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Edited by:
Adrian John Frederick Luty,

Institut de Recherche Pour le
Développement (IRD), France

Reviewed by:
Jo-Anne Chan,

Burnet Institute, Australia
Alister Craig,

Liverpool School of Tropical Medicine,
United Kingdom

*Correspondence:
Samson M. Kinyanjui

skmuchina@kemri-wellcome.org

†These authors have contributed
equally to this work

Specialty section:
This article was submitted to

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Frontiers in Immunology

Received: 12 March 2022
Accepted: 27 April 2022
Published: 30 May 2022

Citation:
Kimingi HW, Kinyua AW,

Achieng NA, Wambui KM,
Mwangi S, Nguti R, Kivisi CA,

Jensen ATR, Bejon P, Kapulu M,
Abdi AI, Kinyanjui SM and
CHMI-SIKA Study Team

(2022) Breadth of Antibodies to
Plasmodium falciparum Variant
Surface Antigens Is Associated
With Immunity in a Controlled

Human Malaria Infection Study.
Front. Immunol. 13:894770.

doi: 10.3389/fimmu.2022.894770

ORIGINAL RESEARCH
published: 30 May 2022

doi: 10.3389/fimmu.2022.894770
Breadth of Antibodies to
Plasmodium falciparum Variant
Surface Antigens Is Associated
With Immunity in a Controlled
Human Malaria Infection Study
Hannah W. Kimingi1,2†, Ann W. Kinyua1†, Nicole A. Achieng1, Kennedy M. Wambui1,3,
Shaban Mwangi1, Roselyne Nguti 1,2, Cheryl A. Kivisi 2,4, Anja T. R. Jensen5,
Philip Bejon1,6, Melisa C. Kapulu1,6, Abdirahman I. Abdi1,4, Samson M. Kinyanjui1,4,6,7*
and CHMI-SIKA Study Team

1 Kenya Medical Research Institute (KEMRI) Wellcome Trust Research Programme, Kilifi, Kenya, 2 Department of Biological
Sciences, Pwani University, Kilifi, Kenya, 3 School of Public Health, Faculty of Health Sciences, University of the
Witwatersrand, Johannesburg, South Africa, 4 Pwani University Bioscience Research Centre, Pwani University, Kilifi, Kenya,
5 Centre for Medical Parasitology, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences,
University of Copenhagen, Copenhagen, Denmark, 6 Centre for Tropical Medicine and Global Health, Nuffield Department of
Medicine, University Oxford, Oxford, United Kingdom, 7 School of Business Studies, Strathmore University, Nairobi, Kenya

Background: Plasmodium falciparum variant surface antigens (VSAs) contribute to malaria
pathogenesis by mediating cytoadhesion of infected red blood cells to the microvasculature
endothelium. In this study, we investigated the association between anti-VSA antibodies
and clinical outcome in a controlled human malaria infection (CHMI) study.

Method:Weused flow cytometry and ELISA tomeasure levels of IgG antibodies to VSAs of
five heterologous and one homologous P. falciparum parasite isolates, and to two PfEMP1
DBLb domains in blood samples collected a day before the challenge and 14 days after
infection. We also measured the ability of an individual’s plasma to inhibit the interaction
between PfEMP1 and ICAM1 using competition ELISA. We then assessed the association
between the antibody levels, function, and CHMI defined clinical outcome during a 21-day
follow-up period post infection using Cox proportional hazards regression.

Results: Antibody levels to the individual isolate VSAs, or to two ICAM1-binding DBLb
domains of PfEMP1, were not associated with a significantly reduced risk of developing
parasitemia or of meeting treatment criteria after the challenge after adjusting for
exposure. However, anti-VSA antibody breadth (i.e., cumulative response to all the
isolates) was a significant predictor of reduced risk of requiring treatment [HR 0.23
(0.10-0.50) p= 0.0002].

Conclusion: The breadth of IgG antibodies to VSAs, but not to individual isolate VSAs, is
associated with protection in CHMI.

Keywords: malaria, Plasmodium falciparum, CHMI, variant surface antigens, anti-VSA antibodies, antibody
breadth, PfEMP1, ICAM1
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Kimingi et al. Role of Anti-VSA Antibodies in CHMI
INTRODUCTION

The number of malaria cases has declined substantially over the
last few decades (1, 2), but 627 000 attributable deaths globally in
the year 2020 (1) still marks malaria out as a major health
concern especially in children under 5 years of age in sub-
Saharan Africa. The looming threat of mosquito resistance to
insecticides (3) and changes in feeding behavior (4), together
with the emergence of malaria parasite resistance to drugs
including artemisinin based combinations (5), has placed a
strong focus on vaccines as potentially the most effective ways
of eradicating malaria. The most advanced malaria vaccine to
date, RTS,S, confers partial and transient immunity to pre-
erythrocytic malaria parasites (6–8), thus pointing to the need
to develop vaccines that induce more potent and long-lasting
immunity. However, this is hampered by the complexity of the
malaria parasite’s life cycle, the variant and polymorphic nature
of many malaria antigens, and the lack of clear correlates of
immunity to malaria (9–12). Nonetheless, immuno-
epidemiological studies showing that humans acquire
immunity through repeated exposure to malaria (13–16) and
passive antibody transfer experiments (17, 18) provide the
incentive for the continued search for more efficacious malaria
vaccines against blood-stage parasites.

