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Impact of malaria pre-exposure on anti-parasite cellular and humoral 1
immune responses after controlled human malaria infection 2
3
Joshua M. Obiero1,a, Seif Shekalaghe2, Cornelus C. Hermsen1, Maxmillian Mpina2, Else M. 4
Bijker1, Meta Roestenberg1,b, Karina Teelen1, Peter F. Billingsley3, B. Kim Lee Sim3, Eric R. 5
James3, Claudia A. Daubenberger4,5, Stephen L. Hoffman3, Salim Abdulla2, Robert W. 6
Sauerwein1,#, Anja Scholzen1,# 7
8
1. Radboud university medical center, Department of Medical Microbiology, Nijmegen, The 9
Netherlands 10
2. Ifakara Health Institute, Bagamoyo Research and Training Center, Bagamoyo, Tanzania 11
3. Sanaria Inc., Rockville, Maryland, USA 12
4. Swiss Tropical and Public Health Institute, Department of Medical Parasitology and 13
Infection Biology, Basel, Switzerland 14
5. University of Basel, Basel, Switzerland 15
16
Present affiliation: 17
(a) University of California Irvine, Department of Medicine, Division of Infectious Diseases, Irvine, 18
California, USA 19
(b) Leiden University Medical Center, Department of Infectious Diseases, Leiden, The 20
Netherlands 21
22
Correspondence (#): Anja Scholzen ([email protected]) and Robert W. Sauerwein, 23
([email protected]) 24
25
Running title: Immune responses after controlled malaria infection 26
27
IAI Accepted Manuscript Posted Online 16 March 2015Infect. Immun. doi:10.1128/IAI.03069-14Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Abstract 28
To understand the effect of previous malaria exposure on anti-parasite immune responses is 29
important for developing successful immunization strategies. Controlled human malaria infections 30
(CHMI) using cryopreserved Plasmodium falciparum (Pf) sporozoites provide a unique 31
opportunity to study differences in acquisition or re-call of anti-malaria immune responses in 32
individuals from different transmission settings and genetic backgrounds. In this study, we 33
compared anti-parasite humoral and cellular immune responses in two cohorts of malaria-naïve 34
Dutch volunteers and Tanzanians from a low endemic area, which were subjected to the identical 35
CHMI protocol by intradermal injection of Pf sporozoites. Samples from both trials were analyzed 36
in parallel in a single center to ensure direct comparability of immunological outcomes. Within the 37
Tanzanian cohort we distinguished one group with moderate levels of pre-existing antibodies to 38
asexual Pf lysate, and another that based on Pf serology resembled the malaria-naïve Dutch 39
cohort. Positive Pf serology at baseline was associated with a lower parasite density at first 40
detection by qPCR after CHMI. Post-CHMI, both Tanzanian groups showed a stronger increase 41
in anti-Pf antibody titers than Dutch volunteers, indicating similar levels of B-cell memory 42
independent of serology. In contrast to the Dutch, Tanzanians failed to increase Pf-specific in 43
vitro re-call IFNγ-production after CHMI, and innate IFNγ-responses were lower in Pf lysate sero-44
positive individuals. In conclusion, positive Pf lysate serology can be used to identify individuals 45
with better parasite control, but weaker IFNγ-responses in circulating lymphocytes, which may 46
help to stratify volunteers in future CHMI trials in malaria endemic areas. 47
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Introduction 49
In 2012, Plasmodium falciparum (Pf) malaria caused an estimated 207 million cases and 627,000 50
deaths, of which 90% occurred in children under five years of age and in pregnant women in sub-51
Saharan Africa (1). Major control efforts have been implemented with some success (2, 3), but 52
malaria eradication will likely require a safe and highly protective vaccine. Subunit vaccines have 53
thus far shown moderate efficacy at best. RTS,S is the only vaccine candidate in phase 3 trials, 54
but despite averting substantial numbers of malaria cases (4), shows only 30-50 % reduction in 55
clinical disease after 12 months depending on both age and malaria endemicity and even less 56
after 18 months (5-7). These results stress the need for more effective second-generation 57
vaccines. Key requirements are not only the identification of novel immunogens, but also a better 58
understanding of protection-related immune responses. This includes the effect of previous 59
malaria exposure on immune responses upon re-exposure or vaccination (8, 9). 60
During the last three decades, Controlled Human Malaria Infection (CHMI) trials have 61
become an indispensable tool not only in assessing the efficacy of candidate vaccines (10, 11), 62
but also in evaluating immune responses induced by exposure to the malaria parasite (12-15). 63
CHMIs have so far been performed in non-endemic countries in previously unexposed individuals 64
(11, 16-19). A logical next step is to study potential differences in the acquisition, maintenance or 65
re-call of immune responses in individuals from different transmission settings and genetic 66
backgrounds (20, 21). The availability of aseptic, purified, cryopreserved live, Pf sporozoites 67
(PfSPZ Challenge) (22) opens up opportunities to carry out CHMI trials in malaria endemic 68
countries, since it bypasses the need for infecting local Anopheles mosquito with Pf or importing 69
Pf-infected mosquitoes to the trial site. The first PfSPZ Challenge trial in malaria-naïve Dutch 70
volunteers demonstrated an infectivity rate of 83% after intradermal injections, independent of the 71
dose given (23). Recently, PfSPZ Challenge was used for the first time during a CHMI trial in 72
healthy adult male Tanzanian volunteers, resulting in similar infection rates (24). As a follow up, 73
we here present results of the malaria-specific humoral and cellular immune responses in 74
Tanzanians and Dutch volunteers who were inoculated intradermally with the same number of 75
live PfSPZs during these CHMI studies. 76
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Methods 78
Human ethics statement 79
The Dutch trial (23) was approved by the Central Committee for Research Involving Human 80
Subjects of The Netherlands (NL31858.091.10) and registered at Clinicaltrials.gov, identifier: 81
NCT 01086917. The Tanzanian trial (24) was approved by institutional review boards of the 82
Ifakara Health Institute (IHI/IRB/No25), National Institute for Medical Research Tanzania 83
(NIMR/HQ/R.8a/Vol.IX/1217), the Ethikkommission beider Basel (EKBB), Basel, Switzerland 84
(EKBB 319/11) and the Tanzanian Food and Drug Administration (Ref. No. CE.57/180/04A/50) 85
