Plasmodium CSP - Based vaccines Past - prsesent - future

PlasmodiumPlasmodium CSPCSP--Based VaccinesBased Vaccines

Past Past -- Present Present -- FutureFuture

Georges SnounouGeorges Snounou

Parasitologie ComparParasitologie Comparéée e et et ModModèèles les

ExpExpéérimentauxrimentaux

MusMusééum um National National dd’’Histoire Naturelle Histoire Naturelle

Paris Paris -- FranceFrance

OVERVIEWOVERVIEW

Malaria & Anti-Malarial Vaccination

CSP-Based Pre-Erythrocytic VaccinesPresent StatusPerspectives

Quest for Novel Protective Antigens

A Simple Life Cycle with ComplexityA Simple Life Cycle with Complexity

Protozoans of the Genus Plasmodium

Transmitted by Mosquitoes of the Genus Anopheles

Established after 100 years of researchEstablished after 100 years of research

Discovery of the PathogenDiscovery of the Pathogen

Alphonse Charles Louis LaveranAlphonse Charles Louis Laveran

Transmission By MosquitoesTransmission By Mosquitoes

MansonMansonLondonLondon

RossRossIndiaIndia

GrassiGrassiRomeRome

TheThe Roman DisputeRoman Dispute

Spot the ErrorSpot the Error

A Single ObservationA Single Observation

SchaudinnSchaudinn Schaudinn 1903 Arb. Kaiserl. Gesundh.

TheThe LiverLiver Fate of Fate of thethe SporozoiteSporozoite

ShorttShortt GarnhamGarnham

Life Cycle Landmark Dates

1903 1924

18801880PlasmodiumAsexual Stages

19481948PlasmodiumLiver Stages

19801980Plasmodium

Liver Hypnozoites

18981898PlasmodiumSexual Stages

18981898AnophelesSporozoites

The Control of MalariaThe Control of Malaria

Former Times

Drainage

Larvicide

Nets

Quinine WHO

Eradication Campaign

Drug & Insecticide Resistance

DDTDDT

ChloroquineChloroquine

1970s

mAb - Cloning - In vitro Culture

CSP Gene Plasmodium Genomes

Scramble for a VaccineScramble for a Vaccine

19551955 19701970 19831983 20012001……

Many Vaccine CandidatesMany Vaccine Candidates……

Erythrocytic Asexual TargetsAMA1 DBP EBA175 EBP2 GLURP GPI MSP1 MSP2 MSP3 PfEMP1 RESA SERP

Erythrocytic Sexual TargetsPfS16 PfS28 PvS25

Pre-erythrocytic TargetsCSP LSA1 LSA3 TRAP

……Little ProtectionLittle Protection

Unknown Mechanisms of Naturally Acquired Immunity

Requires Repeated Lengthy Exposure - Wanes Rapidly

Not Sterilising

Species and Strain-Specificity

VACCINES TARGETING THE VACCINES TARGETING THE P. falciparumP. falciparum CSPCSP

Gold Standard Vaccine : The Attenuated Sporozoite

Sterile Protection Requires Many Doses

Quest for a Subunit Vaccine

1941 & 19661941 & 1966Avian parasites

UV-inactivated sporozoite

1973 1973 -- 19901990Human parasites

Radio-inactivated sporozoite19101910Avian parasites

Heat-inactivated sporozoite

19671967Murine parasites

Radio-inactivated sporozoite

Incrimination of a Sporozoite Surface ProteinIncrimination of a Sporozoite Surface Protein

Circumsporozoite Protein

Normal Serum Immune Serum

PfcspPfcsp -- Repeats and VariationsRepeats and Variations

NANP NVDP

Oocyst Development - Gliding Motility - Salivary Gland Invasion - Interaction with Hepatocyte

1200 - 1400 bp

30 - 50 Repeat Units

Immunological Mechanisms of ActionImmunological Mechanisms of Action

AntibodyAntibody

T CellsT Cells

Liver

Liver StageLiver Stage

KillingKilling

ChallengeChallenge

InvasionInvasion

InhibitionInhibition

Sporozoite

Liver

ArrestedArrested

DevelopmentDevelopment

ImmunisationImmunisation

HepatocyteHepatocyte

InvasionInvasion

SporozoiteBlood

Infection

Blood Infection

PfcspPfcsp--based Vaccines based Vaccines -- Trials and TribulationsTrials and Tribulations

DNAPeptides

Proteins (E. coli, Baculovirus)Recombinant Virus (Pox, Adenovirus)

Recombinant Bacteria (Salmonella)Recombinant Parasite (Leishmania, Toxoplasma)

