Circulating T-follicular helper cells

as correlates of high affinity

antibody response against
Plasmodium falciparum in
malaria-experienced Cameroonian
adults
Thesis presented in partial fulfillment for the award of
the
Medicinae Doctorae Degree by:

SIEWE FODJO Joseph Nelson

Director
Pr. Rose G. F. LEKE

Co-directors
Pr. Wilfred MBACHAM
Dr. Samuel TASSI YUNGA

PLAN

INTRODUCTION
OBJECTIVES
METHODS
RESULTS AND DISCUSSION
CONCLUSION
RECOMMENDATIONS
SUMMARY IN FRENCH

PLAN

INTRODUCTION
OBJECTIVES
METHODS
RESULTS AND DISCUSSION
CONCLUSION
RECOMMENDATIONS
SUMMARY IN FRENCH

 Malaria: Parasitic disease caused by Plasmodium sp.

INTRODUCTION
(1/4)

Public health concern: 584 000 deaths and 3.2 billion

people at risk*. Cameroon: 31% OP, 19% deaths**.

An effective vaccine could alleviate this burden.

Good understanding of immune correlates of malaria
disease and protection needed.

One correlate: Antibody response against P. falciparum.

*WHO 2014
NMCP 2012

**

 High affinity Ab: T-follicular helper cells (TFH) needed.

INTRODUCTION
(2/4)

TFH provide help to B-cells in the germinal centers (GC)

of lymphoid follicles.

IL21
ICO

King
al.
Source: Ma
etet
al.
2012

INTRODUCTION
(3/4)

TFH in GC of lymphoid tissues are not accessible.

Cells expressing similar markers as GC TFH in
peripheral blood: Circulating TFH (cTFH).

cTFH: Circulating counterpart of TFH, may reflect GC

dynamics in peripheral blood*.

cTFH investigated in other infectious diseases (influenza,

hepatitis B, HIV).
*Morita et al. 2011

INTRODUCTION
(4/4)

Relevance of studying cTFH in malaria:

Immune

correlates

of

high-affinity

antibody

against P. falciparum.
Vaccine

development

and

assessment

of

response.

Working hypothesis: There exists an association

between cTFH levels and high affinity antibody
responses in malaria.
7

PLAN

INTRODUCTION
OBJECTIVES
METHODS
RESULTS AND DISCUSSION
CONCLUSION
RECOMMENDATIONS
SUMMARY IN FRENCH

OBJECTIVES
(1/2)

GENERAL OBJECTIVE

To assess the presence and abundance of cTFH in

the peripheral blood of malaria-experienced adults,

and to assess if this abundance correlates with high

affinity anti-malarial antibodies against P. falciparum.

SPECIFIC OBJECTIVES

OBJECTIVES
(2/2)

1. To determine if cTFH expand during P. falciparum

malaria.
2. To explore the kinetics of cTFH during and after
P. falciparum malaria.

3. To investigate whether or not cTFH abundance

correlates with antimalarial IgG titers and avidity.

10

PLAN

INTRODUCTION
OBJECTIVES
METHODS
RESULTS AND DISCUSSION
CONCLUSION
RECOMMENDATIONS
SUMMARY IN FRENCH

 Study type: Cross-sectional

Study duration: 12 months (June 2014 to May 2015)

METHODS
(1/8)

Setting: Nkolbisson Health District

Predictor variable: Malaria exposure
Outcome: Levels of cTFH and antibodies
Sampling: Consecutive
Study population: Cameroonian adults
Sample size: 30 participants
12

 Inclusion Criteria:
1. Cases with Ongoing malaria
- Adults > 18 years, CMR, confirmed malaria.

METHODS
(2/8)

2. Malaria-experienced controls
- Adults > 18 years, CMR, no clinical malaria > 6 months.

3. Malaria-naive controls
- Adults > 18 years, USA, never had malaria.

4. After Malaria
- Same individuals as cases, encountered 2 weeks later.

and all participants who gave signed informed consents.

13

 Exclusion Criteria:
Unconfirmed malaria diagnosis.

METHODS
(3/8)

Ongoing pathologies: Diabetes, HIV.

Pregnancy.