Among the proteins considered potential candidates for
malaria vaccine are the variant surface antigens (VSAs)
displayed on the surface of red blood cells infected by the
mature blood stage of malaria parasites (19–26). P. falciparum
erythrocyte membrane protein 1 (PfEMP1), one of the best
studied VSAs, plays an important role in malaria pathogenesis
by mediating the cytoadhesion and sequestration of infected red
blood cells to the endothelium of host blood vessels as a way of
escaping immune clearance. This adhesion, which is mediated by
host receptors such as ICAM1, EPCR and CD36, results in
vascular occlusion and inflammation, which are hallmarks of
severe malaria (27–31). The wide diversity of VSAs poses a
challenge in their inclusion in malaria vaccines. However, several
longitudinal studies have shown that antibodies to VSAs reduce
the risk of being reinfected by a variant recognized by those
antibodies, but not necessarily to other variants, but there are
also longitudinal studies that suggest that a degree of cross-
variant immunity also exists (24, 25, 32), thus justifying the
continued interest in understanding the role of anti-VSA
antibodies in immunity to malaria.

A complexity in studying immunity to malaria is in
distinguishing between immunological responses that are
simply markers of exposure and those that are causally linked
to immunity, as both increase concurrently with repeated
exposure to malaria. In addition, exposure to infected
mosquito bites in the field is heterogeneous, making it difficult
to distinguish between non-exposure and genuine immunity as
the reason for apparent protection in field-based immunology
studies (33). Controlled human malaria infection study (CHMI)
overcomes these challenges by ensuring homogenous exposure
with subsequent stringent monitoring of parasitemia and
symptoms. We used CHMI studies in Kenya to further explore
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the role of anti-VSA antibodies in immunity to malaria. We
sought to determine whether levels and function of anti-VSA
antibodies were associated with better clinical outcome among
the CHMI participants.
METHODS

Study Design and Population
This study was nested in a larger controlled human malaria
infection (CHMI) study conducted at KEMRI-Wellcome Trust
Research Programme (KWTRP) as described previously (34).
Briefly, healthy adults were recruited for CHMI from Ahero
(high malaria endemicity), Kilifi South (high malaria endemicity)
and Kilifi North (low malaria endemicity) locations in Kenya.
Following initial screening, 161 volunteers were administered
with 3.2×103 NF54 P. falciparum sporozoites and monitored for
parasite growth and clinical outcome for a period of 21 days.
Parasite growth was monitored using qPCR and individuals who
reached a parasite density of 500 parasites/µL or developed fever
were given anti-malarial drugs, while the rest were all treated on
day 22 (34). The blood samples collected from the participants
were processed to separate plasma and cells and stored at -80°C
until use. For this study, we used plasma collected a day before
(C-1) and 14 days after the challenge (C+14).

Parasite Isolates
Six P. falciparum isolates were used for this study, two
laboratory-adapted cultures (SAO75 and A4U) and four
heterologous ex-vivo isolates (6454, 19462, 19477, and NF54).
The ex-vivo parasite isolates (except NF54) were obtained from
patients admitted to the High Dependency Unit at Kilifi County
Hospital with severe malaria and cultured to mature trophozoites
stage before being frozen. The laboratory adapted isolates were
retrieved from liquid nitrogen storage, grown to mature
trophozoite and then frozen. The NF54 parasites isolated from
the study participants and expanded in culture appeared not to
express VSAs as we observed poor antibody recognition by
serum from highly immune positive controls as well as study
participant’s plasma samples (Supplementary Figure 1). This is
in line with previous studies that have shown that continuous
culture decreases var gene transcription and protein expression
(35, 36). We therefore produced an NF54 culture selected on
ICAM1 and CD36 to enhance VSA expression, referred to
hereafter as ‘NF54(ICAM1/CD36)’. All the isolates were
cultured in RPMI 1640 medium (Sigma) supplemented with
2mM L-glutamine, 37.5mM HEPES, 20mM glucose, 50µg/mL
sodium hypoxanthine, 25µg/mL gentamicin and 0.5% albumax
(all from Gibco) (37) and cryopreserved in glycerolyte (38).

Detection of Anti-VSA Antibodies
by Flow Cytometry
The frozen trophozoites were thawed as previously described
(38). A 10% suspension of the thawed infected red blood cells
pellet was prepared using 1XPBS. 2.5µL of the 10% pellet
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Kimingi et al. Role of Anti-VSA Antibodies in CHMI
suspension was added into 8.5µL of 1XPBS/0.5%BSA then
stained with 10µg/µL Ethidium Bromide (EtBr). The
suspension was incubated in a U-bottomed 96 well plate with
1µL of test plasma and malaria naïve control plasma for 30
minutes at room temperature to allow for antigen-antibody
binding. The cells were then washed three times using 1XPBS/
0.5%BSA by centrifugation at 110×g for 3 minutes. 50µL of
fluorescein isothiocyanate (FITC) conjugated sheep anti-human
IgG (Binding Site, UK) was added at 1:50 dilution and this was
incubated for 30 minutes in the dark, then washed 3 times with
1XPBS/0.5%BSA by centrifugation at 100×g for 3 minutes. The
pellet was then suspended in 200µL of 1XPBS. 100µL of the re-
suspended pellet was further diluted with 400µL of 1XPBS in a
FACS tube to bring the pellet volume to 0.05µL and 1000
trophozoite-infected erythrocytes were acquired from each
tube on a FACSCanto flow cytometer (Beckman Coulter, UK).

