Cytotoxic Markers Associate With Protection Against Malaria in Human Volunteers Immunized With Plasmodium falciparum Sporozoites

Abstract

Background. Immunization of healthy volunteers by bites from Plasmodium falciparum–infected mosquitoes during chloroquine chemoprophylaxis (hereafter, chemoprophylaxis and sporozoites [CPS] immunization) induces sterile protection against malaria. CPS-induced protection is mediated by immunity against pre-erythrocytic stages, presumably at least partially by cytotoxic cellular responses. We therefore aimed to investigate the association of CPS-induced cytotoxic T-cell markers with protection.
Methods. In a double-blind randomized controlled trial, we performed dose titration of CPS immunization followed by homologous challenge infection in 29 subjects. Immune responses were assessed by in vitro restimulation of peripheral blood mononuclear cells and flow cytometry.
Results. Dose-dependent complete protection was obtained in 4 of 5 volunteers after immunization with bites from 45 P. falciparum–infected mosquitoes, in 8 of 9 volunteers with bites from 30, and in 5 of 10 volunteers with bites from 15 (odds ratio [OR], 5.0; 95% confidence interval [CI], 1.5–17). Completely protected subjects had significantly higher proportions of CD4 T cells expressing the degranulation marker CD107a (OR, 8.4; 95% CI, 1.5–123; P = .011) and CD8 cells producing granzyme B (OR, 11; 95% CI, 1.9–212; P = .004) after P. falciparum restimulation.
Conclusions. These data underline the efficiency of CPS immunization to induce sterile protection and support a possible role for cytotoxic CD4 and CD8 T-cell responses in pre-erythrocytic immunity.
Clinical Trials Registration. NCT01218893.

Full text

MAJOR ARTICLE
Cytotoxic Markers Associate With Protection
Against Malaria in Human Volunteers
Immunized With Plasmodium falciparum
Sporozoites
Else M. Bijker,1,a Anne C. Teirlinck,1,a,b Remko Schats,3Geert-Jan van Gemert,1Marga van de Vegte-Bolmer,1
Lisette van Lieshout,4,5 Joanna IntHout,2Cornelus C. Hermsen,1Anja Scholzen,1Leo G. Visser,3and Robert W. Sauerwein1
1
Department of Medical Microbiology, and
2
Department for Health Evidence, Section Biostatistics, Radboud university medical center, Nijmegen,
3
Department of Infectious Diseases,
4
Department of Medical Microbiology, and
5
Department of Parasitology, Leiden University Medical Center,
The Netherlands
Background.Immunization of healthy volunteers by bites from Plasmodium falciparum–infected mosquitoes
during chloroquine chemoprophylaxis (hereafter, chemoprophylaxis and sporozoites [CPS] immunization) induces
sterile protection against malaria. CPS-induced protection is mediated by immunity against pre-erythrocytic stages,
presumably at least partially by cytotoxic cellular responses. We therefore aimed to investigate the association of
CPS-induced cytotoxic T-cell markers with protection.
Methods.In a double-blind randomized controlled trial, we performed dose titration of CPS immunization fol-
lowed by homologous challenge infection in 29 subjects. Immune responses were assessed by in vitro restimulation of
peripheral blood mononuclear cells and flow cytometry.
Results.Dose-dependent complete protection was obtained in 4 of 5 volunteers after immunization with bites
from 45 P. falciparum–infected mosquitoes, in 8 of 9 volunteers with bites from 30, and in 5 of 10 volunteers with
bites from 15 (odds ratio [OR], 5.0; 95% confidence interval [CI], 1.5–17). Completely protected subjects had sig-
nificantly higher proportions of CD4 T cells expressing the degranulation marker CD107a (OR, 8.4; 95% CI, 1.5–123;
P= .011) and CD8 cells producing granzyme B (OR, 11; 95% CI, 1.9–212; P= .004) after P. falciparum restimulation.
Conclusions.These data underline the efficiency of CPS immunization to induce sterile protection and support
a possible role for cytotoxic CD4 and CD8 T-cell responses in pre-erythrocytic immunity.
Clinical Trials Registration.NCT01218893.
Keywords.malaria; Plasmodium; immunization; protection; immunity; chloroquine; T cells; degranulation;
granzyme B; cytotoxicity.
Malaria remains a major public health problem, with an
estimated incidence of 207 million clinical cases and ap-
proximately 627 000 deaths every year [1]. Plasmodium
falciparum is the most severe and lethal of 5 species
that can cause malaria in humans. Availability of an
effective vaccine will be critical to fight this disease, but
currently there is no licensed vaccine available, despite
decades of research. Most efforts have focused on the de-
velopment of subunit vaccines, unfortunately showing
Received 13 February 2014; accepted 7 April 2014; electronically published 27
May 2014.
Presented in part: Scientific Spring Meeting of the Royal Netherlands Society for Mi-
crobiology and Netherlands Society for Medical Microbiology, Papendal, the Netherlands,
April 2013; Malaria Vaccines for the World Conference, Lausanne, Switzerland, April
2013; Measuring Antigen Specific Immune Responses, Dubrovnik, Croatia, May 2013;
Vaccine Symposium NWO, Utrecht, the Netherlands, September 2013.
a
E. M. B. and A. C. T. contributed equally to this work.
b
Present affiliation: Center for Infectious Disease Control Netherlands, National
Institute for Public Health and the Environment, Bilthoven, the Netherlands.