and registered at Clinicaltrials.gov, identifier: NCT 01540903. All study teams complied with the 86
Declaration of Helsinki and Good Clinical Practice including monitoring of data, and all volunteers 87
gave written informed consent. 88
89
Clinical trial design 90
Samples for immunological analysis were obtained from two CHMI trials (23, 24). 91
The first trial performed at Radboud university medical center, Nijmegen, The Netherlands was 92
composed of 18 healthy Dutch subjects aged between 19-30 years with no history of malaria. 93
Any volunteer who was positive for Pf serology or had resided in a malaria endemic area within 94
previous six months was excluded from the trial. Three groups (n=6 per group) were infected by 95
intradermal injections of 2,500, 10,000 or 25,000 cryopreserved PfSPZ (NF54 strain). By day 21, 96
15/18 volunteers had developed parasites detectable by positive blood thick smear (TS), 5/6 in 97
each group (23). There were no differences in parasite densities at diagnosis between the three 98
dose groups (23). For immunological analysis, nine Pf positive volunteers of the 10,000 (n=4) and 99
25,000 (n=5) PfSPZ dose groups were selected based on availability of plasma and peripheral 100
blood mononuclear cells (PBMCs). 101
The second trial was carried out in Bagamoyo, Tanzania with volunteers residing in Dar es 102
Salaam (a malaria hypoendemic area). Twenty-four males between 20-35 years were enrolled 103
and confirmed to be free of parasites by real-time quantitative PCR (qPCR). Subjects with a self-104
reported history of clinical malaria in the previous five years were excluded. The volunteers were 105
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divided into two groups with 12 volunteers per group and infected by intradermal injections of 106
either 10,000 or 25,000 PfSPZ (NF54 strain). 21/24 became both qPCR and blood smear positive 107
by day 21 after infection (24). The three Pf negative volunteers were excluded from analysis in 108
the present study. 109
PBMCs, citrate anti-coagulated plasma samples from Dutch volunteers and serum samples from 110
Tanzanian volunteers were collected and cryopreserved one day before challenge (pre-CHMI) 111
and after treatment (post-CHMI, day 35 and 28 after infection, respectively). 112
113
DNA extraction and qPCR analysis 114
5µl Zap-Oglobin II Lytic Reagent (Beckman Coulter) was added to 500µl of EDTA blood, after 115
which the samples were mixed and stored at -80°C. 116
DNA extraction and quantification of parasitemia by qPCR in the Dutch CHMI trial was performed 117
in Nijmegen as described previously (25), with slight modifications. Briefly, after thawing, samples 118
were spiked with murine white blood cells as an extraction control and DNA was extracted with a 119
MagnaPure LC isolation station. For detection of the extraction control and Pf, primers for the 120
murine albumin gene and Pf 18sRNA were used as described previously (25). Additionally, the Pf 121
18sRNA TaqMan MGB probe AAC AAT TGG AGG GCA AG-FAM was used. 122
DNA extraction and qPCR in the Tanzanian trial was carried out at the Leiden University Medical 123
Center, Leiden, The Netherlands as described previously (26). Phocin herpes virus-1 (PhHV-1) 124
was added to the isolation lysis buffer to serve as an internal control. For quantification of PhHV 125
the primers GGGCGAATCACAGATTGAATC, GCGGTTCCAAACGTACCAA and the probe Cy5-126
TTTTTATGTGTCCGCCACCATCTGGATC were used. 127
Pf (NF54 strain) standard curves for both qPCR assays were prepared in Nijmegen by titration of 128
ring-stage infected red blood cells (RBC) in uninfected human blood. The two qPCR assays in 129
both sites were confirmed to yield the same results when quantifying the Pf content in sequential 130
samples from four CHMI volunteers. 131
132
Parasite material for immunological analysis 133
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The Pf NF54 strain used in both CHMI trials is the parental strain of the 3D7 clone (27). Pf (NF54 134
strain) blood-stage parasites were cultured in RPMI-1640 containing 10% human A+ serum and a 135
5% hematocrit erythrocyte suspension in a semi-automated culture system and regularly 136
screened for mycoplasma contamination. For in vitro stimulation assays, asynchronous parasites 137
harvested at a parasitemia of approximately 10-20% were purified by centrifugation on a 63% 138
Percoll density gradient to obtain mature asexual stages. This resulted in concentration of 139
parasitaemia levels to about 80-90% consisting of more than 95% schizonts/mature trophozoites. 140
Cryopreserved Pf infected RBC (PfRBC) were washed twice in RPMI and used in stimulation 141
assays. Mock-cultured uninfected erythrocytes (uRBC) were obtained similarly and served as 142
control. 143
Pf lysate for ELISA was prepared by extracting purified schizonts/mature trophozoites with 1% 144
sodium desoxycholate and 2.5µl phenylmethanesulfonyl fluoride protease inhibitor for 15 min at 145
room temperature (RT). 146
147
Recombinant and synthetic proteins 148
Recombinant proteins of CircumSporozoite Protein (CSP) and liver stage antigen (LSA)-1 were 149
used to probe humoral responses towards pre-erythrocytic stages, while crude Pf lysate were 150
used to assess antibody reactivity towards blood stages. Apical Membrane protein (AMA)-1 and 151
Exported protein (EXP)-1 are expressed in both pre-erythrocytic and asexual stages. 152
Full length P. falciparum NF54 CSP with repeats was produced in Escherichia coli by Gennova 153
Biopharmaceuticals Ltd, Pune, India. A recombinant LSA-1 construct, LSA-NRC, was expressed 154
in Escherichia coli, incorporating the N- and C-terminal regions of the protein and two of the 155
centrally placed 17-amino-acid repeats for the 3D7 LSA-1 sequence (PlasmoDB-156
PF3D7_1036400) (28). Both N- and C-terminal as well as the repeats are highly conserved 157
between NF54 and 3D7. The major difference is the greater number of repeats in the NF54 158
sequence (29), which are the primary target of anti-LSA-1 antibodies (30). Amino acids 25–545 of 159
codon-optimised AMA-1 of the Pf FVO-strain, were expressed in the methylotrophic yeast Pichia 160
pastoris (31, 32). A peptide covering the C-terminal amino acids 73-162 of the integral 161
parasitophorous vacuolar membrane protein EXP-1 (SWISS-PROT database primary accession 162
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number P04926) was chemically synthesized using solid phase F-moc chemistry, and differs only 163
by a single amino acid in position 160 from the 3D7 sequence (33). 164
165
