEGTSGTA1513

SEQUENCEGROUPPEPTIDE

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AOTUS SERA REACTIVITY AGAINST THE STRUCTURALLY

REPLACED IMMUNIZING PEPTIDES

GROUP 1 2024 2027 2033 2042 20811513 0 0 0 0 04863 0 0 0 0 04865 0 0 0 0 1004867 0 0 0 0 0

GROUP 2 2028 2034 2039 2043 20461513 800 0 0 1600 4004863 0 0 0 25600 04865 800 100 0 3200 2004867 0 0 0 6400 6400

GROUP 3 2026 2029 2036 2044 20491513 0 200 0 1600 1004863 0 100 0 6400 04865 200 100 0 6400 04867 200 200 200 12800 0

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DAYS

%

20282034203920432046

GROUP 2

0

5

10

5 7 9 11 13 15 17 19

GROUP 1

%

189920242027203320422081

0

5

105 7 9 11 13 15 17 19 DAYS

GROUP 3

DAYS

%

20262029203620442049

0

5

10

5 7 9 11 13 15 17 19

AOTUS MONKEYS IMMUNIZED WITH MSP-1 STRUCTURALLY MODIFIED PEPTIDES

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STRUCTURALLY REPLACED ANALOGUES OF THE 1598M

PEPTIDE

FPNTIISKLIEGKPQDMPNPSQHQ64879

FPNTIISKLIEGKFQDMPNPSQHQ54873

MPNPIISKLIEGKFQDMLNISQHQ44871

FPNTIISKLIEGKFQDMLNISQHQ144847

SEQUENCEGROUPPEPTIDE

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GROUP 4 1980 2037 2058 2063 2066 21094869 100 200 200 12800 51200 2004871 1600 102400 800 102400 102400 256004873 100 ND 0 ND ND 2004879 3200 12800 6400 51200 6400 3200

GROUP 5 2032 2045 2051 2056 2059 20474869 100 3200 1600 51200 3200 2004871 200 800 200 1600 200 256004873 200 ND ND ND ND ND4879 51200 51200 12800 12800 25600 12800

GROUP 6 2053 2061 2068 2072 20774869 51200 102400 12800 25600 1024004871 25600 51200 3200 6400 128004873 ND ND ND ND ND4879 51200 25600 800 102400 204800

GROUP 14 2087 2092 2096 2102 2108 21174869 0 0 0 0 0 04871 0 0 0 0 0 04873 0 0 0 0 0 04879 0 0 0 0 0 0

AOTUS SERA REACTIVITY AGAINST THE STRUCTURALLY

REPLACED IMMUNIZING PEPTIDES

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GROUP 5

0

5

10

%

20322045205120562059

5 7 9 1 1 13 15 17 19

DAYS

GROUP 4

0

5

10

%

198020372058206320662109

5 7 9 11 13 15 17 19

DAYS

GROUP 14

0

5

10

208720922096210221082117

DAYS

%5 7 9 11 13 15 17 19

GROUP 6

0

5

10

%

204720532061206820722077

DAYS

5 7 9 11 13 15 17 19

AOTUS MONKEYS IMMUNIZED WITH MSP-1 STRUCTURALLY

MODIFIED PEPTIDES

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ANALOGUES OF PEPTIDE 1513FOR BINDING ASSAYS

G Y K E K M V L N E G TG Y G E K M V L N E G TG Y K G K M V L N E G TG Y K E G M V L N E G TG Y K E K G V L N E G T

G Y K E K M V L N E G TG G K E K M V L N E G TG Y K E K M V L N E G TG Y K E K M V L N E G TG Y K E K M V L N E G T

G Y K E K M G L N E G TG Y K E K M V G N E G TG Y K E K M V L G E G TG Y K E K M V L N G G TG Y K E K M V L N E G GG Y K E K M V L N E G TG Y K E K M V L N E G TG Y K E K M V L N E G T

GQQQQ

QQQQQ

QQQQQQQQ

S L FS L FS L FS L FS L F

S L FS L FG L FS G FS L G

S L FS L FS L FS L FS L FS L FS L FS L F

AAAAA

AAAAA

AAAAAAAG

S G TS G TS G TS G TS G T

S G TS G TS G TS G TS G T

S G TS G TS G TS G TS G TG G TS G GS G T

YYYYY

YYYYY

YYYYYYYY

51505151515251535154

15135146514751485149

51555156515751585159516051615162

Urquiza M. et al. 1996. Parasite Immunology 18:515-526

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BINDING OF 1513 PEPTIDE ANALOGUES TO RBCs

1513

5146

5147

5148

5149

5150

5151

5152

5153

5154

5155

5156

5157

5158

5159

5160

5161

5162

Y S L F Q K E K M V L N E T S T A

Thou

sand

sTh

ousa

nds

Thou

sand

s

Urquiza M. et al. 1996. Parasite Immunology 18:515-526

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SPECIFICALLY REPLACED ANALOGUES OF THE 1585

PEPTIDE TO IMMUNIZE AOTUS MONKEYS

EVLYLKPLAGVYRSLKKQLE

PEPTIDE SEQUENCE

1585

5187

5190

EVLYGKPLAGVYRSLKKQLE

EVLYLKPGAGVYRSLKKQLE

Espejo F. et al. 2001. Angewandte Chemie Int. Ed. 40:2631

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AOTUS MONKEYS IMMUNIZED WITH 1585SPECIFICALLY MODIFIED PEPTIDES

GROUP 7

0

5

10

5 7 9 11 13 15 17 19 21

2232 2359 2387 2418

GROUP 8

0

5

10

5 7 9 11 13 15 17 19 21

2256 2360 2388 2423

Espejo F. et al. 2001. Angewandte Chemie Int. Ed. 40:2631

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SPECIFICALLY REPLACED ANALOGUES OF THE 5501