Correspondence: Robert W. Sauerwein, MD, PhD, Radboud university medic al
center, Medical Microbiology 268, PO Box 9101, 6500 HB Nijmegen, the Nether-
lands (robert.sauerwein@radboudumc.nl).
The Journal of Infectious Diseases
®
2014;210:1605–15
© The Author 2014. Published by Oxford University Press on behalf of the Infectious
Diseases Societyof America. Thisis an Open Access article distributed under the terms
of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://
creativecommons.org/licenses/by-nc-nd/ .0/), which permits non-commercial
reproduction and distribution of the work, in any medium, provided the original work
is not altered or tra nsfor med in an y way, and that t he work is properl y cited. For
commercial re-use, please contact journals.permissions@oup.com.
DOI: 10.1093/infdis/jiu293
Markers of Protection Against Malaria •JID 2014:210 (15 November) •1605
4

only limited protective efficacy [2,3]. Immunization strategies
based on whole parasites, however, have repeatedly induced
high levels of protection in experimental settings [4–7]. Previous-
ly, we showed that immunization of healthy, malaria-naive sub-
jects with live sporozoites delivered by 36–45 mosquito bites
during chloroquine chemoprophylaxis (hereafter, chemoprophy-
laxis and sporozoites [CPS] immunization) induces robust, long-
lasting sterile protection against P. falciparum malaria [8,9]. CPS
immunization is about 20 times more efficient than the only al-
ternative approach for complete sterile protection against malaria
in humans, immunization with radiation-attenuated P. falcipa-
rum sporozoites (RAS), which requires bites from >1000 infected
and irradiated mosquitoes [4] or intravenous administration of
675 000 sporozoites [10].
CPS-induced protective immunity targets the earliest stages of
the parasite life cycle (ie, sporozoites and/or liver stages), rather
than the subsequently developing asexual blood stages [11]. The
immune pathways responsible for this pre-erythrocytic protec-
tion, however, remain unknown. In murine malaria models, cy-
totoxic killing of Plasmodium-infected hepatocytes appears to
play a role in protection, but the exact contribution and mecha-
nism of cytotoxicity remain elusive [12,13]. Also, in humans, a
role for both cytotoxic CD4 T cells and CD8 T cells has been sug-
gested, but evidence is scarce and largely circumstantial [14]. We
conducted a double-blind, randomized, controlled CPS immuni-
zation dose titration and challenge study. Subjects, while receiv-
ing chloroquine prophylaxis, were immunized by bites from 45,
30, or 15 infected mosquitoes, followed by a challenge infection,
resulting in dose-dependent protection. Next, we explored mark-
ers of cytotoxic T-cell responses induced by CPS immunization
and identified 2 cytotoxic markers associated with protection.
MATERIALS AND METHODS
Human Ethics Statement
All subjects provided written informed consent before screen-
ing. The study was approved by the Central Committee for
Research Involving Human Subjects of the Netherlands
(NL33904.091.10) and complied with the Declaration of Hel-
sinki and good clinical practice, including monitoring of data.
Clinical Trial Design and Procedures
A single-center, double-blind study was conducted at the Lei-
den University Medical Center from April 2011 until April
2012. Healthy subjects aged 18–35 years with no history of ma-
laria were screened as described previously [11]. Thirty subjects
were randomly divided into 4 groups, using a computer-
generated random-number table. Subjects, investigators, and
primary outcome assessors were blinded to the allocation. All
subjects received 3 CPS immunizations at monthly intervals, as
described previously [8,11], but the number of NF54
P. falciparum–infected versus uninfected mosquitoes varied
per group: 5 subjects were each exposed to bites from 15 infect-
ed mosquitoes 3 times (group 1), 10 subjects were each exposed
to bites from 10 infected and 5 uninfected mosquitoes on three
occasions (group 2), 10 subjects were each exposed to bites from
5 infected and 10 uninfected mosquitoes on three occasions
(group 3), and 5 control subjects were exposed to bites from
15 uninfected mosquitoes on three occasions (group 4). Nine-
teen weeks after the last immunization (15 weeks after the last
chloroquine dose), all subjects were challenged by exposure to
bites from 5 mosquitoes infected with the homologous NF54 P.
falciparum strain, according to previous protocols [8,15]. The
primary outcome was prepatent period, defined as the time be-
tween challenge and first P. falciparum–positive thick blood
smear. Thick blood smears were prepared and read as described
previously [11]. For more details about the immunization and
challenge procedures andfollow-up,seetheSupplementary
Materials.