ELISA to assess antibody reactivity 166
96-well Polystyrene flat-bottom plates (NUNC™ Maxisorp, Thermo Scientific) were coated with 167
2µg/ml of CSP, EXP-1 and AMA-1, 0.25µg/ml of LSA-1 or Pf lysate at the equivalent of 20,000 168
PfRBC/well in phosphate-buffered saline (PBS) and incubated overnight at 4ºC. Plates were 169
blocked with 5% milk in PBS. All following washing steps were carried out with PBS+0.05% 170
Tween (PBST). Using 1% milk in PBST, plasma or serum samples were serially diluted in 171
duplicate starting at 1:50 to 1:800 for protein antigen and 1:250 to 1:4000 for Pf lysate and 172
incubate for 3h at room temperature. Bound IgG was detected using horseradish peroxidase 173
(HRP) conjugated anti-human IgG) (Thermo Scientific, diluted 1:60000 in sample buffer). Plates 174
were developed using TMB peroxidase substrate (tebu-bio). The reaction was stopped using an 175
equal volume of 0.2M H2SO4 and absorbance measured with a spectro-photometer plate reader 176
at 450nm (Anthos 2001 ELISA plate reader). 177
A serial dilution of a pool of sera from 100 HyperImmune Tanzanian (HIT) (20) individuals living in 178
a highly malaria endemic area was used as a reference standard and was included on each 179
plate. Reactivity for each antigen in undiluted HIT serum was defined as 100 arbitrary units (AU). 180
OD values were converted into AUs by four-parameter logistic curve fit using Auditable Data 181
Analysis and Management System for ELISA (ADAMSEL-v1.1, 182
http://www.malariaresearch.eu/content/software). 183
For each antigen, all time points of an individual volunteer were assayed on the same plate. To 184
determine whether Tanzanians had a positive Pf serology (by recognition of Pf lysate), the mean 185
baseline antibody titer against Pf lysate plus 2SD of the Dutch volunteers was used as the cut-off 186
for positivity. 187
188
In vitro PBMC stimulation assay to assess cellular responses 189
Venous whole blood was collected into citrated vacutainer CPT-cell preparation tubes (Becton 190
and Dickinson). PBMCs were obtained by density gradient centrifugation, washed three times in 191
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cold PBS, counted and frozen at 107 cells/ml in fetal calf serum (FCS) with 10% dimethylsulfoxide 192
and stored in vapour-phase nitrogen. After thawing, PBMC were counted and cultured at a 193
concentration of 500,000 cells/well in a 96-well round-bottom plate and stimulated in duplicate at 194
a ratio of 1:2 with 106 Pf NF54-infected RBC or uRBC for either 24 hours or for 6 days in a total 195
volume of 200µl. 196
197
Flow cytometry 198
Cells were stained and analyzed by flow cytometry either directly ex vivo, or after 24 hours or 6 199
days of in vitro stimulation. Cells were stained first for viability with Live/Dead fixable Aqua dead 200
cell stain (Invitrogen) or Fixable viability dye eFluor 780 (eBioscience) and later with three 201
different staining panels for surface markers; For the 24hr stain CD3 PerCP (UCH1, Biolegend), 202
CD56-PE (HCD56, Biolegend), anti-TCR Pan γ/δ-PE (IMMU510, Beckman Coulter), CD4 Pacific 203
Blue (OKT4, Beckman Coulter), CD8 APC-H7 (SK1, BD Pharmigen), CD45RO ECD (UCH1, 204
Beckman Coulter), and CD62L PeCy7 (DREG56, eBioscience). For the ex vivo stain the only 205
changes from previous stain were CD3 V500 (clone SP342, BD Horizon) and CD8 PerCP (RPA-206
T8, Biolegend), and for the third panel 6 days stain CD3 PeCy7 (OKT3, Biolegend) and CD8 207
PerCP (RPA-T8, Biolegend). For intracellular staining, cells were incubated with different mAbs 208
depending on the staining panels. After 30 min of incubation at RT, cells were washed and 209
permeabilized with Foxp3 fix/perm buffer (eBioscience) for 30 min on ice and stained in 210
permeabilization buffer (eBioscience) with IFNγ FITC (4S.B3, eBioscience; 24hr stain), Foxp3 211
eF660 (PCH101, eBioscience; ex vivo stain) or Ki67 FITC (B56, BD Pharmigen) and Foxp3 212
eF660 (PCH101, eBioscience; 6 days stain). Cells were collected on a CyAn ADP 9-color flow 213
cytometer (DAKO/Beckman Coulter) and analyzed using FlowJo software (Tree Star, Inc.) 214
version 9.6. All assays were conducted with the same batches of PfRBC and uRBC, with all time 215
points of one volunteer assayed in one experiment to prevent inter assay variations. Natural killer 216
T-cells (NKT) and gamma delta T-cells (γδT) were analyzed in the same gate and henceforth are 217
referred to as NKT-γδT. 218
219
Statistical analysis 220
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Statistical analysis was performed in GraphPad Prism 5. Differences within the cohorts and 221
between time points were analyzed per volunteer by Wilcoxon matched-pairs signed rank test 222
and those between groups were analyzed by Mann Whitney U-test. Relationships between 223
baseline and increase in antibody titers were analyzed by Spearman correlation, P values <0.05 224
were considered statistically significant. Cellular responses were corrected for background by 225
subtracting responses to uRBC from responses to PfRBC for each sample; resulting negative 226
values were set to zero. 227
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Results 229
Tanzanian volunteers have higher baseline antibody titers than Dutch subjects 230
Pre-CHMI antibody titers were significantly higher in Tanzanian than in the malaria-naïve Dutch 231
volunteers for crude Pf lysate (p<0.03), with 12/21 Tanzanians having titers higher than the mean 232
+ 2SD of Dutch volunteers. This was also true for pre-CHMI antibodies to the individual parasite 233
antigens AMA-1 (p<0.015; 13/21), and CSP (p<0.04; 15/21), and the same trend was found for 234
EXP-1 (p<0.06; 8/21) (Figure 1A). Elevated LSA-1 antibody titers were found in 5/21 235
Tanzanians, but there was no significant difference between Dutch and Tanzanians on group 236
level (p<0.16). Within the Tanzania cohort, there was a wide range of pre-CHMI antibody 237
responses, with some volunteers showing only low responses comparable to the Dutch cohort. 238
To address whether there was a general division into high and low responders to malaria-239
antigens, we stratified Tanzanian individuals based on their reactivity to Pf lysate (containing a 240
large number of late-liver and blood-stage antigens, Figure 1B). Compared to their Pf lysate 241
sero-negative counterparts (n=9), sero-positive Tanzanians (n=12) had significantly higher 242
antibody titers against the cross-stage antigens AMA-1 (p=0.005, 7.6-fold higher median titer) 243
and EXP-1 (p=0.04, 5.4-fold higher). The same trend was found for the sporozoite antigen CSP 244