PEPTIDE TO IMMUNIZE AOTUS MONKEYS

PEPTIDE SEQUENCE

5501

5503

5512

5513

5516

MLNISQHQCVKKQCPQNSY

MGNISQHQCVKKQCPQNSY

MLNISQHQCVGKQCPQNSY

MLNISQHQCVKGQCPQNSY

MLNISQHQCVKKQCGQNSY

Torres M. H. et al. 2003. European J. Biochemistry 270:3946

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AOTUS MONKEYS IMMUNIZED WITH 5501 SPECIFICALLY

MODIFIED PEPTIDES

GROUP 11

0

5

10

15

20

5 7 9 11 13 15 17 19 21

2297 2370 2428

GROUP 12

0

5

10

15

20

5 7 9 11 13 15 17 19 21

2298 2372 2392 2430

GROUP 9

0

5

10

15

20

5 7 9 11 13 15 17 19 21

2273 2361 2389 2424

0

5

10

15

20

5 7 9 11 13 15 17 19 21

2282 2390 2422 2467

GROUP 10

Torres M. H. et al. 2003. European J. Biochemistry 270:3946

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Plasmodia (falciparum & vivax) use CONSERVED as wellas VARIABLE sequences to bind to host cells.

1.

Synthetic vaccines - Emergingprinciples

CONSERVED binding sequences are NOT antigenic, but VARIABLE sequences are

2.

CONSERVED binding sequences are NOT immuno-genicin ANY of the tested species when administe-red as monomers, polymerized, associated with T helperepitopes or presented as MAPs. WhileVARIABLEsequences can be immunogenic they are strain-specific

3.

To overcome this problem the criticalcontact residues in the bindinginteraction have to be identified andchanged

4.

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Polymerizedpeptide Nr. II 15 III 20 PROT1585 E V L Y L KP L AG V Y R S L K K Q L E 0 0 0/55187 - - - - GKP A - - - - S - - - - - - ND. 1(320) 1/45188 - - - - L GP - A - - - - S - - - - - - ND. 1(320) 1/45189 - - - - L KG - A - - - - S - - - - - - ND. 1(1280) 1/46187 - - - - L KPGA - - - S - - - - - - 0 0 06177 - - - - L KP - A - - - - G - - - - - - 0 0 0

13450 - - - - L L D A - - - - S - - - - - - 2(2560) 1(2560) 2/422806 - L L D - A - - - - S - - - - 3(1280) 1(1280) 2/9

10014 - - - - HV P - A - - - - S - - - - - - 2(640) 1(640) 2/422768 - HL P - A - - - - A - - - - 0 1(5120) 1/10

11860 - - - - HMP - G - - - - A - - - - - - 1(1280) 1(2560) 2/622770 - HL P - G - - - - A - - - - 1(1280) 1(1280) 2/9

13454 - - - - L MS - A - - - - S - - - - - - 0 0 0/514448 - - - - HMD - G - - - - V - - - - - - 0 0 0/314496 - - - - HMD - S - - - - V - - - - - - 0 0 0/413724 - - - - HV T - S - - - - S - - - - - - 0 0 0/512896 - - - - HV P - S - - - - A - - - - - - 0 0 0/512898 - - - - HV P - G - - - - A - - - - - - 0 0 0/515954 - - - - L MD - A - - - - S - - - - - - 0 0 0/315956 - - - - L MP - A - - - - S - - - - - - 0 0 0/415958 - - - - L KD - S - - - - S - - - - - - 0 0 0/615960 - - - - L MD S - - - - S - - - - - - 0 0 0/413728 - - - - NL D - G - - - - S - - - - - - 0 0 0/513452 - - - - L I D - A - - - - S - - - - - - 0 0 0/5

CONTROLS 0 0 0/50

IFA TITERS ≥320Peptide Sequence

GROUP C

GROUPB

GROUP A

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IFA IFA No. ofPolymerized ≥1:160 ≥1:160 protectedpeptide No. Peptide Sequence Post 2nd Post 3rd monkeys Group

4325 M I K S A F L P T G A F K A D R Y K S H 0 0 0/6 13486 A S D S P 3(2560) 2(1280) 2/5 A15516 A H M S W 1(1280) 1(160) 1/4 A20034 A M 2(320) 1(160) 2/8 A20032 A 1(160) 1(160) 1/8 A22784 V G D S P 1(2560) ND 1/10 A23404 V S D M S 2(320) ND 1/10 A23406 V S D D M S 1(320) ND 1/10 A14518 V G H S P W 1(320) 0 0/3 B14520 V G H M S P 2(640) 0 0/3 B17934 A S H V M S 2(320) 0 0/7 B17936 A S H V M S P W 2(640) 0 0/5 B9192 G 0 0 0/5 C9196 G 0 0 0/5 C10100 R G 0 0 0/5 C10102 F G 0 0 0/5 C10104 G T 0 0 0/5 C12708 W 0 0 ND C15512 A S W 0 0 0/4 C15510 A S 0 0 0/4 C16000 S 0 0 0/5 C16008 H P 0 0 0/4 C16002 S H 0 0 0/5 C16004 S H P 0 0 0/5 C16006 S P 0 0 0/5 C17932 A S D S P W 0 0 0/7 C23178 V S D G 0 ND 0/9 C23774 V S H D M S 0 ND 0/9 C22424 S S H V P 0 ND 0/8 C23000 V D S P 0 ND 0/7 C

CONTROLS 0/60 0/60 0/60

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IFA IFA No. ofPolymerized ≥1:320 ≥1:320 protectedpeptide No. Peptide Sequence Post 2nd Post 3rd monkeys Group