Gating Strategy
Data obtained from FACSCanto flow cytometer was analyzed
using FlowJo version 10. Ethidium Bromide staining was used to
distinguish between infected and uninfected red blood cells,
while the intensity of the FITC signal was considered a proxy
of the level of anti-VSA IgG antibodies. Background staining of
uninfected red blood cells was corrected by subtracting the
median fluorescent intensity (MdFI) of the uninfected red
blood cells from the MdFI of the infected red blood cells. To
correct for non-specific antibody binding, the background
adjusted MdFI of 13 malaria-naive European plasma was
subtracted from the MdFI test plasma.

Expression of Recombinant
Protein Domains
The ICAM1-binding DBLb domains of PF3D7_0425800
(previous ID PFD1235w) and PF3D7_1150400 (previous ID
PF11_0521) were expressed as HIS-tagged proteins in
Escherichia coli Shuffle C3030 cells (New England BioLabs)
and purified by immobilized metal ion affinity chromatography
using HisTrap HP 1-mL columns (GE Healthcare) as described
previously (28, 39). Recombinant Fc-tagged ICAM-1 was
expressed in HEK293 cells and purified as described
previously (40).

Detection of DBLb Reactive Antibodies
ELISA was used to analyze IgG reactivity to the above PfEMP1
DBLb domains. Briefly, Immunolon plates (Thermo Scientific)
were coated with 2.5µg/mL of the recombinant proteins
PF3D7_1150400 (PF11_0521) and PF3D7_0425800
(PFD1235w) diluted in ELISA carbonate coating buffer
(Thermo Scientific)and incubated overnight at 4°C. The plates
were then washed in PBST (1X PBS with 0.05% Tween 20)
followed by blocking with PBST and 1% skimmed milk for 1h at
room temperature. After washing with PBST, plasma samples
(1:100 in blocking buffer) were added and incubated for 1h at
room temperature. Binding was detected using rabbit anti-
human IgG-HRP (Dako, 1:5000 in blocking buffer). Malaria
naïve plasma samples were used as negative controls.
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Detection of Antibodies to P. falciparum
Schizont Extract
CHMI plasma samples collected a day before the challenge (C-1)
were analyzed for presence of antibodies against P. falciparum
schizont extract using ELISA as previously published (41).
Briefly, P. falciparum 3D7 parasites were cultured to schizont
stage. The harvested culture was sonicated, followed by a series of
freeze-thaw cycles to prepare the schizont extract. Different
dilutions of the schizont extract were coated on ELISA plates
and a pool of plasma from hyper immune individuals was used to
detect antibodies against the extract and determine the optimal
dilution of the extract to use for subsequent assays. Standard
ELISA protocol was followed to measure antibodies against the
schizont extract in the CHMI plasma samples.

Inhibition of Binding of PfEMP1 to ICAM1
To measure the ability of an individual’s plasma to inhibit the
interaction between the recombinant PfEMP1 DBLb domain
(PF3D7_1150400/PF11_0521) and ICAM1, we used competition
ELISA. First, Immunolon plates (Thermo Scientific) were coated
with 5µg/mL of PF3D7_1150400 recombinant protein and
incubated overnight at 4°C. The plates were then washed and
blocked with PBST and 1% skimmed milk for 1h at room
temperature. After washing, ICAM1–Fc (5µg/mL) and plasma
(1:10) were added simultaneously and incubated for 1h at room
temperature (42). The plates were then washed four times with
PBST. Bound ICAM1 was detected by mouse anti-human
ICAM-1 (PE anti-human CD54, cat.no 353106, BioLegend,
1:500) followed by goat anti-mouse IgG-HRP, (Dako, 1:5000).
Plasma samples from malaria naïve individuals from the UK
were used as negative controls.

Statistical Analysis
Data analysis was carried out using R version 4.0.5. For all tests,
P values of <0.05 were considered significant. Graphs were
generated using GraphPad Prism 8. The adjusted MdFI was
log transformed in R and normality of data was checked using
Shapiro test. Cox proportional hazard risk model was used to
assess the association between anti-VSA antibody levels and two
definitions of CHMI outcomes: 1) Time to establishment of a
PCR-detectable infection and 2) time to requiring treatment
during follow up.

To test if CHMI outcome was associated with the breadth of
anti-VSA antibodies across the whole panel of test isolates rather
than responses to individual isolates we developed an antibody
response breadth score for each participant. This was done by
assigning the participant’s response to each isolate a score of 0, 1,
2 or 3 depending on whether the response was in the lowest,
middle, or upper quartile of the responses to that isolate
respectively. The total score across the six isolates (minimum =
0, maximum = 18) was taken as the individual’s anti-VSA
antibodies breadth and was further 3 equal categorized as
“low” (total score 0-6), “medium” (total score 7-12) or “high”
(total score 13-18). The association between the breadth
categories and CHMI outcome was assessed using cox-
proportional hazard model.
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Kimingi et al. Role of Anti-VSA Antibodies in CHMI
Additionally, the association between antibodies to two
PfEMP1 DBLb domains (PF3D7_1150400/PF11_0521 and
PF3D7_0425800/PFD1235w) and CHMI outcome was also
assessed. The antibody-mediated inhibition of ICAM1 binding
of the DBLb domain was analyzed from two independent
experiments carried out in duplicates and the association with
CHMI outcomes assessed using Cox proportional hazard
risk model.