Immunogens

Thirty in MiceFour in Monkeys

Twenty-seven in Humans

Trials

Adequate Levels of Sterile Protection Remain Elusive

NEJM 1997 336:86

The RTS,S VaccineThe RTS,S Vaccine

Naïve Volunteers - Homologous ChallengeAdjuvant-Dependent Efficacy

48% Sterile Protection

The RTS,S VaccineThe RTS,S Vaccine

Strain-Transcending ProtectionShort-lived Sterile Protection

Lancet 2001 357:1927

1998First

1999Boost

≥ One episode Malaria infection

Lancet 2004 364:1411

The RTS,S VaccineThe RTS,S Vaccine

Alonso et al.

Contrasting InterpretationsContrasting Interpretations

Snounou et al.

The vaccine is The vaccine is ineffectiveineffective

29.9 %57.7 %

45.0 %

against first clinical episodeagainst severe malaria

against infection

Contrasting InterpretationsContrasting Interpretations

Placebo is inadequate

Detection by microscopy

Statistical measures obscure

No primary data available

Lancet 2005 366:2012

Contrasting InterpretationsContrasting Interpretations

≥ One episode

35.3 % against first clinical episode48.6 % against severe malaria

More RTS,S TrialsMore RTS,S Trials

About 7 Phase IIb are underway in Africa

About 12 Phase III are planned in Africa

Estimated/Projected costs = > 600 Million USD

Suitability of Suitability of PfcspPfcsp as an Antias an Anti--Infection VaccineInfection Vaccine

Role in Naturally Acquired ProtectionEpidemiological Surveys

Role in Experimentally-Induced Sterile ProtectionExperimental Approach

Role of Role of PfCSPPfCSP in Naturally Acquired Immunityin Naturally Acquired Immunity

Lack of Formal EvidenceLack of Formal Evidence

Exploit Th-Epitope Polymorphisms

Formal Tests in Animal Models

Prevalence of ααααCSP Antibodies

Prevalence of CSP-specific T-Cells

Presence of Th Epitope Polymorphisms

Diversity of Diversity of PfcspPfcsp ThTh EpitopesEpitopes

Th2R Th3R

Th2R*01 P S D H I Y L I S

Th2R*02

Th2R*03

Th2R*04

Th2R*05

Th2R*06

Th2R*07

Th2R*08

Th2R*09

K

T

R

T

T

Th2R*10

Th2R*11

K

Q

Q

Q

Q

Q

Q

Q

Th2R*12

Th2R*13

N

Y

Y

Th2R*14

E

K

T

K

K

N

Q

Q

N

Q

T

Th2R*15

Q

K

E

K

K

K

K

K

K

E

E

E

K

Q

Q

Q

Q

Q

Q

Q

R

Q

Q

Q

Q

Q

I

L

L

L

L

L

L

L

L

L

L

L

L

L

.

.

.

.

Th3R*01 K D L

Th3R*02

Th3R*03

Th3R*04

Th3R*05

Th3R*06

K

R

Th3R*07

Th3R*08

Th3R*09

P

S

Th3R*10

Th3R*11

E

Q

Q

Q

Q

Q

Th3R*12

Th3R*13

N

D

G

D

G

Y DE

A

A

A

A

C

N

D

S

D

D

N

N

E

.

.

.

.

Mae La

EIR 0.1 - 0.5

1996 N = 35

1999 N = 45

2001 N = 20

KanchanaburiRatchaburi

EIR 1 - 5

2001 N = 183

Children and adults

Children and adults

Study Sites in Study Sites in ThailandThailand

ConclusionConclusion

CSPCSP--Induced Natural Immunity is Not SelectiveInduced Natural Immunity is Not Selective

CSP-induced Immunity is Short-Lived

CSP-induced Immunity is Not Protective

Kumkhaek et al. 2005 J. Immunol. 175:3935

HypothesisBiological Selection by the Mosquito

BrazilBrazil*01/*01 *04/*01

23/23 19/23

Papua New GuineaPapua New Guinea*05/*04

22/22

Role in Experimentally Induced Sterile ImmunityRole in Experimentally Induced Sterile Immunity

P. berghei CSP versus P. falciparum CSP

Hypothesis

IFIFCSP plays a role in the acquisition of sterile immunity

THENTHENImmunization with P. berghei/Pbcsp irradiated sporozoites

WILL WILL NOTNOT

Confer protection against a challenge with P. berghei/Pfcsp

Provided anti-CSP cross-reactive immune responses are minimal

ααααααααCSP CSP Antibody Responses after ImmunisationAntibody Responses after Immunisation