14

Procedure

METHODS
(4/8)

FIE
LD

ASSESSMENT
FOR
ELIGIBILITY

RECRUITMENT:
-CRF FILLED
-BLOOD COLLECTED

SAME DAY
COLLECTED
BLOOD

-SLIDES MADE
-PUT IN CULTURE
_(MSP-1/NIL)

LA
BO
RA
TO
RY

-STORAGE
-TRANSPORT
TO THE LAB

NEXT DAY

-PBMC ISOLATION
-PBMC COUNTING
-CD4+ T-CELLS
_SORTED

PLASMA
STORED

ALL
SAMPLES
CD4+ T-CELLS
STORED IN
RNAlater

15

Procedure
ALL SAMPLES
RNA EXTRACTION

ALL
SAMPLES
cDNA

METHODS
(5/8)

FROM CD4+ T-CELLS

SYNTHESIS
cT
F
H
D Quantitative RT-PCR (LightCycler 1.5)
E
Relative &
Absolute
T
quantification
E Initiate run:
C -Amplification
cycles)
TI (50
Real-time
-Dissociation
O & melting.
quantification
of SYBR Green
N

Template cDNA and

qPCR mix loaded in
LC tubes.

ALL
SAMPLES
REAL-TIME
PCR
Relativ
e:
Target
to
referenc
e
pairing
Absolute:
Standard
Curve

fluorescence at
530nm.

16

Procedure
ALL SAMPLES

ALL SAMPLES

STORED PLASMA

MAP (LUMINEX)

1. IgG Titers
2. IgG
Avidities

Multiplex Analyte Platform: (MagPix Luminex)

METHODS
(6/8)

AN
TI
BO
DY
ST
UD
IE
S

Sample with IgG

Incubat
e

MSP1- coupled
beads

MSP-1 IgG

Wash

Detection Antibody +

binding to

PE added & attached

beads

to MSP1-IgG

PE
Bead
Coated
with
MSP1

MSP1
IgG in
sample

Fluorescence
recorded

Incubat
e
&
Read

17

 Data Management:
Data entered in MS Excel 2010 and analyzed using
GraphPad Prism 5.0.

METHODS
(7/8)

Shapiro-Wilk normality test: Normal distribution (t-test,

ANOVA, Pearson) & skewed distribution (Mann-Whitney,
Kruskal Wallis, Spearman).

Wilcoxon paired test for paired samples.

P-value < 0.05: statistically significant.

18

 Ethical Considerations:
Approval: National Ethics Committee & IRB of the
University of Hawaii.

METHODS
(8/8)

Ethical clearance requested from FMBS.

Administrative clearance from the Nkolbisson DMO.
All data treated confidentially.

19

PLAN

INTRODUCTION
OBJECTIVES
METHODS
RESULTS AND DISCUSSION
CONCLUSION
RECOMMENDATIONS
SUMMARY IN FRENCH

 Participants characteristics:

R E S U LT S &
D I S C U SS I O N ( 1 / 7 )

Total of 30 samples:
3 Malaria-naive controls from the United States: US NC
6 Cameroonian negative controls: Cam NC
12 Cameroonian positive controls (active disease): Cam PC
9 Cameroonians 2 weeks after infection: Cam PC+2weeks

Mean age: 27.3 8.57 years.

Mean parasitaemia (Cases): 69,297 17,290
parasites per L of blood.
All cases (Cam PC): Fever, headaches.
21

 Influence of Malaria on cTFH abundance :

ICOS Relative Expression

R E S U LT S &
D I S C U SS I O N ( 2 / 7 )

CXCR5 Relative Expression

200
150
100
50
20

300

200

100
ICOS
Cut-off=76.38

10

CXCR5
Cut-off=6.408

NTC

US NC

Cam NC

Cam PC

CXCR5
Sample relative
Category
expression

NTC

US NC

Cam NC Cam PC

ICOS
relative
Sample Category
expression

66.7% malaria-experienced controls: CXCR5-high.

Tendency of less cTFH in Cam PC vs Cam NC*.
More ICOS than CXCR5 expressed in Cam PC.
*Wykes et al. 2014

22

80

CXCR5 Relative Expression

R E S U LT S &
D I S C U SS I O N ( 3 / 7 )

Influence of Malaria on cTFH abundance :

60
Pearson correlation coefficient
r = 0.911
p=0.0001

40

20

20000

40000

60000

80000

P.falciparum parasitaemia (/L of blood)

CXCR5 expression vs parasitaemia: Positive

correlation (r = 0.911, p = 0.0001).
23

 Influence of in vitro stimulation with MSP-1 on cTFH abundance:

R E S U LT S &
D I S C U SS I O N ( 4 / 7 )