6737 D N I L V K M F K T N E N N D K S E L I 0/10 0/10 0/1012748 * V I * 1(2560) 1(2560) 1/4 A13840 * V I M * 3(640) 1(320) 1/5 A22834 * H V I * 1(2560) ND 2/9 A22796 * V I * 1(5120) ND 2/9 A13844 * H V I M * 1(320) 0/6 1/6 A24096 * H V I M * 1(5120) ND 2/8 A14094 * I V I M * 1(320) 2(320) 0/5 B14096 * H R V I M * 3(640) 1(640) 0/4 B24308 * H P M * 1(640) ND 0/8 B9228 * G * 0/5 0/5 0/5 C9230 * G * 0/5 0/5 0/5 C9234 * G * 0/5 0/5 0/5 C10154 * M I * 0/5 0/5 0/5 C10152 * M L * 0/5 ND 0/5 C10150 * M F * 0/5 0/5 0/5 C16016 * V D * 0/5 0/5 0/5 C17970 * H I * 0/8 0/7 0/7 C22460 * I * 0/10 0/10 0/10 C10148 * R A * 0/5 0/5 0/5 C12754 * H V V I * 0/5 0/5 ND C23418 * H I M * 0/10 ND 0/10 C24392 * H A M * 0/6 ND 0/6 C

CONTROLS 0/50 0/50 0/50

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Synthetic vaccines - Emergingprinciples

Plasmodia (falciparum & vivax) use CONSERVED as wellas VARIABLE sequences to bind to host cells.

1.

CONSERVED binding sequences are NOT antigenic, but VARIABLE sequences are

2.

CONSERVED binding sequences are NOT immuno-genicin ANY of the tested species when administe-red as monomers, polymerized, associated with T helperepitopes or presented as MAPs. While VARIABLE sequences can be immunogenic they are strain-specific

3.

To overcome this problem the critical contact residues in the binding interaction have to be identified and changed

4.

The critical contact residues have to be changed for aminoacids that ARE NOT commonly used during the mutationalprocess in the variable sequences

5.

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Synthetic vaccines - Emergingprinciples

Plasmodia (falciparum & vivax) use CONSERVED as wellas VARIABLE sequences to bind to host cells.

1.

CONSERVED binding sequences are NOT antigenic, but VARIABLE sequences are

2.

CONSERVED binding sequences are NOT immuno-genicin ANY of the tested species when administe-red as monomers, polymerized, associated with T helperepitopes or presented as MAPs. While VARIABLE sequences can be immunogenic they are strain-specific

3.

To overcome this problem the critical contact residues in the binding interaction have to be identified and changed

4.

The critical contact residues have to be changed foraminoacids that ARE NOT commonly used during themutational process in the variable sequences

5.

6.Physicochemical characteristics such as size, charge and volume have to be considered. Therefore A S; P D; F R; H L, K M; N I; Q E; C T; Y W; can be exchanged. G and C have special properties.

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PROTEINS:Structure,Function,and Genetics 50:400 �409 (2003)

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The MHC molecules

the peptide and

the T cell receptor

complex

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P1P9

P4

P6

Class-II Molecule

24.10Å

Stern J. et al., 1994 Nature 368,215

P1P9

P4

P6

Class-II Molecule

24.10Å

Stern J. et al., 1994 Nature 368,215

P1P9

P4

P6

Class-II Molecule

24.10Å

Stern J. et al., 1994 Nature 368,215

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Patarroyo M. E. et al. 2004. ChemBioChem. 5: 1588

E

J

G

E2

G2

J2

19.0 Å 22.2 Å

16.9 Å 20.2 Å

14.3 Å 21.9 Å

E

J

G

E2

G2

J2

19.0 Å 22.2 Å

16.9 Å 20.2 Å

14.3 Å 21.9 Å

B B1

16.4 Å 20.3 Å

17.7 Å 19.6 Å

B B1

16.4 Å 20.3 Å

17.7 Å 19.6 Å

1815 24292

4044 10008

13446

118601585

1522

Native ProtectiveProtective

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13450 15484

Inmunogenic andProtection inducers

Short-lived antibodiesinducers

10000

20.2Å

Y4

D7A9

Y12

13.3Å

Y12

L15 K17

E20

23230

20.0Å

W7

A10

K12

L15

21742

12.7Å

Y9

A12 K14

H17

9782

22.1Å

G11D14 G16

L19

15474

15.2Å

I11

D14 H16

L19

22834

21.0 Å

I11

N14K16

L19

14096

14.2 Å

I11

N14 K16

L19

13492

24.7Å

Y7

S10N12

A15

18.9 Å

V4 Y7 Y9 N12

Patarroyo M. E. et al. 2005. Biochemistry In press

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Salazar L. M. et al. 2004. Biochem. Biophys. R. Comun . 322:119

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Synthetic vaccines - Emergingprinciples

7. There must be a 23.5 Å + 3.5 Å distancebetween those residues fitting intoPockets 1-9 of Class II molecules.

Plasmodia (falciparum & vivax) use CONSERVED as wellas VARIABLE sequences to bind to host cells.

1.

CONSERVED binding sequences are NOT antigenic, but VARIABLE sequences are

2.

CONSERVED binding sequences are NOT immuno-genicin ANY of the tested species when administe-red as monomers, polymerized, associated with T helperepitopes or presented as MAPs. While VARIABLE sequences can be immunogenic they are strain-specific

3.

To overcome this problem the critical contact residues in the binding interaction have to be identified and changed

4.

The critical contact residues have to be changed foraminoacids that ARE NOT commonly used during themutational process in the variable sequences

5.

6. Physicochemical characteristics such as size, charge andvolume have to be considered. Therefore A S; P D; F R; H L, K M; N I; Q E; C T; Y W; can be exchanged. G and C have special properties.