In all cases a univariable and subsequent multivariable
analysis with further stepwise regression was carried out and
the results are reported as “unadjusted” and “adjusted”.

To test if CHMI outcome was associated with changes in
antibody levels between day C-1 and day 14 after challenge,
Mann-Whitney U test was used to assess for significant
difference between the treated and untreated groups as well as
the PCR positive and PCR negative groups.
RESULTS

CHMI Outcome
Of the 161 participants enrolled for CHMI, 19 were excluded
from the final analysis due to infection with P. falciparum isolates
other than NF54 during follow-up or presence of anti-malarial
drug in their plasma as previously described (41, 43). The 142
participants included in the final analysis were grouped as either
untreated PCR negative (n=33), untreated PCR positive (n=53),
treated non-febrile (n=30), or treated febrile (n=26) based on
parasite growth monitored by qPCR or development of fever
during follow-up. Further details of the outcomes are described
elsewhere (43).

Anti-VSA Antibodies Response to a Panel
of Parasite Isolates
All the participants showed varied levels of anti-VSA antibodies
to all the test isolates. The ex-vivo isolates were better recognized
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with highest levels of antibodies being against isolate 19477 and
6454 and the lowest against NF54 (ICAM1/CD36) (Figure 1A).
Participants from Kilifi South and Ahero, which are high malaria
transmission areas, had higher levels of antibodies compared to
those from Kilifi North which is a low malaria transmission
area (Figure 1B).

Anti-VSA Antibodies and Risk of
Developing PCR-Detectable
Infection During CHMI
Antibody levels to each of the parasite isolates except NF54
(ICAM1/CD36), overall antibody breadth, binding inhibition
levels and anti-schizont antibodies were all significantly
associated with reduced risk of the endpoint of a PCR
detectable infection in univariable analysis (Table 1). However,
there was marked collinearity of the different responses with
varying degrees of positive cross-correlations (Supplementary
Figure 2). Antibodies to the specific parasite isolates were not
independently associated with reduced risk of acquiring PCR
detectable infection in a multivariable analysis model that
adjusted for antibodies to schizont extract as indicated
in Table 1.

Anti-VSA Antibodies and Risk of
Meeting a Threshold for Anti-Malarial
Treatment During CHMI
Similarly, univariable analysis showed that antibody levels to
each of the parasite isolates, overall antibody breadth, binding
inhibition levels and anti-schizont antibodies were all associated
with a significant reduction in the risk of meeting a threshold for
malaria treatment during follow-up (Table 2). However, only
breadth of antibody responses and anti-schizont antibodies were
significant predictors of reduced risk of reaching a threshold for
anti-malarial treatment on multivariable regression analysis
(Table 2 and Supplementary Table 1). Further analysis
showed that the untreated group had significantly higher levels
A

B

FIGURE 1 | Anti-VSA antibodies response and malaria endemicity. (A) The median and interquartile range of anti-VSA antibodies levels (expressed as log median
fluorescent intensity) against the panel of isolates. (B) Comparison of anti-VSA IgGs (expressed as log median fluorescent intensity) among individuals from low and
high malaria transmission areas. (**** - P-value = <0.0001).
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Kimingi et al. Role of Anti-VSA Antibodies in CHMI
of anti-VSA antibodies compared to those who required
treatment (Supplementary Figure 3). Similarly, the untreated
group showed a higher binding inhibition ability compared to
the treated group ((Supplementary Figure 4).

We categorized the participant’s anti-VSA antibody breadth
into three categories, low, medium, or high. Having either medium
or high antibody breadth was associated with significantly longer
time to treatment compared to having low antibody breadth
(Figure 2). In addition, individuals who remained PCR negative
and those that did not require treatment during follow up had
significantly higher antibody breadth compared to those that
required treatment (Supplementary Figures 5A, B).

Boosting of Heterologous Anti-VSA
Antibodies After NF54 Challenge
Next, we assessed the effect of NF54 challenge on the levels of
anti-VSA antibodies to the test isolates. Comparing the response
one day prior to challenge with responses 14 days (C+14) after
the challenge, we observed significant boosting of antibodies
against two field isolates 19462, 19477 and the NF54 (ICAM1/
CD36) lab isolate. By contrast, the antibody levels to isolate 6454
dropped significantly by C+14, while there were no significant
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changes in the levels of antibody to the remaining two parasite
isolates (Supplementary Figure 6). We then stratified the
participants by outcome and compared the changes in
antibody responses between the groups. The untreated group
showed a significantly larger drop in antibodies to isolate 6454
compared to the treated group while there was no difference in
the change between the two groups for all the other isolates. The
same pattern of change was observed whether the outcome
considered was requiring treatment or establishment of a PCR
detectable infection (Figures 3A, B respectively).
DISCUSSION

In this study, we assessed the role of anti-VSA antibodies in
immunity against malaria in CHMI participants using three
approaches. We looked at the association between either
having anti-VSA antibodies to individual isolates or the
cumulative antibodies, or having antibodies to specific PfEMP1
domains, or having functional anti-VSA and CHMI outcome
defined by either establishment of a PCR detectable infection or
meeting the threshold for requiring treatment.
TABLE 1 | Univariable and multivariable cox regression models for risk of developing detectable parasitemia after the challenge.