(p=0.09, 2.4-fold higher) and the liver-stage antigen LSA-1 (p=0.06, 1.7-fold) (Figure 1C). 245
246
Pf lysate sero-positivity prior to CHMI is associated with reduced initial blood-stage 247
parasitemia 248
We next assessed whether pre-existing humoral responses might be associated with control of 249
parasites in Tanzanian volunteers. In line with stronger humoral responses in the Tanzanian 250
cohort at baseline, Tanzanian volunteers became qPCR positive significantly later than the Dutch 251
volunteers (Figure 2A), with a median pre-patent period of 11.0 days [IQR: 11.0-13.5] in 252
Tanzanians and 10.0 days [9.5-11.0] in Dutch volunteers (p=0.009). Similarly, pre-patency by TS 253
was also longer in Tanzanians (median with IQR: 13.7 days [12.75-16.7]) compared to the Dutch 254
(12.6 [12.3-14]) (p=0.035). The time between detection by qPCR and TS was comparable 255
between both cohorts (median with IQR: Tanzanians 2.6 days [2.2-3.15] vs Dutch 3.0 [2.0-3.15], 256
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respectively; p=0.88), but Dutch volunteers had a significantly higher peak parasite density 257
(parasites/ml, median with IQR: Tanzanians 12,000 [6,800-15,000] vs Dutch 74,000 [26,000-258
190,000], respectively; p=0.02), possibly due to slight differences in the thick smear protocol 259
between the two sites. Within the Tanzanian cohort, there was no significant difference in time to 260
qPCR-detectable parasitemia between volunteers that were either Pf lysate sero-positive or -261
negative at baseline (p=0.16; Figure 2B), nor was there a difference in pre-patency by TS 262
(p=0.41), or time between detection by qPCR and TS (p=0.15). However, sero-positive 263
Tanzanian volunteers had a significantly lower parasite load at the time of first qPCR-detectable 264
parasitemia than their sero-negative counterparts (p=0.033; Figure 2C). This difference remained 265
evident, but became smaller, by the time when the first peak in parasite load was reached 266
(p=0.05; Figure 2D). Across all Tanzanian volunteers, pre-CHMI antibody titers for CSP (Pearson 267
r=0.45, p=0.04), but no other antigens, correlated significantly with prepatancy by qPCR (Figure 268
S1). 269
270
Pf-specific antibody responses are more efficiently increased in Tanzanians after CHMI 271
Post-CHMI, antibody responses increased significantly in the Tanzanian volunteers for Pf lysate 272
(p<0.001; 15/21 with >3-fold increase in titers), AMA-1 (p=0.002, 6/21), EXP-1 (p<0.0001, 14/21), 273
LSA-1 (p=0.001, 5/21) and CSP (p<0.001, 11/21; Figure 3A). In contrast, the Dutch volunteers 274
showed significant increased titers only against EXP-1 (p=0.004, 7/9), CSP (p=0.008, 6/9) and Pf 275
lysate (3/9) (p=0.008), while responses to LSA-1 and AMA-1 remained low and unaltered (Figure 276
3B). Compared to the Dutch, Tanzanians showed a trend for stronger induction or boosting of 277
responses based on overall fold increase in titers to Pf lysate, AMA-1 and LSA-1 (Figure 3C), 278
and significant higher absolute increases in titers for these antigens (Figure 3D). Volunteers in 279
both cohorts were subjected to CHMI with either 10,000 or 25,000 PfSPZ. However, the only 280
significant differences in antibody responses between the two dose groups was a slightly higher 281
response in the 25,000 versus 10,000 dose group for Pf lysate in Dutch volunteers (p=0.04), and 282
a similar trend for the Tanzanians for CSP (p=0.07; Figure S2). Post-CHMI, Pf lysate sero-283
negative Tanzanians had a significantly stronger fold increase in antibody titers against AMA-1 284
(p=0.02) and EXP-1 (p=0.04) than sero-positive individuals, with a similar trend for Pf lysate 285
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(p=0.082) and CSP (p=0.095), but no difference in LSA-1 responses (p=0.80; Figure 3E). For the 286
entire cohort, the fold increase of Pf lysate (p=0.01) and AMA-1 (p=0.001) responses upon CHMI 287
correlated negatively with the baseline response. The absolute increase in titers, however, was 288
not different for any of the antigens between the two groups (Figure 3F). Notably, Pf lysate sero-289
negative Tanzanians showed thereby also a greater absolute increase in antibody titers than 290
malaria-naïve Dutch volunteers. 291
292
Dutch, but not Tanzanian volunteers show cellular re-call responses induced by CHMI 293
To analyze the acquisition of cellular responses after CHMI, we investigated in vitro IFNγ-294
responses upon incubation with PfRBC for 24 hours (Figure 4A, B). At baseline, Pf-specific 295
IFNγ-responses was comparable between Dutch and Tanzanians for all cell subsets except for 296
NKT-γδT cells, which showed slightly higher responses in the Dutch (p=0.077; Figure 4C). Post-297
CHMI, previously malaria-naïve Dutch volunteers showed significant increases in Pf-specific IFNγ 298
production by all lymphocyte subsets analyzed (CD4+ p=0.004; CD8+ p=0.009; CD56dim 299
p=0.004; CD56bright NK p=0.008; NKT-γδT p=0.004; Figure 4E-I). In contrast, Tanzanian 300
volunteers showed little or no increase in IFNγ-producing cells (CD4+ p=0.74; CD8+ p=0.065; 301
CD56dim p=0.49; CD56bright NK p=0.75; NKT-γδT p=0.92). In both cohorts and at both time 302
points, CD56bright NK cells showed significantly higher IFNγ-responses than CD56dim NK cells 303
(Dutch pre/post-CHMI p=0.004; Tanzanian pre/post-CHMI p≤0.0001). Significantly increased 304
IFNγ-responses were found both in both the effector (CD4+ p=0.004, CD8+ p=0.008) and central 305
memory T-cell compartment (CD4+ p=0.004, CD8+ p=0.012) in the Dutch cohort, while the 306
Tanzanians showed no such increase (Figure S3). As a result, post-CHMI IFNγ-responses in the 307
Dutch were significantly higher for all cell subsets than in the Tanzanian cohort (Figure 4D). 308
Within the Tanzanian cohort, there was an overall trend for higher Pf-specific pre-CHMI IFNγ-309
responses in those volunteers that had particularly short pre-patency by qPCR (Figure S1). 310
CD8+ T-cells from Dutch (p=0.04), but not Tanzanian volunteers (p=0.47), showed increased 311
proliferative responses post-CHMI (Figure S4A). Proliferation of CD4+ T-cells in response to 312
PfRBC was not significantly altered post-CHMI in either cohort (p=0.25 in Dutch and p=0.11 in 313
Tanzanians, respectively; Figure S4B). 314
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Analyzing the lymphocyte subset composition in both cohorts, we found that those were largely 315
stable between pre- and post-CHMI time points, with the exception of a prominent and significant 316