Immunological Methods
Peripheral blood mononuclear cells (PBMCs) were collected at
the following time points: before initiation of chloroquine pro-
phylaxis (baseline [B]), 27 days after immunization 1 (I1; 1 day
before the second immunization), 27 days after immunization 2
(I2; 1 day before the third immunization), 27 days after immu-
nization 3 (I3), the day before the challenge infection (C −1),
and 20 weeks after the challenge infection (C + 140). For the as-
sessment of P. falciparum–specific immune responses, PBMCs
were restimulated in vitro with P. falciparum–infected red blood
cells (RBCs) as described before [16]. Expression of the degra-
nulation marker CD107a, the cytotoxic molecule granzyme B,
and the cytokine interferon γ(IFN-γ) by CD4, CD8, and γδ
T cells was assessed by flow cytometry. For a detailed descrip-
tion of these methods, see the Supplementary Materials.
Statistical Analysis
The dose-dependent induction of protection was tested by logistic
regression, using SPSS 20. Comparison of CD107a expression and
granzyme B and IFN-γproduction by T-cell subsets between im-
munized unprotected and protected volunteers after CPS immu-
nization was done per selectedcellular response by means of Firth
penalized logistic regression [17,18], resulting in Pvalues, odds
ratios (ORs) related to a change of 1 interquartile range (IQR),
and 95% profile likelihood confidence intervals (CIs) for the
OR. Analyses were performed using R, version 3.0.1 [19], with
the packages logistf, version 1.21 [20]; rms, version 4.1–3[21];
and penalized, version 0.9–42 [22,23]. The ability of (a combina-
tion of) markers to discriminate between protected and unpro-
tected volunteers was assessed with the area under the receiver
operator curve (ROC), based on leave-one-out cross-validation,
using R and pROC, version 1.7.1 [24]. For further details about
these methods, see the Supplementary Materials.
1606 •JID 2014:210 (15 November) •Bijker et al

RESULTS
CPS Immunization
Thirty volunteers were included (median age, 21 years; range,
19–31 years), out of 63 subjects screened for eligibility (Supple-
mentary Figure 1). Volunteers were randomly assigned to one of
the 4 groups described in Materials and Methods. After each con-
secutive immunization, the number of subjects with parasitemia,
as retrospectively detected by quantitative polymerase chain
reaction (qPCR) analysis, steadily decreased in groups 1 and
2. In group 3, however, 5 volunteers still showed parasitemia
after the second and third immunization (Figure 1). Remarkably,
in 4 immunized subjects (3 in group 2 and 1 in group 3), para-
sitemia was never detectable by qPCR. One subject from group 2
withdrew consent after the first immunization for reasons unre-
lated to the trial and was excluded from the analysis.
Challenge Infection
Nineteen weeks after the last immunization, volunteers were
challenged by standard exposure to bites from 5 homologous
strain NF54-infected mosquitoes [5]. Protection by CPS immu-
nization was dose-dependently induced in 4 of 5 subjects in
group 1, 8 of 9 subjects in group 2, and 5 of 10 subjects in
group 3, while all control subjects became thick smear positive
(OR, 5.0; 95% CI, 1.5–17; P= .01). The median prepatent period
was 2.5 days longer in CPS-immunized unprotected subjects,
compared with controls, both by thick smear and qPCR. Al-
though these differences were not statistically significant
(P= .22 for thick smear findings and P= .31 for qPCR findings),
this delay suggests the presence of partial protection at least in
some of the unprotected CPS-immunized subjects (Figure 2and
Table 1). In retrospect, all 6 volunteers with detectable parasite-
mia by qPCR after the third immunization were not completely
protected from challenge infection, while 17 of 18 subjects with
a negative qPCR result after the third immunization were fully
protected.
Platelet levels decreased below the reference value (150 × 10
9
platelets/L) in 8 of 12 thick-smear-positive subjects (ie, both
controls and CPS-unprotected) at any point after challenge
(median for all thick-smear-positive subjects, 134 × 10
9
plate-
lets/L;range,79×10
9
–213 × 10
9
platelets/L). D-dimer levels
were elevated in all thick-smear-positive subjects after challenge
(median peak concentration, 2431 ng/mL; range, 1014–5000
ng/mL). Parameters normalized in all subjects after treatment
without complications. All thick-smear-positive subjects expe-
rienced solicited adverse events (AEs) during challenge
Figure 1. Parasitemia after the first, second and third chemoprophylaxis and sporozoites immunization. Parasitemia was determined once daily by quan-
titative polymerase chain reaction (qPCR) analysis from day 6 until day 10 after each immunization. Each line represents an individual subject. Panels show
data for volunteers from group 1 (A; exposure to bites from 15 infected mosquitoes on three occasions), group 2 (B; exposure to bites from 10 infected and 5
uninfected mosquitoes on three occasions), group 3 (C; exposure to bites from 5 infected and 10 uninfected mosquitoes on three occasions), and group 4 (D;
exposure to bites from 15 uninfected mosquitoes on three occasions; control). Values shown as 10 on the log scale were negative (ie, half the lower
detection limit of the qPCR, 20 parasites/mL). Proportions along the x-axes denote subjects with a positive qPCR result/total number of subjects immunized.