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13450

24292

24224

23230

10014

22834

14044

9782 13492

24112

22814

10022

20.2 Å

19.6 Å

20.3 Å

20.0 Å

22.1 Å

20.9 Å

21.0 Å

20.2 Å

24.7 Å

26.5 Å

25.3 Å

25.0 Å

DRβ1*0301 DRβ1*1101 DRβ1*0401

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Immunogenic and Protection Inducer

Immunogenic and NON-Protection Inducer

Patarroyo M. E. et al. 2004, Submitted to Science

13494

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Patarroyo M. E. et al. 2005. Submitted to Angewandte Chemie

13766

13492

13494

20034 20032

24922 24296

14044 14048

10022

24148 23754

24166 23390

21.28Å24.73Å

20.62Å25.50Å

20.12Å25.23Å

17.90Å23.10Å

19.27Å25.04Å

19.29Å25.10Å

20.30Å24.90Å

ImmunogenicNON- Protection inducer

Immunogenicand Protection inducer

13766

13492

13494

20034 20032

24922 24296

14044 14048

10022

24148 23754

24166 23390

21.28Å24.73Å

20.62Å25.50Å

20.12Å25.23Å

17.90Å23.10Å

19.27Å25.04Å

19.29Å25.10Å

20.30Å24.90Å

ImmunogenicNON- Protection inducer

Immunogenicand Protection inducer

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Synthetic vaccines - Emergingprinciples

7. There must be a 23.5 Å + 3.5 Å distance between thoseresidues fitting into Pockets 1-9 of Class II molecules.

Plasmodia (falciparum & vivax) use CONSERVED as wellas VARIABLE sequences to bind to host cells.

1.

CONSERVED binding sequences are NOT antigenic, but VARIABLE sequences are

2.

CONSERVED binding sequences are NOT immuno-genicin ANY of the tested species when administe-red as monomers, polymerized, associated with T helperepitopes or presented as MAPs. While VARIABLE sequences can be immunogenic they are strain-specific

3.

To overcome this problem the critical contact residues in the binding interaction have to be identified and changed

4.

The critical contact residues have to be changed foraminoacids that ARE NOT commonly used during themutational process in the variable sequences

5.

6. Physicochemical characteristics such as size, charge andvolume have to be considered. Therefore A S; P D; F R; H L, K M; N I; Q E; C T; Y W; can be exchanged. G and C have special properties.

8. All the changes made to theCONSERVED binding peptides are designed to achieve a perfect fit with theMHC molecules and the TCR

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Reinherz E. L. et al., 1999 Science 286, 1973

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Synthetic vaccines - Emergingprinciples

7. There must be a 23.5 Å + 3.5 Å distance between thoseresidues fitting into Pockets 1-9 of Class II molecules.

Plasmodia (falciparum & vivax) use CONSERVED as wellas VARIABLE sequences to bind to host cells.

1.

CONSERVED binding sequences are NOT antigenic, but VARIABLE sequences are

2.

CONSERVED binding sequences are NOT immuno-genicin ANY of the tested species when administe-red as monomers, polymerized, associated with T helperepitopes or presented as MAPs. While VARIABLE sequences can be immunogenic they are strain-specific

3.

To overcome this problem the critical contact residues in the binding interaction have to be identified and changed

4.

The critical contact residues have to be changed foraminoacids that ARE NOT commonly used during themutational process in the variable sequences

5.

6. Physicochemical characteristics such as size, charge andvolume have to be considered. Therefore A S; P D; F R; H L, K M; N I; Q E; C T; Y W; can be exchanged. G and C have special properties.

8. All the changes made to the CONSERVED binding peptidesare designed to achieve a perfect fit with the MHC moleculesand the TCRStructural considerations together withaffinity, biological and Immunologicalactivities should guide the design ofsynthetic peptide vaccines.

9

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FIDICFIDIC

Bozedech Z et al August 18 2003. PLOS Biology 1, 001

PHASEOGRAM OF PUTATIVE VACCINE TARGETS

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C : CONSERVED

V: VARIABLE

High RBC binding activity peptides

FIDICFIDIC

PROTEIN C V Reference

MSP1 3 6 Urquiza M. et. al . 1996. Parasite Immunology . 18: 515-526

RESA 2 − Vera R. et al . 2000. Vaccine. 18: 1289-1293

SERA 6 1 Puentes A. et. al . 2000. Parasitology International. 49: 105-117

MSA2 1 2 Ocampo M. et. al . J. Peptide Research. 55: 216-233

EBA175 6 − Rodríguez L.E et. al. 2000. Parasitology. 120: 225-235

GBP130 1 − Suárez J. et. al . 2000. Mem. Inst. Oswaldo Cruz . 95: 495-501

HRP I, II, II 3 1 López R. et a l. 2000. Acta Tropica. 75: 349-359

ABRA 5 − Curtidor H. et. al . 2001. Vaccine. 19: 4496-4504

AMA1 4 3 Urquiza M. et. al . 2001. Vaccine. 19: 508-513

EBA140 6 − Rodríguez L.E.. et. al . 2003. J. Peptide Research. 62: 175-184

NBP1 2 − Valbuena J. et al . 2003. Peptides . 24: 1007-1014

MSP8 5 − Puentes A. et. al. 2003. Peptides .24: 1015-1023

MAEBL 9 − Ocampo M. et al . 2004. Biochem. Biophys. Res. Comm. 315: 319-329

RAP2 4 − López R. et al. 2004. Biochemie . 86: 1-6

RBP2 Ha/Hb 9 3 Ocampo M. et. al . 2004. Parasitol. Int. 53: 77-88

RAP1 4 − Curtidor H. et. al . 2004. Vaccine . 22: 1054-1062

EBA160 5 − Valbuena J. et al. 2004. Biochem. Biophys. Res. Comm . 321: 835-844

CLAG3 5 − Ocampo M. et. al. 2005. Proteins Science . 14: 504-513

EBL1 5 − Curtidor H. et. al. 2005. Protein Science . 14: 464-473

JESEBL 5 − Vera R. et al . 2004. Biochemie. In press

STEVOR − 3 García J. et al. 2004. Peptides. In press

MSP10 3 − Puentes A. et al . 2004. Biochemie. In press

MSP3 3 − Rodríguez L.E. et. al. 2004. Submitted to Protein Science

SORTILIN 6 − Vera R. et al. 2004. Submitted to Biochemistry

RESA-LIKE 3 − Rodríguez L.E. et. al. 2004. Submitted to J. Int. Parasitol.

TryThrA 4 − Curtidor H. et. al. 2004. Submitted to Chembiochem

HAP 2 − Valbuena J. et. al . 2004. Submitted to Biol. Chem.