Variable Univariable Multivariable (all variables) Multivariable (restricted)

HR (95% CI) P value HR (95% CI) P value HR (95% CI) P value

Anti-19462 0.74 (0.62-0.90) 0.0019 0.81 (0.56-1.18) 0.28 NA NA
Anti-19477 0.80 (0.65-0.99) 0.04 1.23 (0.84-1.79) 0.28 NA NA
Anti-6454 0.72 (0.60-0.87) 0.0005 0.83 (0.57-1.21) 0.34 0.92 (0.68-1.24) 0.59
Anti-A4U 0.80 (0.67-0.96) 0.01 0.89 (0.67-1.19) 0.44 NA NA
Anti-SAO75 0.72 (0.61-0.85) 0.0002 0.87 (0.64-1.19) 0.38 0.89 (0.66-1.20) 0.44
Anti-NF54 (ICAM1/CD36) 0.87 (0.72-1.04) 0.12 NA NA NA NA
Antibody breadth 0.75 (0.61-0.92) 0.01 1.22 (0.74-2.02) 0.43 NA NA
Anti-PF3D7_1150400 0.96 (0.84-1.10) 0.58 NA NA NA NA
Anti-PF3D7_0425800 1.03 (0.88-1.21) 0.70 NA NA NA NA
Binding inhibition 0.79 (0.64-0.99) 0.04 0.89 (0.70-1.14) 0.36 NA NA
Anti-Schizont 0.57 (0.44-0.74) <0.0001 0.70 (0.51-0.96) 0.03 0.67 (0.49-0.92) 0.01
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Multivariable (restricted): Stepwise regression including the factors indicated in the table.
NA, Indicates factors not included in the model.
TABLE 2 | Univariable and multivariable cox regression models for risk of meeting a threshold for treatment after the challenge.

Variable Univariable Multivariable (all variables) Multivariable (restricted)

HR (95% CI) P value HR (95%CI) P value HR (95%CI) P value

Anti-19462 0.57 (0.46-0.70) <0.0001 0.65 (0.39-1.09) 0.10 NA NA
Anti-19477 0.58 (0.47-0.72) <0.0001 1.52 (0.89-2.59) 0.12 NA NA
Anti-6454 0.51 (0.42-0.63) <0.0001 0.88 (0.49-1.60) 0.68 NA NA
Anti-A4U 0.62 (0.48-0.79) 0.0001 1.63 (1.06-2.53) 0.03 NA NA
Anti-SAO75 0.54 (0.45-0.66) <0.0001 1.00 (0.67-1.49) 0.99 NA NA
Anti-NF54 (ICAM1/CD36) 0.61 (0.48-0.78) 0.0001 1.81 (1.07-3.03) 0.03 NA NA
Antibody breadth 0.33 (0.24-0.46) <0.0001 0.23 (0.10-0.50) 0.0002 0.46 (0.32-0.67) <0.0001
Anti-PF3D7_1150400 0.83 (0.71-0.96) 0.01 1.27 (0.69-2.32) 0.44 NA NA
Anti-PF3D7_0425800 0.98 (0.79-1.22) 0.85 NA NA NA NA
Binding inhibition 0.58 (0.40-0.83) 0.0028 0.71 (0.43-1.16) 0.17 NA NA
Anti-Schizont 0.13 (0.21-0.34) <0.0001 0.31 (0.17-0.55) 0.0001 0.40 (0.25-0.64) 0.0001
Multivariable (restricted): Stepwise regression including the factors indicated in the table.
NA, Indicates factors not included in the model.
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Kimingi et al. Role of Anti-VSA Antibodies in CHMI
The notable finding in this study is that having a wide breadth
of anti-VSA antibodies to a panel of randomly selected isolates is
associated with a reduced risk of meeting the threshold for
treatment in a controlled human malaria infection with isolate
NF54. Although anti-VSA antibodies to individual isolates and
to two PfEMP1 DBLb domains were significantly associated with
reduced risk of treatment in a univariable analysis, the
association was not observed in a multivariable analysis. On
the other hand, none of the anti-VSA antibodies or related
parameters were associated with immunity against establishing
a PCR-detectable infection following a multivariable analysis.

There are two potential explanations for these findings: The
first is that all the apparent associations between anti-VSA
Frontiers in Immunology | www.frontiersin.org 6
antibodies and CHMI outcomes are confounded, and high anti-
VSA antibodies are just markers of immunity mediated by other
mechanisms which increase co-incidentally with exposure. To
account for this possibility, we used a multivariable analysis
model that included antibodies to schizont extract. The extract
represents potentially all blood-stage malaria parasite antigens and
therefore should correct for confounding protection by
mechanisms targeted at other antigens that were not specifically
tested here. The fact that having a wide antibody breadth remained
significantly associated with reduced risk of reaching a threshold
for treatment even after adjusting for schizont extract suggest that
cumulative anti-VSA antibodies, by their own right, can provide
protection against high parasitemia and/or symptoms in CHMI.
FIGURE 2 | Kaplan Meier survival analysis of time to treatment stratified by individuals’ breadth of anti-VSA antibodies at day C-1. The dotted line denotes the
median survival time where the survival probability is below 50%. Medium breadth score: HR= 0.38(95%CI 0.21-0.67, p=0.0008). High breadth score: HR= (0.08
(95%CI 0.03-0.24,p=<0.0001).
A