Markers of Protection Against Malaria •JID 2014:210 (15 November) •1607

infection consistent with uncomplicated malaria (median num-
ber/subject, 9.5 AEs [range, 4–14 AEs); median duration of each
AE,1.1days[range,0.0–12.3 days]). As expected, protected
subjects presented with fewer AEs: 15 of 17 subjects experienced
solicited AEs possibly or probably related to the challenge
(median number/subject, 2 AEs [range, 0–15 AEs]; median
duration, 0.7 days [range, 0.00–15.9 days]). One subject from
group 2 was preliminarily treated with atovaquone/proguanil
on day 10.5 after challenge because of unrelated exertional rhab-
domyolysis after extensive sports activity (weightlifting) fol-
lowed by sauna visits. No other severe AEs occurred. One
volunteer from group 1 was treated for reasons unrelated to
the trial at day 19. Both of these volunteers remained parasite
negative by qPCR analysis after the third immunization and
at any time point after challenge and were considered protected
in further analysis.
Figure 2. Parasitemia after challenge infection. Parasitemia was assessed retrospectively by real-time quantitative PCR (qPCR) from day 5 after challenge
onward, until day 21, at 2 time points per day for thick-smear-positive volunteers and at 1 time point per day for protected volunteers. Each line represents
an individual subject. Gray dotted lines show chemoprophylaxis and sporozoites–immunized volunteers from group 1 (exposed to bites from 15 infected
mosquitoes on three occasions; n = 5), gray dashed lines subjects from group 2 (exposed to bites from 10 infected and 5 uninfected mosquitoes on three
occasions; n = 9), gray solid lines represent subjects from group 3 (exposed to bites from 5 infected and 10 uninfected mosquitoes on three occasions;
n = 10), and black lines represent malaria-naive control subjects (exposed to bites from 15 uninfected mosquitoes on three occasions; n = 5). Values shown
as 10 on the log scale were negative. The 2 thick-smear-positive subjects from groups 1 and 2 became qPCR positive on days 9.5 and 8.5 respectively; both
became thick smear positive on day 12.0.
Table 1. Protection Against Challenge Infection After Chemoprophylaxis and Sporozoites Immunization
Group
Protection
Day of Positivity After Challenge, Median
(Range)
c
Proportion
a
Percentage (95% CI
b
) Thick Smear qPCR
Group 1 4/5 80 (36.0–98.0) 12.0 9.5
Group 2 8/9 89 (54.3 to >99.9) 12.0 8.5
Group 3 5/10 50 (23.7–76.3) 11.0 (9.0–15.0) 9.0 (6.5–13.0)
Group 4 0/5 0 (0.0–48.9) 9.5 (9.0–13.5) 6.5 (6.5–10.5)
Subjects were exposed to bites from 15 infected mosquitoes on th ree occasions ( group 1), to bites from 10 infected and 5 uninfected mosquit oes on three
occasions (group 2), to bites from 5 infected and 10 uninfected mosquitoes on three occasions (group 3), or to bites from 15 uninfected mosquitoes on three
occasions (group 4; control).
a
Data are no. of protected subjects/total no. of subjects.
b
95% confidence intervals (CIs) were calculated by the modified Wald method.
c
Data are for thick-smear-positive subjects.
1608 •JID 2014:210 (15 November) •Bijker et al

Table 2. Cytotoxic T-Cell Markers, Relative to Immunization Time, for 24 Volunteers Who Received Chemoprophylaxis and Sporozoites (CPS) Immunizations and 5 Controls
Study Group, Marker
a
CD4 T Cells CD8 T Cells γδ T Cells
BI1
P
Value I2
P
Value I3
P
Value C−1
P
Value B I1
P
Value I2
P
Value I3 PC−1
P
Value B I1
P
Value I2
P
Value I3
P
Value C−1
P
Value
CPS
CD107a
Expression, %, mean 0.26 0.53 <.001 0.64 <.001 0.59 <.001 0.53 <.001 0.08 0.09 0.15 0.14 0.19 26.6 36.5 <.001 41.2 <.001 40.3 <.001 33.8 <.01
iMFI 15.7 38.5 <.001 46.0 <.001 41.0 <.001 37.0 <.001 7.6 14.5 19.2 <.05 17.5 <.05 20.9 <.01 4472 6925 <.001 7925 <.001 7594 <.001 6262 <.01
Granzyme B
Production, %, mean 0.25 0.49 0.41 0.38 0.43 0.50 0.89 0.92 1.03 0.94 6.06 7.35 8.27 8.96 8.79
iMFI 1.40 9.80 11.3 7.50 3.24 5.11 71.3 <.05 40.5 26.7 19.9 2.54 15.3 <.001 15.0 <.001 12.4 <.001 8.18
IFN-γ
Production, %, mean 0.08 0.43 <.001 0.54 <.001 0.42 <.001 0.47 <.001 0.04 0.11 0.13 <.05 0.08 0.13 <.01 6.01 13.6 <.001 16.3 <.001 14.9 <.001 12.0 <.01
iMFI 5.16 32.2 <.001 37.7 <.001 25.7 <.05 37.2 <.001 2.96 9.8 9.3 6.4 12.5 <.01 407 1020 <.01 1173 <.001 1029 <.01 982 <.05
Control
CD107a
Expression, %, mean 0.25 0.27 0.16 0.20 0.42 0.02 0.06 0.02 0.00 0.05 26.3 22.6 22.6 21.1 29.2
iMFI 15.6 17.2 10.6 13.4 27.3 3.38 7.04 3.28 1.56 7.92 4438 4439 4191 4512 5608
Granzyme B
Production, %, mean 0.31 0.18 0.20 0.10 0.60 0.95 0.43 0.96 0.72 1.16 5.68 2.41 1.00 1.73 8.56
iMFI 1.06 4.04 2.98 −0.36 11.1 −23.2 −3.02 14.6 −25.2 25.3 45.1 −77.4 −258 −326 302
IFN-γ
Production, %, mean 0.09 0.02 0.00 0.02 0.12 0.02 0.02 0.01 0.01 0.07 5.58 3.51 3.40 3.13 7.06
iMFI 4.62 0.86 0.36 0.70 6.98 1.44 0.38 0.22 0.26 5.44 356 161 146 143 486
Plasmodium falciparum–infected red blood cell (RBC)–specific responses were corrected for the value for the uninfected RBC background. Pvalues were calculated using 1-way analysis of variance with the Dunnett post
hoc test, with the B value serving as a control.