MSP6 2 1 López R. et al. 2004. Submitted to Peptides

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Functional profile of proteinsexpressed in different P.

falciparum life-cycle stages

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Plasmodium sporozoite surface

and/or apical organelleproteome

Adapted of :Baldacci P et al. 2004 Mol. Microbiol. 54:298

Protein Other stages Subcellular Localization Conserved pepCs - Surface 2

TRAP - Surface, micronemes 7

MAEBL - Surface, micronemes

SPECT - Micronemes

EBA175 Mz Surface, micronemes

P235 Mz

AMA1 Mz Surface, micronemes

STARP RBC, LS Surface 5SALSA RBC, LS Surface 2

PEMP3 RBC, LS Surface

LSA3 LS Surface 11LSA1 LS 1

P52 - ?

MCP1 MZ ?

SPATR RBC Surface

PPLP1 - Micronemes

PfEMP1 ¡RBCSTEVOR ¡RBCRIFINS ¡RBC

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PEPTIDE SEQUENCE BINDING ACTIVITYNUMBER 2 4 6 8 10

3239 1 M N H L G N V K Y L V I V F L 15 S.S.3241 11 V I V F L I F F D L F L V N G 25

3243 21 F L V N G R D V Q N N I V D E 35 R I3245 31 N I V D E I K Y R E E V C N D 45 N-103247 41 E V C N D E V D L Y L L M D C 55

3249 51 L L M D C S G S I R R H N W V 65

3251 61 R H N W V N H A V P L A M K L 75

3253 71 L A M K L I Q Q L N L N D N A 85

3255 81 L N D N A I H L Y A S V F S N 95 A3257 91 S V F S N N A R E I I R L H S 105

3259 101 I R L H S D A S K N K E K A L 115 D3261 111 K E K A L I I I K S L L S T N 125 o3263 121 L L S T N L P Y G K T N L T D 135 m3265 131 T N L T D A L L Q V R K H L N 145 a3267 141 R K H L N D R I N R E N A N Q 155 i3269 151 E N A N Q L V V I L T D G I P 165 n3271 161 T D G I P D S I Q D S L K E S 175

3273 171 S L K E S R K L S D R G V K I 185

3275 181 R G V K I A V F G I G Q G I N 195

3277 191 G Q G I N V A F N R F L V G C 205

3279 201 F L V G C H P S D G K C N L Y 215 R-II3281 211 gf C N L Y A D S A W E N V K N 225

3283 221 E N V K N V I G P F M K A V C 235

3285 231 M K A V C V E V E K T A S C G 245

3287 241 T A S C G V W D E W S P C S V 255 R-II3289 251 S P C S V T C G K G T R S R K 265 Plus.

3291 261 T R S R K R E I L H E G C T S 275

3293 271 E G C T S E L Q E Q C E E E R 285 S.Py.3295 281 C E E E R C L P K R E P L D V 295 SSP23297 291 E P L D V P D E P E D D Q P R Y 305

3299 301 D D Q P R P R G D N F A V E K Y 315

3301 311 F A V E K P N E N I I D N N P 325 D3303 321 I D N N P Q E P S P N P E E G 335 i3305 331 N P E E G K G E N P N G F D L 345 R III v3307 341 N G F D L D E N P E N P P N P Y 355 e3309 351 N P P N P P N P P N P P N P P Y 365 r3311 361 P P N P P N P P N P D I P E Q 375 g3313 371 D I P E Q E P N I P E D S E K 385 e3315 381 E D S E K E V P S D V P K N P Y 395 n3317 391 V P K N P E D D R E E N F D I 405 t3319 401 E N F D I P K K P E N K H D N 415 R.3321 411 N K H D N Q N N L P N D K S D Y 425

3323 421 N D K S D R Y I P Y S P L S P 435 R. 3325 431 S P L S P K V L D N E R K Q S Y 445 B3327 441 E R K Q S D P Q S Q D N N G N Y 455 i3329 451 D N N G N R H V P N S E D R E Y 465 n3331 461 S E D R E T R P H G R N N E N Y 475 d3333 471 R N N E N R S Y N R K H N N T 485

nd i3335 481 K H N N T P K H P E R E E H E Y 495 R IV n3337 491 R E E H E K P D N N K K K A G Y 505 g3339 501 K K K A G S D N K Y K I A G G 515

3341 511 K I A G G I A G G L A L L A C 525 S.Py. 3343 521 A L L A C A G L A Y K F V V P 535 SSP23345 531 K F V V P G A A T P Y A G E P 545 T.D3347 541 Y A G E P A P F D E T L G E E 555 R V3349 551 T L G E E D K D L D E P E Q F 565 C.D.3351 561 E P E Q F R L P E E N E W N 575

Control PLGFFPDHQLDPAFGANSNNPDWDFNP

Ramsés López et al 2001, J. Pept. Res, 58: 285

Plasmodium falciparum binding studiesOf peptide derived from the sporozoite surface protein 2

to Hep G2 cell

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PEPTIDENUMBER Sequence BINDING ACTIVITY

20 30 40

4370 1 MMRKLA I L SVSSF L F VEAL FQEY 22

4371 8 L SVSS F L F VEAL FQEYQCYG 27

4372 13 F L F VEAL FQEYQCYGSSSNT 32.