B

FIGURE 3 | Change in heterologous anti-VSA antibodies levels after the challenge. Anti-VSA antibodies to the test isolates a day before and on day 14 after the
challenge stratified by: (A) treatment outcome and (B) parasitaemia (*- P<0.05).
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Kimingi et al. Role of Anti-VSA Antibodies in CHMI
The lack of association between any of the parameters relating to
anti-VSA antibodies and protection against establishment of a PCR-
detectable parasitemia is consistent with observations in immuno-
epidemiological studies suggesting some individuals acquire a form
of immunity that protect from clinical symptoms but not infection
(44, 45). On the other hand, the observation that specific anti-VSA
responses to individual isolates were not associated with protection,
but that breadth of anti-VSA antibodies to a randomly selected
panel of isolates protects against high parasitemia and symptoms is
interesting, given that prior work reports variant specificity of anti-
VSA antibodies (25, 46). It is possible that in addition to the
predominantly variant specific anti-VSA antibodies responses,
malaria infections also generates a small amount of partially
cross-reactive anti-VSA antibodies (47). With repeated infections,
the partially cross-reactive antibodies accumulate sufficiently to
provide significant protection against symptomatic infection by a
wide range of variants including NF54.

We further explored the possibility that the heterologous anti-
VSA antibodies were cross-reactive with the NF54 specific anti-VSA
antibodies by looking at the changes in the levels of the antibodies
14 days after challenge. Significant boosting was apparent for
antibodies to two of the clinical isolates (19477 and 19462). There
was also a slight boost in the levels of anti-NF54 (ICAM1/CD36),
but a significant drop was observed in the case of anti-6454
antibodies. There was no change in the level of antibodies to the
lab isolates. The observed changes were similar whether all
participants were considered together or stratified by CHMI
outcome. The boosting of antibodies to field isolates by the
challenge infection suggest that there may be some level of cross-
reactivity with NF54. By extension, the lack of boosting of
antibodies by the lab isolates may be reflecting the narrowness of
the antibodies specific to these clonal isolates. However, the NF54
(ICAM1/CD36) parasites that we use have been selected in vitro for
specific VSAs binding to ICAM1/CD36, and in vivo NF54 is likely
to express a greater variety of VSAs. Furthermore, it is then perhaps
not surprising that no single anti-VSA response predicts immunity,
since individuals with a single anti-VSA specificity will result in
parasite selection for alternative VSA expression. On the other hand,
individuals with a broad range of anti-VSA responses are protected
against parasite growth due to protection against cytoadherence.We
speculate that protection against low-level parasitemia requires
other immunological responses.

This study was limited by inability to measure the levels of anti-
VSA antibodies to the actual infecting NF54 isolate. Parasites
isolated from the participants appeared not to express VSAs upon
culturing and were not recognised by antibodies from any of the
participants. Altered or loss of expression of VSAs upon in vitro
culturing is a commonly observed phenomenon (48). This is due to
many factors, including use of Albumax instead of human serum to
supplement the culture media which influences knobs and VSA
expression (49). To circumvent this problem, we used NF54 that
was selected to express VSAs binding to the endothelial receptors
ICAM1 and CD36 by panning on recombinant ICAM1 and CD36
proteins and confirmed to select for expression of VSAs that bind to
these proteins (data not shown). Although homologous to the
isolate used to infect the CHMI participants, the selected isolate
Frontiers in Immunology | www.frontiersin.org 7
may represent only a portion of the full NF54 VSA repertoire.
Second, recent findings have highlighted the importance of
persistent IgM responses against blood stage antigens (50). In this
study, we only measured IgG antibodies and we cannot make any
conclusion regarding IgM antibodies to VSAs among the study
participants. We therefore recommend further studies to examine
the role of pre-existing anti-VSA IgM in parasite growth control and
disease outcome in CHMI.

Previous longitudinal studies have shown that VSAs are
associated with variant-specific immunity to malaria, but most of
the studies are either in children or pregnant women (51–53). This
study suggests that, despite VSA diversity, there is some level of
cross-protection (albeit small) between variants. Understanding the
mechanism behind this cross-protection, for example, if it is driven
by conserved, but poorly immunogenic epitopes, could form the
basis for further studies on VSA as potential vaccine candidates.
MEMBERS OF THE CHMI-SIKA
STUDY TEAM