The Majority of Induced Antibodies are Directed against the CSP

Fluorescence (AU x 10-3)

P. falciparum

P. berghei

0 100 200 300 4000 100 200 300 400 0 100 200 300 400

N-Terminus Repeats C-Terminus

Sterile Protection Induced by ImmunisationSterile Protection Induced by Immunisation

None

P. berghei/Pbcsp

Immunising Parasite

Sterile protection (%)

Challenge Parasite

0 20 40 60 80 100

P. berghei/Pfcsp

0 20 40 60 80 100

P. berghei/Pbcsp

ConclusionConclusion

Immune Responses Induced against CSP Immune Responses Induced against CSP

Do not Confer Sterile ImmunityDo not Confer Sterile Immunity

Other Antigens from Irradiated Sporozoites must have a

Determining Role in Sterile Protection

Grüner et al. 2007 PLoS ONE e-1371

Quest for Novel Protective PreQuest for Novel Protective Pre--Erythrocytic AntigensErythrocytic Antigens

A strategy for IdentificationA strategy for Identification

Discriminating Immunological ReagentsDiscriminating Immunological Reagents

Animal Models of MalariaEthical ConsiderationsCollection of Reagents

Suboptimal ImmunisationIrradiated Sporozoite

Challenge

ProtectedProtected InfectedInfected

AntibodiesT Cells

AntibodiesT Cells

Liver Stage TranscriptomesLiver Stage Transcriptomes

mRNA PurificationmRNA Purification

cDNA ConstructioncDNA Construction

Normal Sporozoite Hepatic Trophozoite Hepatic Schizont

Irradiated Sporozoite Hepatic Trophozoite Arrested Growth

High Efficiency High Efficiency In vitroIn vitro InfectionInfectionCD81-HepG2 Cells

Gene Identification by Microarray AnalysesGene Identification by Microarray AnalysesDNA Chip for the DNA Chip for the P. yoeliiP. yoelii GenomeGenome

Immunological ScreeningImmunological Screening

Bioinformatic SelectionBioinformatic Selection

Confirmation of Confirmation of in vivoin vivo ExpressionExpressionRTRT--PCRPCR

Cloning in Appropriate Expression VectorsCloning in Appropriate Expression Vectors

Quantify B Cell and T Cell ResponsesQuantify B Cell and T Cell Responses

Correlates of ProtectionCorrelates of Protection

ProofProof--ofof--Principle Validation of Protective RolePrinciple Validation of Protective Role

Immunisation / Challenge in Mice

Reagents from Humans

Volunteers Immunised with Sporozoite under Chloroquine Cover

Naturally Protected Endemic Residents following Radical Cure

Identification of Homologues in P. falciparum

PfcspPfcsp Th Th Repertoire Diversity Repertoire Diversity -- Age and ExposureAge and Exposure

Age

Para

sit

es

RepTh Non-Immunes RepTh Immunes>?

Prompt & Effective Treatment

Relatively Low Endemicity

Mosquito Bites Lead to Clinical Symptoms

Blood Parasites Reflect Sporozoite InoculumBlood Parasites Reflect Sporozoite Inoculum

An Unexpected Biased DistributionAn Unexpected Biased Distribution

0.80.8

0.15Th2R Th3R

Th2R*5 Th3R*1

n % %

144 78 81Ratchaburi

43 84 91Kanchanaburi

100 82 84

*5/*1

%

76

84

82Mae La

A Dominant A Dominant PfcspPfcsp HaplotypeHaplotype

Frequencies did not Vary with Age or Time of Collection

Fixation of the Fixation of the PfcspPfcsp HaplotypeHaplotype

BDACBCBFCFCBB

CDBD

CDBA

AB

F

C

F

E

A

C

A

C

C

C

C

A

C

C

C

A

DF

AB

C

BC

C

glurpmsp1

C

C

C

D

A

C

A

BC

E

D

D

C

D

C

C

C

K1

D

G

D

C

D

F

F

D

E

D

BD

F

C

D

C

C

AC

D

D

EG

C

D

D

E

BD

D

DE

E

MAD20

A

A

A

RO33

msp2

B

B

B

B

B

B

AB

B

B

AB

B

B

B

AB

B

B

B

B

B

FC27

EECH

DK

DK

GD

DG

CEG

EEF

D

J

C

E

I

C

E

D

I

F

EK

EK

CK

H

D

C

C

G

IC

Children

Adults

Children

Adults

Children

Adults

1996

1999

2001

*5/*1*5/*1