4373 18 AL FQEYQCYGSSSNTRV LNE 37

4374 23 YQCYGSSSNTRVLNE LNYDN 42

4375 28 SSSNTRV LNE LNYDNAGTNL 47

4376 33 RV LNE LNYDNAGTNL YNE L E 52

4377 38 LNYDNAGTNL YNE L EMNYYG 57

4378 43 AGTNL Y EL EMNYYGKQENW 62

4379 48 YNE L EMNYYGKQENWYS LKK 67

4380 53 MNYYGKQENWYSLKKNSRS L 72

4381 58 KQENWYS LKKNSRS LGENDD 77

4382 63 YS LKKNSRS LGENDDGNNED 82

4383 68 NSRSLGENDDGNNEDNEKLRY 87

4384 73 GENDDGNNEDNEKLRKPKHKY 92

4525 78 GNNEDNEKLRKPKHKKLKQPY 97

4386 83 NEKLRKPKHKKLKQPADGNPY 102

4592REPEATDPNANPVDPNANPNVY4593REPEATNANPNANPNANPY nd

4387 281 NKNNQGNGQGHNMPNDPNRNY 300

4388 286 GNGQGHNMPNDPNRNVDENAY 305

4389 291 HNMPNDPNRNVDENANANSAY 310

4390 296 DPNRNVDENANANSAVKNNNY 315

4391 301 VDENANANSAVKNNNNEEPSY 320

4392 306 NANSAVKNNNNEEPSDKH I KY 325

4393 311 VKNNNNEEPSDKH I KEY LNK 330

4394 316 NEEPSDKH I KEYLNK I QNS L 335

4395 321 DKH I KEY LNK I QNS L ST EWS 340

4396 326 EY LNK I QNS L S TEWSPCSV T 345

4397 331 I QNSL S T EWSPCSV TCGNG I Y 350

4398 336 S T EWSPCSV TCGNG I QVR I KY 355

4399 341 PCSVTCGNG I QVR I KPGSANY 360

4400 346 CGNG I QVR I KPGSANKPKDEY 365

4401 351 QVR I KPGSANKPKDE LDYAN 370

4402 356 PGSANKPKDE LDYAND I EKK 375

4403 361 KPKDE LDYAND I EKK I CKMEKCS 380

4404 366 LDYAND I EKK I CKMEKCSSV 385

4405 371 D I EKK I CKMEKCSSV FNVVNY 390

4406 376 I CKMEKCSSV FNVVNSS I GL Y 395

4407 381 KCSSVFNVVNSS I GL I MV L SY 400

4408 386 FNVVNSS I GL I MV L S F L F LN 405

83

383

RTS

-S

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Polimorphic sequence found in different P. falciparum isolatesCorresponding to amino acids 326 to 345

Of the NF54 P. falciparum

Moreno A. et al. 1993 J Immunol. 151:489.

Isolates OriginNF54,XP8,9, X10,11,4271-4,6-10 3D7 Wets Africa, Honduras, The Netherlands E Y L N K I Q N S L S T E W S P C S V TltG2G1 Brazil K KMS2,SO6 Thailand, Brazil K K RLE5, 3669 Liberia, Gambia K K T366t, 3991-10, 4061-9, 41910 Gambia K K T K366 8,10 Gambia K Q406 10, 419 1-9 Gambia K Q K366 2-7 Gambia K Q RXs,XP 12,13 HB3, D10, T4, T9-101, Honduras, The Netherlands, Papua New Guinea, Q KWEL, B11, PA, FCR3 Thailand, West Africa, Uganda, GambiaS24, T9-98 Angola, Thailand Q K T K7G8 Brazil Q K K I

aAmino acids numbering is based on the CS protein of the NF54 strain of P. falciparum . The polimorphic residues are underline, and only amino acids that differ from the NF54 sequence are shown for each strain or isolate. The subscript numbers correspond to the isolate name described in materials and methods

Sequence

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SOME OTHER

VACCINES

IN PROGRESS

AT FIDIC

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PEPTIDO 1%

11175 1 20

11176 21 40

11177 41 60

11178 61 80

11179 81 100

11180101 120

11181121 140

11182141 160

11183161 180

11184181 200

11185191

SEQUENCE

MI ATTRDREGATMI TFRLRLY

PCRTI L RVF SRNPL VRGTDRY

LEAVVMLL AVTVSLLT I PFAY

AAAGTAVQDSRSHVYAHQAQ

TRHPAT AT VI DHEGVI DSNTY

TAT SAPPRT KI TVPARWVVNY

GI ERSGEVNAKPGT KSGDRVY

GI WVDSAGQL VDEPAPPARAY

I ADAAL AAL GLWLSVAAVAGY

ALL ALT RAI L I RVRNASWQHY

I RVRNASWQHDI DSLFCTQRY 210

PERFIL DE UNION DE PEPTIDOS PERFIL DE UNION DE PEPTIDOS DERIVADOS DEL CLON DERIVADOS DEL CLON

MTCY04C12.18c DE M.MTCY04C12.18c DE M. tuberculosistuberculosis

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L

16nM

-8K

0

8K

16K

24K

32K

* A E I L V K Y AQ L A D K R A R V Y V L

UN

ION

ESP

ECIF

ICA

400nM

0

9K

18K

27K

36K

* A E I L V K Y A QL A D K R A R V Y V

UN

ION

ESP

ECIF

ICA

cpm

s

4µM

-10K

-5K

0

5K

10K

15K

20K

25K

30K

35K

* A E I L V K Y A QL A D K R A R V Y V L

UN

ION

ESP

ECIF

ICA

cpm

s

40µM

0

9K

18K

27K

36K

45K

54K

* A E I L V K Y AQ L A D K R A R V Y V L

UN

ION

ESP

ECIF

ICA

cpm

s

MYCOBACTERIUM TUBERCULOSIS: MYCOBACTERIUM TUBERCULOSIS: ANALOGOS DEL PEPTIDO 11060 DE LA ANALOGOS DEL PEPTIDO 11060 DE LA PROTEINA PUTATIVA DE MEMBRANA PROTEINA PUTATIVA DE MEMBRANA