Abdirahman I Abdi, KEMRI-Wellcome Trust Research
Programme, Kilifi, Kenya; Yonas Abebe, Sanaria Inc.,
Rockville, MD, USA; Agnes Audi, Centre for Clinical Research,
Kenya Medical Research Institute, Kisumu, Kenya; Philip Bejon,
KEMRI-Wellcome Trust Research Programme, Kilifi, Kenya;
Centre for Tropical Medicine and Global Health, Nuffield
Department of Medicine, University Oxford, Oxford, UK;
Peter Billingsley, Sanaria Inc., Rockville, MD, USA; Peter C
Bull, Department of Pathology, University of Cambridge,
Cambridge, UK; Primus Che Chi, KEMRI-Wellcome Trust
Research Programme, Kilifi, Kenya; Zaydah de Laurent,
KEMRI-Wellcome Trust Research Programme, Kilifi, Kenya;
Susanne H Hodgson, The Jenner Institute, University of
Oxford, Oxford, UK.; Stephen Hoffman, Sanaria Inc.,
Rockville, MD, USA; Eric James, Sanaria Inc., Rockville, MD,
USA; Irene Jao, KEMRI-Wellcome Trust Research Programme,
Kilifi, Kenya; Dorcas Kamuya, KEMRI-Wellcome Trust
Research Programme, Kilifi, Kenya; Gathoni Kamuyu, KEMRI-
Wellcome Trust Research Programme, Kilifi, Kenya; Silvia
Kariuki, KEMRI-Wellcome Trust Research Programme, Kilifi,
Kenya; Nelson Kibinge, KEMRI-Wellcome Trust Research
Programme, Kilifi, Kenya; Sam Kinyanjui, KEMRI-Wellcome
Trust Research Programme, Kilifi, Kenya; Centre for Clinical
Research, Kenya Medical Research Institute, Kisumu, Kenya;
Pwani University, P. O. Box 195-80108, Kilifi, Kenya; Cheryl
Kivisi, Pwani University, P. O. Box 195-80108, Kilifi, Kenya;
Nelly Koskei, Centre for Clinical Research, Kenya Medical
Research Institute, Kisumu, Kenya; Mallika Imwong, Faculty of
Tropical Medicine, Department of Molecular Tropical Medicine
and Genetics, Mahidol University, Bangkok, Thailand; Brett
Lowe, KEMRI-Wellcome Trust Research Programme, Kilifi,
Kenya; Centre for Tropical Medicine and Global Health,
Nuffield Department of Medicine, University Oxford, Oxford,
UK; Johnstone Makale, KEMRI-Wellcome Trust Research
Programme, Kilifi, Kenya; Kevin Marsh, KEMRI-Wellcome
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Kimingi et al. Role of Anti-VSA Antibodies in CHMI
Trust Research Programme, Kilifi, Kenya; Centre for Tropical
Medicine and Global Health, Nuffield Department of Medicine,
University Oxford, Oxford, UK; Vicki Marsh, KEMRI-Wellcome
Trust Research Programme, Kilifi, Kenya; Centre for Tropical
Medicine and Global Health, Nuffield Department of Medicine,
University Oxford, Oxford, UK; Khadija Said Mohammed,
KEMRI-Wellcome Trust Research Programme, Kilifi, Kenya;
Moses Mosobo, KEMRI-Wellcome Trust Research Programme,
Kilifi, Kenya; Sean C Murphy, Departments of Laboratory
Medicine and Microbiology, University of Washington, Seattle,
Washington, USA; Jennifer Musyoki, KEMRI-Wellcome Trust
Research Programme, Kilifi, Kenya; Michelle Muthui, KEMRI-
Wellcome Trust Research Programme, Kilifi, Kenya; Jedidah
Mwacharo, KEMRI-Wellcome Trust Research Programme,
Kilifi, Kenya; Daniel Mwanga, KEMRI-Wellcome Trust
Research Programme, Kilifi, Kenya; Joyce Mwongeli, KEMRI-
Wellcome Trust Research Programme, Kilifi, Kenya; Francis
Ndungu, KEMRI-Wellcome Trust Research Programme, Kilifi,
Kenya; Maureen Njue, KEMRI-Wellcome Trust Research
Programme, Kilifi, Kenya; George Nyangweso, KEMRI-
Wellcome Trust Research Programme, Kilifi, Kenya; Domitila
Kimani, KEMRI-Wellcome Trust Research Programme, Kilifi,
Kenya; Joyce M. Ngoi, KEMRI-Wellcome Trust Research
Programme, Kilifi, Kenya; Janet Musembi, KEMRI-Wellcome
Trust Research Programme, Kilifi, Kenya; Omar Ngoto, KEMRI-
Wellcome Trust Research Programme, Kilifi, Kenya; Edward
Otieno, KEMRI-Wellcome Trust Research Programme, Kilifi,
Kenya; Bernhards Ogutu, Centre for Clinical Research, Kenya
Medical Research Institute, Kisumu, Kenya; Center for Research
in Therapeutic Sciences, Strathmore University, Nairobi, Kenya;
Fredrick Olewe, Centre for Clinical Research, Kenya Medical
Research Institute, Kisumu, Kenya; James Oloo, Centre for
Clinical Research, Kenya Medical Research Institute, Kisumu,
Kenya; Donwilliams Omuoyo, KEMRI-Wellcome Trust
Research Programme, Kilifi, Kenya; John Ongecha, Centre for
Clinical Research, Kenya Medical Research Institute, Kisumu,
Kenya; Martin O Ongas, Centre for Clinical Research, Kenya
Medical Research Institute, Kisumu, Kenya; Center for Research
in Therapeutic Sciences, Strathmore University, Nairobi, Kenya;
Michael Ooko, KEMRI-Wellcome Trust Research Programme,
Kilifi, Kenya; Jimmy Shangala, KEMRI-Wellcome Trust
Research Programme, Kilifi, Kenya; Betty Kim Lee Sim, Centre
for Tropical Medicine and Global Health, Nuffield Department
of Medicine, University Oxford, Oxford, UK; Joel Tarning,
Centre for Tropical Medicine and Global Health, Nuffield
Department of Medicine, University Oxford, Oxford, UK;
Mahidol-Oxford Tropical Medicine Research Unit, Mahidol
University, Bangkok, Thailand; Juliana Wambua, KEMRI-
Wellcome Trust Research Programme, Kilifi, Kenya; Thomas
N Williams, KEMRI-Wellcome Trust Research Programme,
Kilifi, Kenya; Department of Medicine, Imperial College,
London, UK; Markus Winterberg, Centre for Tropical
Medicine and Global Health, Nuffield Department of
Medicine, University Oxford, Oxford, UK; Mahidol-Oxford
Tropical Medicine Research Unit, Mahidol University,
Bangkok, Thailand
Frontiers in Immunology | www.frontiersin.org 8
DATA AVAILABILITY STATEMENT