post-CHMI increase of NKT-γδT proportion in the Dutch volunteers and a clear decrease of 317
CD56dim NK proportions in both cohorts. There was further no significant difference in the 318
proportion of CD4+CD45RO+Foxp3+ T-cells between the two cohorts, which could have 319
explained differences in responsiveness (Table S1). For the Tanzanian cohort, we observed a 320
significant reduction of Foxp3+ T-cells post-CHMI (p=0.008), while for the Dutch volunteers there 321
was no change (p=0.44). Stimulation with the mitogens PMA/ionomycin showed that lymphocytes 322
of both Dutch and Tanzanian volunteers were fully functional and able to produce IFNγ at 323
comparable levels both pre- and post-CHMI (Figure S4C). 324
325
Reduced innate IFNγ responses in Tanzanian volunteers are associated with pre-existing 326
humoral immunity and thus potential pre-exposure 327
Finally, we analyzed whether the degree of pre-exposure reflected by differences in malaria-328
specific humoral immunity might affect cellular re-call responses to PfRBCs. Cells from Pf lysate 329
sero-negative Tanzanian volunteers had significantly higher responses to PfRBC (pre-CHMI) 330
than those from sero-positive Tanzanian volunteers (NKT-γδT p=0.05; CD56dim p=0.01 and 331
CD56bright NK p=0.04;), and were of comparable magnitude as those observed in the Dutch 332
volunteers (Figure 5). CD4+ and CD8+ T-cells showed a similar pattern, although the difference 333
between sero-positive and -negative Tanzanians did not reach statistical significance (Figure 334
5A,B). Post-CHMI, these differences remained, and there was no increase in these responses in 335
either of the two groups. 336
337
338
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Discussion 339
In the present study we performed a side-by-side comparison of anti-malarial immune responses 340
initiated or re-called by CHMI in two cohorts of young adults with different malaria exposure 341
histories and genetic background, in the Netherlands and Tanzania. Recruitment of Tanzanian 342
volunteers took place in the hypo-endemic urban area of Dar es Salaam. Inclusion criteria into 343
this CHMI trial were tailored to ensure minimal recent exposure to Pf: Tanzanian volunteers had 344
not experienced a clinical episode of malaria for five years, as self-reported, and were confirmed 345
free of parasites by qPCR before CHMI took place. Nevertheless, more than 50% of the 21 346
Tanzanian volunteers had a positive Pf lysate serology before CHMI based on Pf lysate ELISA, 347
which is a standard exclusion criterion in Dutch CHMI trials. In line with this, increased baseline 348
antibody titers for the Pf antigens CSP, LSA-1, EXP-1 and AMA-1 were found in the Tanzania 349
cohort and particularly in those with positive serology for Pf lysate. This clearly indicates previous 350
exposure to the malaria parasite in this cohort. Asymptomatic infections, which can often occur in 351
people living in low endemic areas (34), also in the 5 years of no self-reported clinical malaria 352
preceding CHMI, might have led to a maintenance of higher antibody responses. 353
354
As might be expected, pre-existing antibodies to Pf antigens appeared to have an effect on the 355
outcome of CHMI. Tanzanians had a significantly longer pre-patency based on qPCR detection 356
than the previously malaria-naïve Dutch. Furthermore, Pf lysate sero-positive Tanzanians had a 357
lower parasite load at time of first detection by qPCR and first peak of parasite load. The first 358
peak of blood-stage parasitemia can be used as a proxy for parasite liver-load (16, 35). Our 359
results might therefore indicate emergence of fewer parasites from the liver and hence better 360
control of either initiation or progression of liver-stage infection in the Tanzanian cohort. In line 361
with such an effect of pre-existing anti-sporozoite immunity would be the observation that those 362
Tanzanian volunteers with a longer prepatancy by qPCR also had higher baseline antibody titers 363
against the sporozoite antigen CSP. However, first detection by qPCR in both the Dutch and 364
Tanzanians after intradermal PfSPZ injection was uncharacteristically late compared to infection 365
by mosquito bite routinely conducted in the Netherlands and elsewhere (11, 16, 18, 19, 23, 24, 366
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35). This is likely due to less efficient liver-stage infection by this route, and hence a lower initial 367
blood-stage load that only reaches the qPCR-detection limit later. A lower first detectable 368
parasitemia in sero-positive Tanzanians might therefore additionally be attributed to control of 369
blood-stage replication prior to qPCR-detection. Antibodies directed against AMA-1, for instance, 370
are known to interfere with blood-stage multiplication (36), but can also confer protection against 371
liver-stage infection (37). Since recognition of both cross-stage and liver-stage antigens was 372
stronger in the Tanzanian cohort, and particularly the sero-positive individuals, both possibilities 373
remain open. Our data would, however, only support antibody control of blood-stage replication 374
during the initial phase of sub-qPCR detectable parasitemia: While parasite multiplication based 375
on PCR data could not be directly compared due to the high variability of the amplification 376
dynamics (24), there was no difference between pre-patency by TS or qPCR between the two 377
Tanzanian groups. 378
379
Upon CHMI, Dutch volunteers showed a slight but significant induction of humoral responses 380
against CSP, Pf lysate and EXP-1, while Tanzanians boosted responses to all antigens 381
examined. Reactivity to the sporozoite antigen CSP is expected after PfSPZ inoculation, and 382
consistent with previous findings (38, 39). Increased antibody responses against Pf lysate and 383
EXP-1 likely reflect cross-stage reactivity between late liver-stage and blood-stage merozoites, 384
while exposure to developing liver-stages (LSA-1) in naïve volunteers appears insufficient to 385
induce a detectable antibody response. Similarly, limited exposure to blood-stage expressed 386
AMA-1, due to early curative treatment, appears to prevent induction of detectable titers in naïve 387
volunteers, which is consistent with results even after multiple CHMIs (39). Consistent with pre-388
exposure, Tanzanians showed on group level a greater increase in titers for Pf lysate, AMA-1 and 389
LSA-1 compared to the previously malaria-naive Dutch. Within the Tanzanian cohort, Pf lysate 390
sero-negative Tanzanians showed a similar absolute and accordingly much greater fold increase 391