MTCY277.12 MTCY277.12

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PEPTIDO SEQUENCE 1%

11199 1MTNWMLRGLAFAAAMVVLRL 20

1120021FQGAL I NAWQMLSGL I SLVL 40

1120141LLLFAI GGVVWGVMDGRADA 60

1120261KASPDPDRRQDLAMTWLLAGY 80

1120381LVAGALSGAVAWL I SLFYKA 100

11204101I YTGGP I NELTTFAAFTAL I 120

11205121VFLVGI VGVAVGRWLVDRQLY140

11206141AKAPVRHHGLAAEHERAADTY160

11207161DVFSAVRADDSPTGEMQVAQY180

11208181PEAQTAAVATVEREAPTEV I Y200

11209201RTTESDTPTEV I RTDTEADQY220

11210211V I RTDTEADQTKPGDEPKKDY230

PERFIL DE UNION DE PEPTIDOS PERFIL DE UNION DE PEPTIDOS DERIVADOS DEL CLON DERIVADOS DEL CLON

MTCY159.20c DE M.MTCY159.20c DE M. tuberculosistuberculosis

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MYCOBACTERIUM TUBERCULOSIS: MYCOBACTERIUM TUBERCULOSIS: ANALOGOS DEL PEPTIDO 11076 DE LA ANALOGOS DEL PEPTIDO 11076 DE LA PROTEINA PUTATIVA DE MEMBRANA PROTEINA PUTATIVA DE MEMBRANA

MTCY277.12 MTCY277.12

16nM

-10K

6K

22K

38K

54K

70K

86K

* Y V S A V P V R T F F A E L R N L A L

UN

ION

ESP

ECIF

ICA

cpm

s

400nM

-20K

0

20K

40K

60K

80K

* Y V S A V P V R T F F A E L R N L A L

UN

ION

ESP

ECIF

ICA

cpm

s

4µM

-32K

0

32K

64K

96K

128K

160K

* Y V S A V P V R T F F A E L R N L A L

UN

ION

ESP

ECIF

ICA

cpm

s

40µM

0

48K

96K

144K

192K

* Y V S A V P V R T F F A E L R N L A L

UN

ION

ESP

ECIF

ICA

cpm

s

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PEPTIDO

11059110601106111062110631106411065110661106711068110691107011071110721107311074110751107611077110781107911080

20

40

60

80

101

120

140

160

180

200

220

240

260

280

300

320

341

360

380

400

420

21

41

61

81

101

121

141

161

181

201

221

241

261

281

301

321

341

361

381

401

416

SEQUENCE1M S Q C F A V K G I G G A D Q A T L G S YA E I L V K Y A Q L A D K R A R V Y V LV S T WL V V WG I WH V Y F V E A V FP N A I L WL H Y Y A A S Y E F G F V RR G L G G E L I R M L T G D H F F A G A YY T V L WT S I T V WL I A L A V V V WL I L S T G N R S E R R I M L A L L V P YV L P F A F S Y A I Y N P H P E L F G MT A L V A F S I F L T R A H T S R T R V YI L S T L Y G L T M A V L A L I H E A IP L E F A L G A V L A I I V L S K N A T YG A T R R I C T A L A I G P G T V S V L YL L A V V G R R D I A D Q L C A H I P H YG M V E N P WA V A T T P Q R V L D Y IF G R V E S H A D Y H D WV C E H V T PWF N L D WI T S A K L V A V V G F R A YL F G A F L L G L L F F V A T T S MI R YY V S A V P V R T F F A E L R G N L A LP V L A S A L L V P L F I T A V D WT R YWWV MI T L D V A I V Y I L Y A I D RP E I E Q P P S R R N V Q V F V C V V L YV C V V L V L A V I P T G S A N N I G R Y 435

1%

PERFIL DE UNION DE PEPTIDOS PERFIL DE UNION DE PEPTIDOS DERIVADOS DEL CLON DERIVADOS DEL CLON

MTCY277.12 DE M.MTCY277.12 DE M. tuberculosistuberculosis

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16nM

0

39K

78K

117K

156K

* F R V E S H A D Y H DWV CE H V T P

UN

ION

ESP

ECIF

ICA

cpm

s

400nM

0

58K

116K

174K

232K

290K

* F R V E S H A D Y H DWV C E H V T P

UN

ION

ESP

ECIF

ICA

cpm

s

4µM

0

66K

132K

198K

264K

330K

* F R V E S H A D Y HDWV C E H V T P

UN

ION

ESP

ECIF

ICA

cpm

s

40µM

0

90K

180K

270K

360K

450K

* F R V E S H A D Y H DWV C E H V T P

UN

ION

ESP

ECIF

ICA

cpm

s

MYCOBACTERIUM TUBERCULOSIS: MYCOBACTERIUM TUBERCULOSIS: ANALOGOS DEL PEPTIDO 11073 DE LA ANALOGOS DEL PEPTIDO 11073 DE LA PROTEINA PUTATIVA DE MEMBRANA PROTEINA PUTATIVA DE MEMBRANA

MTCY277.12 MTCY277.12

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PEPTIDE SEQUENCE High Specific Binding Activity (VERO CELLS) High Specific Binding Activity (HeLa CELLS)NUMBER 2 2

18306 1 M C L Y T R V L I L H Y H L L P L Y G P 2018307 21 L Y H P R P L P L H S I L V Y M V H I I 4018308 41 I C G H Y I L F L R N V N V F P I F L I 6018309 61 Q M A L W R P S D N T V Y L P P P S V A 8018310 81 R V V N T D D Y V T P T S I F Y H A G S 10018311 101 S R L L T V G N P Y F R V P A G G G N K 12018312 121 Q D I P K V S A Y Q Y R V F R V Q L P D 14018313 141 P N K F G L P D T S I Y N P E T Q R L V 16018314 161 W A C A G V E I G R G Q P L G V G L S G Y 18018315 181 H P F Y N K L D D T E S S H A A T S N V 20018316 201 S E D V R D N V S V D Y K Q T Q L C I L 22018317 221 G C A P A I G E H W A K G T A C K S R P Y 24018318 241 L S Q G D C P P L E L K N T V L E D G D Y 26018319 261 M V D T G Y G A M D F S T L Q D T K C E 28018320 281 V P L D I C Q S I C K Y P D Y L Q M S A 30018321 301 D P Y G D S M F F C L R R E Q L F A R H 32018322 321 F W N R A G T M G D T V P Q S L Y I K G 34018323 341 T G M P A S P G S C V Y S P S P S G S I 36018324 361 V T S D S Q L F N K P Y W L H K A Q G H 38018325 381 N N G V C W H N Q L F V T V V D T T P S Y 40018326 401 T N L T I C A S T Q S P V P G Q Y D A T 42018327 421 K F K Q Y S R H V E E Y D L Q F I F Q L 44018328 441 C T I T L T A D V M S Y I H S M N S S I 46018329 461 L E D W N F G V P P P P T T S L V D T Y 48018330 481 R F V Q S V A I T C Q K D A A P A E N K Y 50018331 501 D P Y D K L K F W N V D L K E K F S L D 52018332 521 L D Q Y P L G R K F L V Q A G L R R K P 54018333 541 T I G P R K R S A P S A T T S S K P A K Y 56018334 561 A P S A T T S S K P A K R V R V R A R K Y 580