Abbreviations: B, baseline; C −1, 1 day before challenge; I1, 27 days after immunization 1; I2, 27 days after immunization 2; I3, 27 days after immunization 3.
a
The geometric mean fluorescence intensity (iMFI) is calculated as the percentage of positive cells multiplied by the MFI of this positive population.
Markers of Protection Against Malaria •JID 2014:210 (15 November) •1609

Analysis of Cytotoxic T-Cell Markers After In Vitro
P. falciparum Stimulation
Next, we tested a panel of representative cytotoxic T-cell mark-
ers, including surface expression of degranulation marker
CD107a and granzyme B and IFN-γproduction, in CD4,
CD8, and γδ T cells after in vitro restimulation with P. falcipa-
rum–infected RBCs in all immunized subjects (Table 2). CPS im-
munization induced a significant increase in both the percentage
and integrated geometric mean fluorescence intensity (iMFI) of
CD107a
+
CD4 and γδ T cells from the first immunization until
challenge. Similarly, CD8 T cells expressed a significantly higher
CD107a iMFI after the second immunization. The proportion of
granzyme B–producing cells did not change after immunization,
but the granzyme B iMFI was significantly increased in both CD8
and γδ T cells, returning to baseline on C −1. Production of IFN-
γwas induced in all T-cell subsets, but induction was most
pronounced in CD4 and γδ T cells. There were only weak corre-
lations between cellular responses on C −1 and total blood-stage
parasite exposure, as calculated by the number of parasites per
milliliter after all 3 immunizations (data not shown; Spearman
rho for all, <0.5). None of the responses in the control group
changed significantly from baseline at any point of time (Table 2),
suggesting that chloroquine alone did not affect P. falciparum–
specific T-cell responses.
We next assessed the association of these markers with pro-
tection after challenge (Figure 3). Indeed, complete protection
associated with the proportion of CD107a
+
CD4 T cells (OR,
8.4; 95% CI, 1.5–123; P=.011; Figure 3A), the iMFI of
CD107a on CD4 T cells (OR, 11; 95% CI, 1.6–188; P= .011;
data not shown), and production of granzyme B by CD8
T cells (OR, 11; 95% CI, 1.9–212; P= .004; Figure 3E)at
C−1. A subgroup analysis of data from group 3 confirmed
these findings: the only markers with higher levels in protected
subjects were the proportion of both CD107a
+
CD4 T cells (OR,
4.2; 95% CI, .9–140; P= .081) and granzyme B–producing CD8
T cells (OR = 27; 95% CI, 1.5–27 687; P= .019). While expres-
sion of CD107 on CD4 T cells and granzyme B in CD8 T cells
predicted protection, with areas under the ROC of 0.73 (95% CI,
.48–.98) and 0.81 (95% CI, .63–.99), respectively, combining
both markers resulted in only a slight improvement of the
area under the ROC (0.82; 95% CI, .61–1).
P. falciparum–specific IFN-γproduction by CD4, CD8, or γδ
T cells could not distinguish protected volunteers (Figure 3G,
3H, and 3I). Also pluripotent (IFN-γ
+
IL-2
+
) effector memory
T-cell (CD4
+
CD62L
−
CD45RO
+
) responses, previously shown
to be significantly increased by CPS immunization [8], were
again induced (P= .013) but did not differentiate between pro-
tected and unprotected volunteers (OR, 1.6; 95% CI, .5–4.9;
P= .41; data not shown).
CD107a
+
CD4 T cells presented as the clearest marker asso-
ciated with protection, with values consistently higher in fully
protected subjects from I1 onward (Figure 4A), and was
independent of immunization dose (Figure 4B). A significant
correlation was found between CD107a expression by CD4
T cells after 1 immunization and prepatent period after chal-
lenge infection in all thick-smear-positive subjects (Spearman
rho, 0.69; P= .013; Figure 4C). The proportion CD107a
+
CD4
T cells in the control subject who developed parasitemia signifi-
cantly later than the other controls (ie, day 13.5 vs days 9–10.5)
was, at baseline, on average 2.8-fold higher than in the other
subjects. Possibly, the inherently higher response in this volun-
teer contributed to delayed patency after challenge.