Catego Code
ry

Cam
PC

Cam
NC

M24
M25
M26
M27
M28
M29
M30
M31
M32
M35
M39
M41
M33
M34
M36
M37
M38
M40

[CXCR5]
Without
Stimulation
(ng/L)
5.00E-36
6.93E-34
4.35E-66
1.06E-30
8.15E-68
5.28E-40
1.06E-33
1.31E-36
1.68E-30
1.57E-72
1.29E-55
4.35E-31
2.19E-59
1.35E-53
4.4E-57
3.87E-68
9.01E-52
7.99E-37

[CXCR5]
Stimulated
with MSP1(ng/L)
3.22E-43
3.36E-32
7.97E-57
6.31E-44
1.34E-28
5.25E-39
2.03E-32
4.06E-34
1.67E-43
1.62E-50
1.53E-61
1.68E-27
7.48E-51
2.92E-51
5.96E-72
6.77E-64
4.73E-65
7.19E-33

Increment in
[CXCR5]
(ng/L)
-5.00E-36
3.29E-32
7.97E-57
-1.1E-30
1.34E-28
4.72E-39
1.92E-32
4.05E-34
-1.7E-30
1.62E-50
-1.3E-55
1.68E-27
7.48E-51
2.91E-51
-4.4E-57
6.77E-64
-9E-52
7.19E-33

Increment in absolute [CXCR5] upon stimulation: 12/18 samples*.

24
*Riccio et al. 2013

 Kinetics of cTFH during and after malaria :

CXCR5 levels during infection and 2 weeks later.

CXCR5 Relative Expression

R E S U LT S &
D I S C U SS I O N ( 5 / 7 )

60

50

40

Wilcoxon
paired test:
p = 0.0391

30

20

10

CXCR5 Cam PC

CXCR5 CamPC+2weeks

Sample Category

Significantly higher cTFH levels after infection.

Shulman et al. 2013

25

 MSP-1 Antibody profiles:

p=
0.0056

10000

1000

NetIgG
MFI 500
cut-off=302.72
0

100

MSP1 IgG Avidity ( %)

20000

Net MSP1-IgG MFI

R E S U LT S &
D I S C U SS I O N ( 6 / 7 )

30000

US-NC

CamNC
CamPC CamPC+2weeks
Sample Category

MSP-1 IgG
titers

80

60

40

20

CamNC

CamPC

CamPC+2weeks

Sample Category

MSP-1 IgG
avidity

Lower titers in inexperienced vs experienced (p=0.0056).

Titers and avidity higher after the disease than in the
malaria-experienced controls.
26

 cTFH and MSP-1 antibodies:

100

MSP1 IgG Avidity ( %)

R E S U LT S &
D I S C U SS I O N ( 7 / 7 )

Net MSP1-IgG MFI

15000

10000

5000

80

60

40

20

ICOS
low

ICOS
high

CXCR5-Low

ICOS
low

ICOS
high

CXCR5-high

MSP-1 IgG
titers

ICOS
low

ICOS
high

ICOS
low

CXCR5-low

CXCR5-high

MSP-1 IgG
avidity

Low cTFH (CXCR5-low, ICOS-low): Low IgG titers.

High cTFH (CXCR5-high, ICOS-high): High avidity.

Correlation CXCR5 vs MSP-1 IgG titers: r = +0.061.

Spensieri et al. 2013

ICOS
high

Locci et al. 2013

27

PLAN

INTRODUCTION
OBJECTIVES
METHODS
RESULTS AND DISCUSSION
CONCLUSION
RECOMMENDATIONS
SUMMARY IN FRENCH

Our results suggest that:

CO NCLUSI ON
(1/1)

1.

P. falciparum malaria is associated with an expansion of

cTFH in Cameroonian adults.

2. The kinetics of cTFH during and after the disease reveal

that the effect of malaria on cTFH expansion is more
pronounced outside the active phase of the disease.

3. There exists an association between cTFH abundance

and anti-MSP1 IgG titers and avidity.

29

R E C O M M E N D AT I O N S
(1/1)

We therefore recommend:

To the research community:

Evaluate long term kinetics of cTFH with repeated
infections

Stimulating cTFH proliferation in healthy subjects to obtain

acquired immunity against P. falciparum.

Conduct a similar study with larger sample size and

supplementary techniques for cTFH identification.

To the Biotechnology Center

Continue to train researchers on malaria and other
domains.
30

PLAN

INTRODUCTION
OBJECTIVES
METHODS
RESULTS AND DISCUSSION
CONCLUSION
RECOMMENDATIONS
SUMMARY IN FRENCH

 Le

paludisme:

Maladie

parasitaire,

cause

par

INTRODUCTION
(1/2)

Plasmodium sp.