in antibody titers compared to their sero-positive counterparts. At baseline, these volunteers had 392
significantly lower responses to most parasite antigens and largely resembled the malaria-naïve 393
Dutch cohort. The fact that this group showed a greater increase in antibody titers than the Dutch 394
strongly suggests that despite largely negative Pf serology, these individuals have a stable Pf-395
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specific memory B-cell repertoire (40, 41). Given the same increase in absolute antibody titers, 396
their memory B-cell repertoire appears to be of a similar magnitude as that of the Pf lysate sero-397
positive Tanzanians, despite lower circulating plasma antibody levels. 398
399
Of note, humoral responses were assessed using a number of recombinant or synthetic proteins, 400
which are not fully identical to the sequences of these proteins expressed by the NF54 strain 401
used in both CHMI trials. While most antigens used in this study were of 3D7 sequence and thus 402
closely resemble the NF54 sequence, AMA-1 responses were assessed using the FVO strain 403
sequence. AMA-1 is known to for its extensive antigenic polymorphism and to cause strain-404
specific immunity (42, 43). Nevertheless, there is a significant antigenic overlap between AMA-1 405
alleles allowing for cross-reactivity across different strains, both in terms of functional activity and 406
recognition, which mainly affects the magnitude of the response (42, 44-46). Based on these 407
data, we consider it unlikely that assessment of AMA-1 responses using the NF54 or 3D7 408
sequence would have yielded different qualitative results. However, absolute titers would likely 409
have been higher when using the AMA-1 sequence homologous to the CHMI strain. 410
Another potential confounder is the fact that the Pf strains, which Tanzanian volunteers were 411
exposed to prior to CHMI with Pf NF54, are naturally unknown. As any study examining pre-412
exposed individuals, analysis of antigen-specific responses against polymorphic antigens has 413
therefore the additional limitation that it is difficult to match this unknown exposure history. This 414
has to especially be taken into account when investigating CHMI-induced boosting of potential 415
pre-existing responses in this cohort, which might be masked by using antigens of different strain 416
origin. However, despite the fact that Tanzanians likely experienced exposure to a variety of Pf 417
strains prior to CHMI, there was (i) a clear division into sero-positive and negative individuals not 418
just by total Pf NF54 lysate, but also reflected by all individual antigens analyzed and (ii) a clearly 419
stronger increase in antibody titers in Tanzanians compared to malaria-naïve Dutch volunteers to 420
several antigens including AMA-1. We cannot exclude, however, that this might have even been 421
more pronounced when using antigens of different strain origin for analysis. 422
423
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Although the Tanzanian volunteers in the present study showed evidence for humoral immune 424
memory, parasite-specific IFNγ-production by adaptive T-cell subsets was not higher than in 425
malaria-naïve Dutch volunteers at baseline and remained unchanged after CHMI. In contrast, 426
previously naïve Dutch volunteers showed a significant increase in IFNγ-production by CD4 and 427
CD8 T-cell subsets after a primary infection, consistent with what has been shown previously (47, 428
48). The lack of increased proliferative and Th1 responses one month after CHMI in Tanzanian 429
volunteers could be partially due to immunosuppression following exposure to blood-stage 430
parasites during CHMI. Such T-cell immunosuppression is well described for malaria (49-58), and 431
although usually resolved within two weeks (52, 53) can persist for more than 4 weeks (50). That 432
Dutch volunteers are not equally affected by this might be due to the fact that such 433
immunosuppressive effects appear to be more pronounced in immune than non-immune donors, 434
as shown elsewhere (59). One potential mediator of suppressed IFNγ-production by adaptive 435
cells are T regulatory cells (Tregs). Increased Treg numbers during or after malaria are a well 436
reported phenomenon (60), and malaria parasites can enhance the suppressive activity of Tregs 437
(61). While Dutch and Tanzanian volunteers had similar Treg proportions, we cannot exclude that 438
Tregs in Tanzanian volunteers might be functionally more active, potentially due to past priming 439
in malaria infections. That this apparent lack of a Th1 immune response in the Tanzanian cohort 440
is malaria-specific is supported by the fact Dutch and Tanzanian volunteers showed similar Th1 441
responses to mitogen stimulation both pre- and post-CHMI. Nevertheless, an additional influence 442
of genetic background, which may explain differential Pf-specific responses as described in 443
endemic settings, cannot be excluded (62, 63). 444
445
NK cells are rapidly activated by malaria parasites, contribute to the early IFNγ-response during 446
blood-stage infection (64) and can eliminate infected erythrocytes in vivo in a contact-dependent 447
manner (65). Importantly, there seems is a functional dichotomy: the rarer CD56bright cells are 448
more prominent in lymphatic tissues and are superior cytokine producers, while the CD56dim 449
subset harbors a stronger cytotoxic potential (66-68). However, NK cells have usually been 450
examined as one population and the exact contribution of CD56dim and CD56bright NK cell 451
subsets to malaria immunity thus remains to be established. An in vitro study on PBMCs from 452
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malaria-naïve donors found greater IFNγ-production by CD56bright compared to CD56dim NK 453
cells only in response to cytokines, but not upon PfRBC stimulation (69). However, consistent 454
with their generally reported greater cytotoxic potential, only CD56dim cells showed de-455
granulation upon PfRBC stimulation (69). Our findings that CD56bright NK cells show a greater 456
IFNγ-response than CD56dim NK cells to PfRBC both before and after a primary malaria 457
infection, as well as a memory-like effect of this response is in line with findings from a previous 458
CHMI trial (70). Tanzanian volunteers showed the same functional difference between the two 459
NK cell subsets, but no memory-effect in either subset after CHMI. It was previously shown that 460