CD107a
+
CD4 T cells expressed proportionally more gran-
zyme B (7.4% vs 0.39% on C −1; P< .0008) in protected subjects,
indicative of their cytotoxic phenotype, and proportionally more
IFN-γ(13.3% vs 0.39% on C −1; P< .0001) than CD107a
−
CD4
T cells (Figure 4Dand 4E). CD8 T cells, traditionally considered
the cytotoxic subclass of T cells, contained a larger proportion of
CD107a
+
cells at baseline than CD4 T cells when uninfected
(0.39% vs 0.19%; P< .0001 [all volunteers]). However, the pro-
portion of P. falciparum–specific degranulation of CD8 T cells
was not notably increased by CPS immunization (P= .44), in
contrast to CD4 T cells (P<.0001; Supplementary Figure 2A
and 2B).
Both CD107a expression by CD4 T cells and granzyme B
production by CD8 T cells remained significantly elevated up
to 20 weeks after the challenge infection (C + 140; P< .05 and
P< .01, respectively; Figure 5Aand 5B), demonstrating longev-
ity of the CPS-induced T-cell response.
DISCUSSION
We show that CPS immunization reproducibly and dose-
dependently induces protection against a homologous
challenge infection. With exposure to a total number of P. fal-
ciparum–infected mosquito bites as low as 30, CPS immuniza-
tion still induces 89% protection in healthy volunteers. We
furthermore demonstrate that markers of cytotoxic T-cell
responses are associated with protection against malaria after
whole-sporozoite immunization.
This study provides further support for the remarkable po-
tency of the CPS-protocol to induce complete protection by
using even lower numbers of P. falciparum–infected mosquitoes
than before [8]. The observed dose-dependent protection is in
line with results from RAS immunization trials with sporozoites
administered either intravenously by needle and syringe [10]or
by bites from irradiated infected mosquitoes [4]. Although the
delay of patency in unprotected CPS-immunized subjects was
not statistically significant, the patterns of parasitemia indicate
partial protection in some subjects. The unexpectedly delayed
control subject hampered statistical significance but could be
considered an outlier, possibly because of the inherently high
baseline immune response. The establishment of a suboptimal
CPS immunization regimen inducing protection in 50% of the
1610 •JID 2014:210 (15 November) •Bijker et al

volunteers immunized with bites from 5 mosquitoes on three
occasions will facilitate further studies of protective immune
mechanisms against P. falciparum malaria.
Our data provide evidence for a role of cytotoxic T-cell re-
sponses in pre-erythrocytic immunity in humans. Because of
obvious practical limitations, we only assessed immune cells
Figure 3. Cytotoxic immune responses upon in vitro Plasmodium falciparum–infected red blood cell stimulation 1 day before challenge infection (C −1).
Each symbol represents a single protected (black symbols) or chemoprophylaxis and sporozoites–immunized unprotected (gray symbols) individual from
group 1 (exposed to bites from 15 infected mosquitoes on three occasions; dots), group 2 (exposed to bites from 10 infected and 5 uninfected mosquitoes
on three occasions; triangles), or group 3 (exposed to bites from 5 infected and 10 uninfected mosquitoes on three occasions; squares). Horizontal bars and
whiskers represent means and standard errors of the mean (SEM). Panels show CD107a
+
CD4 T cells (A), CD8 T cells (B), and γδ T cells (C); granzyme B
expression by CD4 T cells (D), CD8 T cells (E), and γδ T cells (F); and interferon γ(IFN-γ) expression by CD4 T cells (G), CD8 T cells (H), and γδ T cells (I).
Values are corrected for uninfected red blood cell (uRBC) background and for baseline response before immunization. Mean background responses (±SEM) to
uRBC stimulation for CD4, CD8, and γδ T cells were 0.19 ± 0.01, 0.41 ± 0.02, and 0.61 ± 0.05, respectively, for CD107a; 1.65 ± 0.50, 15.34 ± 1.46, and
64.56 ± 1.74, respectively, for granzyme B; and 0.09 ± 0.00, 0.07 ± 0.00, and 0.14± 0.01, respectively, for IFN-γ(calculated for all volunteers at both baseline
and C −1). High uRBC granzyme B responses in CD8 and γδ T cells indicate that a significant percentage of these cells contain granzyme B even in a resting
situation. uRBC responses did not change significantly from baseline for any of the readouts. The differences between responses of protected and unpro-
tected volunteers that are not indicated with a Pvalue are nonsignificant. The differences between protected and unprotected volunteers were evaluated
using logistic regression.
Markers of Protection Against Malaria •JID 2014:210 (15 November) •1611

in the peripheral blood, which may not necessarily reflect
responses in the liver but rather represent a surrogate. The re-
sults of this exploratory analysis will have to be confirmed in
future trials, and the functional relevance remains to be
investigated.