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100 Å100 Å

18283

18294

18301

Vera, R. et al. 2003. Int. J. Cancer. 107:416

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Herpes Virus SaimiriPeptide Sequence and Binding Activity of Probable Antigen 75

Curtidor H. et al. Manuscript in preparation

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VHTVHG-VHG+THG+THG-THTIHTIHG-IHG+LHG+LHG-LHTQHG+EHTEHG-EHG+QHG-QHTDHTDHG+NHTNHG-NHG+DHG-SHTSHG+AHSHG-CHTCHG+CHG-MHTMHG+MHG-KHTKHG+KHG-VBTVBG-VBG+TBTTBG-TBG+SBG+SBG-SBTABLBTLBG-LBG+IBTIBG-IBG+CBTCBG-CBG+QBG-EBG-EBTQBG+EBG+QBTDBTDBG-DBG+NBTNBG-MBTMBG-MBG+KBG+KBG-KBTHHTHHG-HHG+PHPBYHTFHTYHG+FHG+YHG-FHG-YBTYBG-FBTFBG-YBG+FBG+HBTHBG-HBG+WHTWHG-WHG+WBG-WBG+WBTRHG-RHG+RHTRBTRBG-RBG+GHGB

Conformación Alfa

Conformación Beta

Aminoácidos Pi

Prolina

Glycina

ϕ

φ

21 índices de mecánica quántica19 índices grafo-teóricos

Ala-X-AlaNH2-X-CHO

• 2 conformaciones (alfa y beta)• 3 rotámeros (g+, g-, t)540 moléculas

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Asn +

Asn -

Ile +

Ile -

Glu +

Glu -

Met +

Met -

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Synthetic vaccines - Emergingprinciples

7. There must be a 23.5 Å + 3.5 Å distance between thoseresidues fitting into Pockets 1-9 of Class II molecules.

Plasmodia (falciparum & vivax) use CONSERVED as wellas VARIABLE sequences to bind to host cells.

1.

CONSERVED binding sequences are NOT antigenic, but VARIABLE sequences are

2.

CONSERVED binding sequences are NOT immuno-genicin ANY of the tested species when administe-red as monomers, polymerized, associated with T helperepitopes or presented as MAPs. While VARIABLE sequences can be immunogenic they are strain-specific

3.

To overcome this problem the critical contact residues in the binding interaction have to be identified and changed

4.

The critical contact residues have to be changed foraminoacids that ARE NOT commonly used during themutational process in the variable sequences

5.

6. Physicochemical characteristics such as size, charge andvolume have to be considered. Therefore A S; P D; F R; H L, K M; N I; Q E; C T; Y W; can be exchanged. G and C have special properties.

8. All the changes made to the CONSERVED binding peptidesare designed to achieve a perfect fit with the MHC moleculesand the TCRStructural considerations together with affinity, biological andImmunological activities should guide the design of syntheticpeptide vaccines.

9.

This methodology elicits protective immunityagainst NON ANTIGENIC, NON IMMUNOGENIC CONSERVED BINDING PEPTIDES, providing a new and powerful tool for the rationaldevelopment of synthetic vaccines

10.

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RECEPTOR LIGANDOMarisol OcampoRamsés López

Hernando CurtidorAlvaro Puentes

Luis Eduardo RodríguezRicardo VeraJavier García

John ValbuenaErika Vega

RMN

BIOCATALISISJosé Manuel Lozano

Andrés López

SINTESIS DE PEPTIDOS

IMMUNOLOGIA

Jaiver rosas Zuly Rivera

Elizabeth TorresMagnolia Vanegas

Luis RamírezClaudia ReyesMartha Pinto

Carolina VargasPaola PachecoMiguel Navarro

Jesús LeitonRubén Quiroz

Sandra Calderón

Carlos ParraGabriela DelgadoEsperanza Trujillo

Luis Eduardo VargasEdith Hernández

Juan BaqueroDarnel Marchena

Fabiola EspejoLuz Mary SalazarGladys Cifuentes

Patricia AlbaAdriana Bermúdez

FISIOLOGIAJean Paul Vernot

Hernando del CastilloAlberto Pérez

Ana Maria Perdomo

VIROLOGIARoberto AmadorMauricio Urquiza

Tatiana RozoLuz Helena Patiño

INMUNOQUIMICAYolanda SilvaMartha ForeroJimena Cortes

BIOLOGIA MOLECULARManuel Alfonso Patarroyo

Carlos BarreroAdriana Sierra

Diana TovarPilar Martínez

Paula CárdenasCamilo MoncadaYolanda López

Rosalba AlfonsoOscar Pérez

BIOMATEMATICASMateo Obregón

Constanza CárdenasCarlos Suárez

Alejandro BalbinMarisol Ortiz

Marcia ArdilaRaúl Rodríguez

Manuel Elkin Patarroyo

ESTACION PRIMATES

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Inmunología MolecularReceptor-Ligando

Inmunoquímica

Síntesis Quimica de

Macro-moleculas

NMR y Estructura 3DBiologia Molecular

Quimica Cuantica