depletion of αβ T-cells abrogates memory-like IFNγ responses of innate cells otherwise observed 461
after CHMI (47, 70). Therefore, the absence of increased PfRBC-specific αβ T-cell responses 462
post-CHMI in Tanzania volunteers might be one reason why Pf-specific IFNγ-production by NK 463
cells and other innate lymphocytes (NKT and γδ T-cells) was also not increased post-CHMI. The 464
fact that Pf lysate sero-negative Tanzanians had higher innate IFNγ-responses already at 465
baseline is a further indication that reduced Th1 responses are likely linked to the degree of 466
previous malaria-exposure. Of note, the proportion of CD56bright cells remained unaltered after 467
CHMI, while the CD56dim subset had a smaller contribution to the PBMC compartment post-468
CHMI in both cohorts. Whether this means that in contrast to memory-like IFNγ-production, other 469
NK cell functions such as migration out of the circulation and engagement in anti-malaria immune 470
responses at other sites such as the spleen is unaffected and fully functional in pre-exposed 471
individuals remains to be established. 472
473
Within the Tanzanian cohort, there was no association of pre-existing Pf-specific IFNγ-responses 474
by T-cell subsets or innate lymphocytes with the parasiological outcome of CHMI, i.e. pre-patency 475
or parasite load at first detection by qPCR. If at all, those with higher IFNγ-responses to blood-476
stage PfRBC had a shorter pre-patency. This does not, however, exclude that cellular responses 477
play a role in the prolonged pre-patency of Tanzanian volunteers and specifically those with 478
positive Pf-serology. One possible reason for the lack of such an association is that PfRBC were 479
chosen as a stimulus for Pf-specific responses. This was done due to the relatively large 480
antigenic overlap between blood-stage and (late) liver-stage parasites (71, 72). However, 481
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responses to sporozoite and early liver-stage antigens, which may be more relevant when 482
assessing responses responsible for reducing the parasite load by targeting the earlier stages of 483
infection, are likely to be missed using PfRBC as a stimulus. Moreover, our analysis was 484
restricted to Pf-specific IFNγ-production, and it is likely that other responses, for instance de-485
granulation as a proxy for cytotoxicity may be more relevant readouts (71). Finally, the only 486
accessible compartment for analysis of cellular responses in these human trials was peripheral 487
blood. Pf-specific responses and particularly those responsible for reducing liver-infection, might, 488
however, be enriched or primarily located in other sites, for instance as tissue-resident memory 489
cells in the liver (73, 74). This might be even more pronounced in pre-exposed individuals, where 490
such a tissue-resident memory population might have already been established. 491
492
Noteworthy, the differential immune responses described herein may explain why Tanzanian 493
volunteers reported fewer clinical symptoms such as fever compared to their Dutch counterparts 494
during CHMI after PfSPZ injection (24). On the one hand, pre-existing antibody responses may 495
reduce the initial parasite load and mediate anti-disease immunity. Additionally, the relatively 496
lower Pf-specific cellular Th1 responses observed in the Tanzanian cohort might also be 497
beneficial. Dutch volunteers pre-exposed to infected mosquito-bites under chloroquine 498
prophylaxis exhibited stronger IFNγ-production and earlier clinical symptoms when re-exposed to 499
blood-stage parasites than malaria-naïve volunteers during their first infection (15). Thus, a shift 500
away from Th1 responses in pre-exposed volunteers may make them less vulnerable to fever 501
and other inflammation-induced symptoms. 502
503
In conclusion, our data show that previous malaria exposure is associated with some degree of 504
parasite control during liver or early blood-stage infection after CHMI, humoral immune memory, 505
and reduced anti-parasite Th1 responses in the circulating lymphocyte compartment. Positive Pf 506
lysate serology can be used to identify individuals with better parasite control but weaker 507
peripheral blood Th1 responses, which may help to stratify volunteers in future CHMI trials in 508
endemic areas. However, assessment of memory B-cell responses might be explored as a 509
potentially better tool than serology to define pre-exposure per se. Important questions to be 510
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addressed in future studies include (i) which readouts other than Th1 responses should be used 511
to determine immunization-induced cellular immunity, and (ii) how differences in pre-existing 512
malaria-specific immune responses affect the outcome of whole parasite immunization and 513
vaccination approaches in malaria-endemic areas. 514
515
516
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Acknowledgements 517
We thank all the trial volunteers and the staff from the Radboud university medical center, the 518
Ifakara Health Institute/Bagamoyo Research and Training Centre, Sanaria Inc. and the Swiss 519
Tropical and Public Health Institute, all of whom made this study possible. We thank S. Singh 520
(Gennova Biopharmaceuticals), D. Lanar and G. Corradin, for providing recombinant and 521
synthetic CSP, LSA-1 and EXP-1 proteins, respectively. We thank M. van de Vegte-Bolmer, R. 522
Siebelink-Stoter and W. Graumans for culture and isolation of PfRBCs. 523
524
S.L.H. is Chief Executive and Scientific Officer at Sanaria Inc., B.K.L.S., P.F.B. and E.R.J. are 525
employees of Sanaria Inc. which manufactured and provided PfSPZ Challenge, and do thus have 526
a potential conflict of interest. There are no other conflicts of interest for all other authors. 527
528
This work was supported by Top Institute (TI) Pharma (grant number T4-102),the FP7-founded 529
European Virtual Institute of Malaria Research (EVIMalaR, grant agreement number 242095), the 530
Tanzanian Commission on Science and Technology (COSTECH), the Ifakara Health Institute, 531
and the Swiss Tropical Public Health Institute. A.S. received a long-term post-doctoral fellowship 532
from the European Molecular Biology Organization (EMBO). The development and manufacturing 533
of cryopreserved Pf sporozoites PfSPZ Challenge was further supported by Small Business 534
Innovation Research (SBIR) (grants R44AI058375-03, 04, 05, 05S1) from the National Institute of 535
Allergy and Infectious Diseases at the National Institute of Health (NIAID/NIH), USA and through 536