So-called classic cytotoxic CD8 T cells can be activated by
malaria parasite antigen on infected hepatocytes via major his-
tocompatibility complex (MHC) class I [25] and are associated
with protection in a number of (animal) models [13,14,26].
CD8 T cells are involved in protection in the murine CPS and
RAS models [27–29], but their precise effector mechanisms re-
main subject of debate. They might either require direct contact
with infected hepatocytes [13] or be indep endent of granzyme B
and/or other cytotoxic molecules, suggesting a more indirect cy-
tokine-mediated effect by CD8 T cells [12] or other hepatic im-
mune cells [30]. In additi on, a functional role for cytotoxic CD4
T cells is also conceivable because these cells can use cytolytic
pathways, such as those involving granulysin, perforin,
Figure 4. Cytotoxic profile of chemoprophylaxis and sporozoites (CPS)–induced CD4 T cells. Aand B, Induction of Plasmodium falciparum–specific
CD107a
+
CD4 T cells was determined in protected and unprotected CPS-immunized subjects (A) and in protected subjects separated for each immunization
dose (B) over the course of immunization. Horizontal bars and whiskers represent mean responses and standard errors of the mean. C, The relationship
between P. falciparum–specific CD107a CD4 T cells on day 27 after immunization 1 (I1) and the prepatent period after challenge for all thick-smear-positive
volunteers (CPS-immunized individuals and controls). Dand E, Among protected CPS-immunized subjects, granzyme B (D) and interferon γ(IFN-γ) production
(E) by CD107a
+
(black dots) and CD107a
−
(gray dots) CD4 T cells were analyzed at baseline (B) and after CPS immunization (on the day before the challenge
infection [C −1]). Horizontal bars show the mean response. All data were corrected for uninfected red blood cell background for every volunteer at each time
point. *P< .05, **P< .01, and ***P< .001. I2, 27 days after immunization 2; I3, 27 days after immunization 3.
1612 •JID 2014:210 (15 November) •Bijker et al

granzymes, and FAS-L, as shown mostly in viral infections [31,
32]. The protective role of CD4 T cells in murine malaria has
been suggested, using in vitro experiments [33]andin vivo
depletion [12] or passive transfer [34].Furthermore, functional cy-
totoxic CD4 T cells derived from RAS-immunized or synthetic-
peptide-immunized volunteers are able to lyse autologous B
cells pulsed with a peptide from the circumsporozoite protein
[35–37]. We used surface expression of CD107a (LAMP-1), a
marker for cytotoxic degranulation, to phenotypically identify cy-
totoxic CD4 T cells [31]. To directly kill a P. falciparum–infected
hepatocyte, parasite antigens should be presented in the context of
MHC class II to the cytotoxic CD4 T cells. Although hepatocytes
do not express MHC class II in noninflammatory circumstances,
the presence of MHC class II on human hepatocytes has been
shown in a small number of patients with chronic hepatitis [38]
and immune-mediated liver disorders [39,40]. Functionally, over-
expression of MHC class II on hepatocytes in a transgenic mice
model showed their capacity for costimulation, antigen presenta-
tion, and CD4 T-cell activation [41]. Only indirect evidence sug-
gests that MHC class II expression on mice hepatocytes may play
a role in murine malaria [33,42], and the presence of MHC class
II on hepatocytes in human malaria has never been studied. Here,
we show for the first time that degranulating CD4 T cells are as-
sociated with protection in human malaria and are significantly
induced after 1 immunization.
The observed lack of boosting by the second and third immu-
nizations may reflect a saturated response of antigen-specific
memory cells. This raises the possibility that fewer immuniza-
tions may be sufficient to induce protection, supported by the
increased proportion of volunteers without parasitemia after the
second and third immunizations in groups 1 and 2. Moreover,
the observed longevity of the immune response is in line with
long-term protection after CPS immunization in a previous
study [9].
The T-helper type 1 (Th1) cytokine IFN-γhas been repeat-
edly shown to be an important effector molecule in protection
against malaria parasites [43], and the clear induction of Th1
responses in our study corroborates earlier findings in both an-
imals and humans after whole-sporozoite immunization [8,10,
12,26,27]. We previously showed that a broad range of both in-
nate and adaptive cellular subsets contribute to CPS-induced
P. falciparum–specificIFN-γproduction [16], which is sus-
tained at least up to 2.5 years after immunization [9].IFN- γpro-
duction alone, however, does not correlate with protection in
the RAS model [10] or our CPS model. Also, production of
both IFN-γand IL-2 by effector memory CD4 T cells and pro-
duction of IFN-γby γδ T cells, although clearly increased in im-
munized volunteers [8,16], were not different between
protected and unprotected volunteers.
During CPS immunization, 4 protected subjects did not show
parasitemia by qPCR at any measured time point, not even after
the first immunization. A possible explanation is that the num-
ber of merozoites released from the liver is too low for qPCR
detection. A strong primary innate immune response may be
responsible for clearing sporozoites and/or killing infected he-
patocytes upon first encounter. Previous studies in mice indeed
showed that the inflammatory cytokines interleukin 1 (IL-1)
and interleukin 6 (IL-6) block pre-erythrocytic development
in mice [16,44]. Alternatively, chloroquine may have contribut-
ed to the decreased (ie undetectable) number of parasites re-
leased from the liver either by direct killing or, indirectly, by
stimulating the immune system.