The datasets presented in this study can be found in online
repositories. The names of the repository/repositories and
accession number(s) can be found below: Harvard Dataverse,
URL: https://doi.org/10.7910/DVN/AYWX0Y.
ETHICS STATEMENT

Ethical approval was obtained from KEMRI Scientific and Ethics
Review Unit (KEMRI//SERU/CGMR-C/029/3190) and the
University of Oxford Tropical Research Ethics Committee
(OxTREC 2-16). The patients/participants provided their
written informed consent to participate in this study.
AUTHOR CONTRIBUTIONS

SK, AA, and CK designed the study. HK, AK, KM, and SK
analyzed the data. HK, AK, and SK and wrote the first draft of the
manuscript. PB, MK, SK, AA, and CK edited the manuscript.
HK, AK, RN, NA, and SM performed flow cytometry
experiments. NA performed binding inhibition assays. AJ
provided the PfEMP1 recombinant proteins and edited the
manuscript. All authors contributed to interpretation of the
analyses and revised the draft manuscript.
FUNDING

This work was, in part, supported by the Tackling Infections to
Benefit Africa (TIBA, NIHR Project no. 16/136/33) and in part,
by The Wellcome Trust (grant no.107769/Z/10/Z) and the UK
Foreign, Commonwealth and Development Office, with support
from the Developing Excellence in Training Science and
Leadership in Africa DELTAS Africa Initiative [DEL-15-003].
CAK was funded by the Wellcome Trust (grant 080883). ATRJ is
supported by a Lundbeck Foundation grant (R313-2019-322).
The views expressed in this publication are those of the author(s)
and not necessarily those of NHS, NIHR, Wellcome Trust,
Lundbeck Foundation, or the UK government.
ACKNOWLEDGMENTS

We would like to thank all the CHMI study participants. This
manuscript is published with permission of the Director of the
Kenya Medical Research Institute.
SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fimmu.2022.
894770/full#supplementary-material
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https://www.frontiersin.org/articles/10.3389/fimmu.2022.894770/full#supplementary-material
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Kimingi et al. Role of Anti-VSA Antibodies in CHMI
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.

Publisher’s Note: All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations, or those of
the publisher, the editors and the reviewers. Any product that may be evaluated in
this article, or claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.

Copyright © 2022 Kimingi, Kinyua, Achieng,Wambui, Mwangi, Nguti, Kivisi, Jensen,
Bejon, Kapulu, Abdi, Kinyanjui and CHMI-SIKA Study Team. This is an open-access
article distributed under the terms of the Creative Commons Attribution License
(CC BY). The use, distribution or reproduction in other forums is permitted, provided
the original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No
use, distribution or reproduction is permitted which does not comply with these terms.
May 2022 | Volume 13 | Article 894770

https://doi.org/10.4049/jimmunol.1202578
https://doi.org/10.4049/jimmunol.1202578
https://doi.org/10.1371/journal.pone.0069999
https://doi.org/10.1186/s12879-022-07044-8
https://doi.org/10.1186/s12879-022-07044-8
https://doi.org/10.1128/IAI.00622-17
https://doi.org/10.1172/jci.insight.146443
https://doi.org/10.1093/infdis/jiy591
https://doi.org/10.1016/S0140-6736(97)02038-2
https://doi.org/10.1128/IAI.67.2.733-739.1999
https://doi.org/10.1128/IAI.67.2.733-739.1999
https://doi.org/10.1038/nature02486
https://doi.org/10.1371/journal.pone.0049540
https://doi.org/10.1038/srep16766
https://doi.org/10.1126/sciadv.aax4489
https://doi.org/10.1128/IAI.70.6.2982-2988.2002
https://doi.org/10.1128/IAI.70.6.2982-2988.2002
https://doi.org/10.4269/ajtmh.2002.67.597
https://doi.org/10.4269/ajtmh.2002.67.597
https://doi.org/10.1086/599841
http://creativecommons.org/licenses/by/4.0/
http://creativecommons.org/licenses/by/4.0/
https://www.frontiersin.org/journals/immunology
http://www.frontiersin.org/
https://www.frontiersin.org/journals/immunology#articles

	Breadth of Antibodies to Plasmodium falciparum Variant Surface Antigens Is Associated With Immunity in a Controlled Human Malaria Infection Study
	Introduction
	Methods
	Study Design and Population
	Parasite Isolates
	Detection of Anti-VSA Antibodies by Flow Cytometry
	Gating Strategy
	Expression of Recombinant Protein Domains
	Detection of DBLβ Reactive Antibodies
	Detection of Antibodies to P. falciparum Schizont Extract
	Inhibition of Binding of PfEMP1 to ICAM1
	Statistical Analysis

	Results
	CHMI Outcome
	Anti-VSA Antibodies Response to a Panel of Parasite Isolates
	Anti-VSA Antibodies and Risk of Developing PCR-Detectable Infection During CHMI
	Anti-VSA Antibodies and Risk of Meeting a Threshold for Anti-Malarial Treatment During CHMI
	Boosting of Heterologous Anti-VSA Antibodies After NF54 Challenge

	Discussion
	Members of the CHMI-SIKA Study Team
	Data Availability Statement
	Ethics Statement
	Author Contributions
	Funding
	Acknowledgments
	Supplementary Material
	References



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