agreement 07984 from the Program for Appropriate Technology in health (PATH) Malaria 537
Vaccine Initiative (with funds from the Bill and Melinda Gates Foundation). The funders had no 538
role in study design, data collection and analysis, decision to publish, or preparation of the 539
manuscript. 540
541
Author contributions 542
J.M.O., S.S., C.C.H., E.M.B., M.R, C.D.A., S.L.H., S.A., R.S. and A.S. designed the studies and 543
experiments. S.S., E.M.B. and M.R. performed the clinical studies and collected clinical data. 544
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J.M.O., C.C.H., K.T. and A.S. conducted experiments. J.M.O., C.C.H. and A.S. analyzed the 545
data. M.M., P.F.B., B.K.L.S., E.R.J., S.L.H. and A.S. collected/prepared/contributed vital 546
reagents. J.M.O., C.C.H., R.W.S. and A.S. interpreted the data. J.M.O., C.C.H., R.W.S. and A.S. 547
wrote the manuscript. All authors read and approved the final manuscript. 548
549
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Figures 550
Figure 1: Baseline malaria-specific antibody titers indicate previous exposure in Tanzanian 551
volunteers. Antibody reactivity against crude Pf lysate and the Pf antigens AMA-1, EXP-1, LSA-1 552
and CSP was tested prior to malaria infection. A pool of sera from 100 hyperimmune Tanzanians 553
(HIT) was used as a reference. Reactivity for each antigen in undiluted HIT serum was set at 100 554
arbitrary units (AU). (A) Responses between Dutch (D; circles; n=9) and Tanzanian (T; squares; 555
n=21) cohorts were compared using Mann-Whitney U-test. (B) Tanzanian volunteers were 556
stratified based on Pf lysate recognition at baseline as Sero+ (n=12; black box plots) or Sero- 557
(n=9; white box plots), using the mean +2SD of Dutch volunteers (grey circles) as a cut-off for 558
positivity. (C) Pre-CHMI responses of sero+ and sero- Tanzanians were analyzed by ELISA for 559
individual Pf antigens and compared by Mann Whitney U-test. Scatter plots show individual data 560
points, horizontal lines indicate the median of the group and error bars the interquartile range 561
(IQR). Dashed lines indicate the mean + 2SD of antibody titers in Dutch volunteers for each 562
respective antigen. 563
564
Figure 2: Pre-exposure to malaria is associated with difference in pre-patency and 565
parasitemia after CHMI. Parasitemia after CHMI was determined by qPCR. The day of first 566
parasite detection by qPCR after PfSPZ injection is shown for (A) Dutch (D, grey circles; n=9) 567
versus Tanzanians (T, grey squares; n=21) and (B) for Pf sero-positive (n=12; black squares) 568
and sero-negative (n=9; white squares) Tanzanian volunteers. The parasite load (number of Pf 569
parasites per ml blood) at (C) the first day of qPCR-detectable parasitemia and (D) the time of 570
first peak parasite density is shown for Pf sero-positive (n=12; black squares) and sero-negative 571
(n=9; white squares) Tanzanian volunteers. Data are shown as median ± IQR. Groups were 572
compared by Mann Whitney U-test. 573
574
Figure 3: Increased humoral responses in Dutch and Tanzanians after CHMI. Antibody titers 575
were measured (A) four weeks (Tanzanians; squares, n=21) and (B) five weeks (Dutch; circles, 576
n=9) after intradermal infection with PfSPZ. Graphs show the antibody reactivity (AU) pre-CHMI 577
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(black) versus post-CHMI (white) against crude Pf lysate, AMA-1, EXP-1, LSA-1 and CSP. Plots 578
show individual data points, with lines connecting the two time points for each volunteer. 579
Responses in the two cohorts pre-versus post-CHMI were compared using Wilcoxon matched-580
pairs signed rank test. The (C, E) fold increase (ratio AU post-CHMI/AU pre-CHMI) and (D, F) 581
absolute increase in antibody reactivity ( AU post-CHMI minus AU pre-CHMI) was calculated for 582
(C, D) Dutch versus Tanzanian volunteers, and (E, F) in Tanzanian volunteers classified as Pf 583
lysate sero-positive or sero-negative at baseline. Data are shown as whisker plots with boxes 584
indicating the median and IQR, and whiskers the min/max values. Responses were compared 585
using Mann-Whitney U-test. 586
587
Figure 4: Dutch, but not Tanzanian volunteers show increased Pf-specific in vitro IFNγ-588
production after CHMI. PBMCs collected pre- and post-CHMI from Dutch (D; circles; n=9; 589
post=35 days after CHMI) and Tanzanian (T; squares; n=21; post=28 days after CHMI) 590
volunteers were stimulated with PfRBC for 24h. After stimulation cells were stained for (A) 591
surface expression of CD3, CD4, γδTCR, CD8 and CD56 to gate lymphocyte subsets. NKT and 592
γδT were gated as a combined population. (B) Intracellular IFNγ is shown for total lymphocytes 593
after 24h uRBC or PfRBC stimulation. Graphs show the proportions of cells with Pf-specific IFNγ-594
production, comparing Dutch and Tanzanian volunteers (C) pre- and (D) post-CHMI, and 595
comparing pre- and post-CHMI responses within each cohort for (E) CD4+ T-cells, (F) CD8+ T-596
cells, (G) CD56dim NK cells, (H) CD56bright NK cells and (I) NKT-γδT-cells. For each volunteer 597
all time points were measured in a single experiment. Data are shown as (C, D) whisker plots 598
with boxes indicating the median with IQR and whiskers the min/max values or (E-I) individual 599
data points with lines connecting the two time points for each volunteer. All responses were 600
corrected for background by subtracting responses to uRBC. Responses pre- versus post-CHMI 601
were compared using Wilcoxon matched-pairs signed rank test. 602
603
Figure 5: Pf-specific in vitro IFNγ-production by innate lymphocytes from Tanzanian 604
volunteers is dependent on baseline serological status. PBMC collected from Tanzanian 605
volunteers who either had a positive Pf (Sero+; n=12; black squares) or negative Pf serology 606
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(Sero-; n=9; white squares) prior to challenge (baseline) were stimulated with PfRBC for 24h and 607
stained for intracellular IFNγ. Panels show IFNγ-production by lymphocyte subsets; (A) CD4+ T-608
cells, (B) CD8+ T-cells, (C) NKT-γδT-cells, (D) CD56dim and (E) CD56bright NK cells. Data are 609
shown as median ± IQR of responses corrected for background by subtracting responses to 610
uRBC. Differences in responses between the sero+ and sero-groups were analyzed by Mann 611
Whitney U-test. Pf-specific IFNγ-responses of pre-CHMI PBMCs from malaria-naïve Dutch 612
volunteers (D; n=9; grey circles), who had negative Pf serology, are shown as a comparator. 613
614
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