Antigen recognition and immune cell activation are essential
for an effective response. To investigate pre-erythrocytic cellular
Figure 5. Longevity of cellular immune responses after chemoprophylaxis and sporozoites (CPS) immunization. Plasmodium falciparum–specific cellular
immune responses (corrected for uninfected red blood cell background) were assessed in protected (black dots) and unprotected (gray squares) CPS-
immunized volunteers before CPS immunization (B), and before (C −1) and 20 weeks after (C + 140) challenge infection. Data are shown as mean ± standard
error of the mean for CD107a expression on CD4 T cells (A) and granzyme B production by CD8 T cells (B). Tests are performed separately for protected and
immunized unprotected volunteers, by repeated-measures analysis of variance (including all time points before and after immunizations) and the Dunnett
multiple comparison post test, using B as control column. Only the test results for C + 140, compared with those for baseline, for protected volunteers are
displayed. For immunized unprotected volunteers, all results were nonsignificant. *P< .05 and **P< .01.
Markers of Protection Against Malaria •JID 2014:210 (15 November) •1613

immune responses, stimulation with cultured liver-stage P. fal-
ciparum would be preferred, but this is currently impossible.
We therefore used asexual blood-stage parasites for our experi-
ments, and although responses to purely pre-erythrocytic anti-
gens may be missed, the majority of potential memory
responses are likely detected upon P. falcip aru m–infected
RBC stimulation, given the large overlap between liver-stage
and blood-stage antigens [45]. Future antigen screening by
stimulation with a comprehensive library of pre-erythrocytic
and cross-stage proteins or peptides, and subsequent functional
studies focusing on cytotoxic T cells, will further identify and
delineate the specificity of protective responses [33,46].
In conclusion, we identified 2 in vitro cellular cytotoxic im-
mune markers that are associated with protection against ma-
laria in a controlled clinical setting. Furthermore, this study
confirms the robustness of CPS immunization as a highly effi-
cient and reproducible immunization strategy for complete ho-
mologous protection. Further exploration of immune responses
induced by CPS immunization will make important contribu-
tions to pre-erythrocytic malaria vaccine development and clin-
ical testing.
Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases
online (http://jid.oxfordjournals.org/). Supplementary materials consist of
data provided by the author that are published to benefit the reader. The
posted materials are not copyedited. The contents of all supplementary
data are the sole responsibilityof the authors. Questions or messages regard-
ing errors should be addressed to the author.
Notes
Acknowledgments. We thank all of the trial volunteers, for their partic-
ipation in this study; K. Suijk-Benschop, J. Fehrmann-Naumann, C. Prins,
E. Jonker, G. Hardeman, and S. ten Velden-Schipper, for blood collection
and their good care of the volunteers; the LUMC Department of Medical
Microbiology, for facilitating parasitological diagnosis; M. Erkens,
T. Arens, J. van der Slot, H. Gerritsma, F. van de Sande, J. van Schie,
E. Brienen, J. Schelfaut, J. Verweij, J.Kromhout, E. van Oorschot, and
M. Beljon, for reading many thick smears; C. Janse, for his unlimited hos-
pitality;M.Bootsma,forhercardiacmonitoringofthetrialvolunteers;
W. Graumans and R. Siebelink-Stoter, for culturing parasites; J. Klaassen,
L. Pelser-Posthumus, J. Kuhnen, and A. Pouwelsen, for generating infected
mosquitoes and for assistance with immunization of and providing chal-
lenge infection to the volunteers; G. Bastiaens and J. Wiersma, for assistance
with the immunizations and the challenge infections; P. Houzé, for chloro-
quine measurements; members of the Safety Monitoring Committee—
J. A. Romijn, M. de Boer, and M. Laurens—for their participation, guidance,
and safety recommendations throughout the trial; J. Verweij, for the set up
and supervision of the DNA isolation and qPCR activities; E. Brienen, for
performing DNA isolation and qPCR activities; CromSource, for clinical
monitoring; the staff from the LUMC Central Clinical Biochemistry and
Haematology Laboratories and the staff from the LUMC Pharmacy, for
making this study possible; A. Jansens, and D. Zimmerman, for their ad-
ministrative support; K. Teelen, for help with sample logistics; M. Vos
and B. van Schaijk, for molecular analysis of the parasite batches; and
W. Nahrendorf, for critically reading the manuscript.
Financial support. This work was supported by The Netherlands Or-
ganisation for Health Research and Development (ZonMw) (project
95110086), the Dioraphte Foundation (project 12010100), and a European
Malaria Vaccine Development Association fellowship (to A.C.T.). The re-
search leading to these results has furthermore received funding from the
European Union Seventh Framework Programme (FP7/2007-2013; grant
242095 [EviMalar]).
Potential conflicts of interest. All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the con-
tent of the manuscript have been disclosed.
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