Sant publique: 584 000 dcs, 3,2 milliards risque.

Mesures de prvention: Importance dun vaccin.
Rponse immunitaire au paludisme: Importance des
anticorps haute affinit contre P. falciparum.

32

INTRODUCTION
(2/2)

Intervention des cellules T-auxiliaires folliculaires (TFH)

dans la production des anticorps haute affinit.

TFH circulantes (cTFH): plus accessibles dans le sang.

Identification par lexpression de CXCR5 et ICOS.

Etude des cTFH dans le paludisme.

33

OBJECTIFS
(1/2)

Objectif Gnral:
Apprcier

le

taux

de

cTFH

dans

le

sang

priphrique des adultes Camerounais sensibiliss

au P. falciparum, et dtudier la relation entre les
cTFH et les anticorps haute affinit contre
P. falciparum.

34

OBJECTIFS
(2/2)

Spcifiquement:
1. Dterminer si il ya prolifration des cTFH lors du
paludisme P. falciparum.
2. Explorer la cintique des cTFH pendant et aprs
linfection au P. falciparum.
3. Investiguer si les taux de cTFH corrlent avec les taux
danticorps ainsi quavec lavidit de ces anticorps.

35

 Etude transversale, District de sant de Nkolbisson.

Adultes > 18 ans, consentants.

MTHODES
(1/3)

4 catgories de participants:
- Nafs au P. falciparum (Etats-Unis)
- Sensibiliss, pas de paludisme > 6 mois
- Sensibiliss, cas de paludisme en cours
- Mmes cas de paludisme, 2 semaines plus tard

36

 7 mL de sang priphrique pour:

- Lames: Goutte paisse, frottis mince.

MTHODES
(2/3)

- Plasma: Etude des anticorps.

- Mise en culture (37C, 5% CO2, 15 heures).

Cellules mononuclaires spares du sang cultiv.

Isolement des lymphocytes T-CD4+.
Extraction de lARN ADNc.

37

 Quantification des cTFH via lexpression gntique de

CXCR5 et ICOS par PCR en temps rel (quantification

MTHODES
(3/3)

relative/absolue).
Plasma: Anticorps quantifis par MAP (Luminex).
Donnes: Microsoft Excel 2010; GraphPad Prism 5.0.
Tests paramtriques/non-paramtriques; p<0,05.
Ethique, autorisations, confidentialit.

38

 30 chantillons:
- 3 nafs au P. falciparum (Etats-Unis)

R S U LTAT S
(1/3)

- 6 sensibiliss, pas de paludisme > 6 mois

- 12 sensibiliss, cas de paludisme en cours
- 9 de ces cas de paludisme, 2 semaines plus tard

Age moyen: 27,3 ans 8,57 ans

39

 CXCR5 lev dans 66,7% Camerounais vs 0% nafs.

R S U LTAT S
(2/3)

Diminution de cTFH au cours du paludisme actif, et

augmentation en convalescence.

CXCR5 vs parasitmie: corrlation positive (r=0,911;

p=0,001).

40

 Taux dIgG anti-MSP-1: Significativement plus lev

R S U LTAT S
(3/3)

chez les Camerounais vs sujets nafs (p=0,0056).

Association cTFH et taux dIgG: Corrlation positive
(r=0,061).

Association cTFH et avidit des IgG: Corrlation

positive (r=0,291).

41

CO NCLUSI ON
(1/1)

En Conclusion:
1. Le paludisme P. falciparum est associ une
prolifration des cTFH chez les adultes Camerounais
sensibiliss.
2. La cintique des cTFH montre que leffet du paludisme
sur lexpansion des cTFH est plus marqu en dehors
de la phase active de la maladie.
3. Il existe une association entre le taux de cTFH et le
taux dIgG anti-MSP1, ainsi quavec lavidit de ces
anticorps.
42

R E C O M M A N D AT I O N S
(1/1)

Nous recommandons:

A la Communaut Scientifique:
- Evaluer la cintique des cTFH long terme, avec des
infections rptes P. falciparum.
- Pouvoir stimuler sans risque la prolifration des cTFH
chez des sujets sains afin de btir une immunit
acquise contre le paludisme.
- Confirmer ces trouvailles avec une taille dchantillon
plus large et des techniques supplmentaires.

43

MERCI
44

Special Thanks to!!!

All participants in the study
Directors
Staff of the Immunology Laboratory, BTC UY1,
for the technical support
FOGARTY and IMPM for financial support
University of Hawaii
Staff and teachers of FMBS
Parents and friends

45