Ba uae al. Mala J (2019) 18:11
h ps://doi.o g/10.1186/s12936-019-2647-8
RESEARCH
The impac o ea ly li e exposu e
oPlasmodium alcipa um on hede elopmen
o na u ally acqui ed immuni y omala ia
inyoung Malawian child en
P iyanka Ba ua1,6, James G. Beeson1,2,3, Kenne h Male a4, Pe Asho n5,7 and S ephen J. Roge son1*
Abs ac
Backg ound: An ibodies a ge ing mala ia blood-s age an igens a e impo an a ge s o na u ally acqui ed immu-
ni y, and may ac as aluable bioma ke s o mala ia exposu e.
Me hods: Six-hund ed and one young Malawian child en om a andomized ial o p ena al nu ien supplemen-
a ion wi h i on and olic acid o p e- and pos na al mul iple mic onu ien s o lipid-based nu ien supplemen s
we e ollowed up weekly a home and eb ile episodes we e in es iga ed o mala ia om bi h o 18 mon hs o age.
An ibodies we e measu ed o 601 child en agains me ozoi e su ace p o eins (MSP1 19kD, MSP2), e y h ocy e bind-
ing an igen 175 (EBA175), e iculocy e binding p o ein homologue 2 (Rh2A9), schizon ex ac and a ian su ace
an igens exp essed by Plasmodium alcipa um-in ec ed e y h ocy es (IE) a 18 mon hs o age. The an ibody measu e-
men da a was ela ed o concu en mala ia in ec ion and o documen ed episodes o clinical mala ia.
Resul s: A 18 mon hs o age, an ibodies we e signi ican ly highe among pa asi aemic han apa asi aemic child en.
An ibody le els agains MSP1 19kD, MSP2, schizon ex ac , and IE a ian su ace an igens we e signi ican ly highe
in child en who had documen ed episodes o mala ia han in child en who did no . An ibody le els did no di e
be ween child en wi h single o mul iple mala ia episodes be o e 18 mon hs, no be ween child en who had mala ia
be o e 6 mon hs o age o be ween 6 and 18 mon hs.
Conclusions: An ibodies o me ozoi e and IE su ace an igens inc eased ollowing in ec ion in ea ly childhood, bu
nei he age a i s in ec ion no numbe o mala ia episodes subs an ially a ec ed an ibody acquisi ion. These ind-
ings ha e implica ions o mala ia su eillance du ing ea ly childhood in he con ex o elimina ion.
T ials egis a ion Clinical T ials Regis a ion: NCT01239693 (Da e o egis a ion: 11-10-2010). URL: h p://www.ilins .o g
Keywo ds: Episodes, Nu ien supplemen s, Randomized con olled ial, Me ozoi e an igens, Va ian su ace
an igens, Se op e alence
© The Au ho (s) 2019. This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License
(h p://c ea i eco mmons .o g/licen ses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium,
p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license,
and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i eco mmons .o g/
publi cdoma in/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed.
Open Access
Mala ia Jou nal
*Co espondence: s oge @unimelb.edu.au
1 The Depa men o Medicine (RMH), Pe e Dohe y Ins i u e o In ec ion
and Immuni y, The Uni e si y o Melbou ne, Melbou ne, VIC 3000,
Aus alia
Full lis o au ho in o ma ion is a ailable a he end o he a icle
Page 2 o 12
Ba uae al. Mala J (2019) 18:11
Backg ound
Mala ia is one o he leading causes o childhood mo bid-
i y and mo ali y, and Plasmodium alcipa um in ec ion
is esponsible o nea ly all mala ia dea hs. The Wo ld
Heal h O ganiza ion es ima ed ha in 2017 he e we e
435,000 dea hs due o mala ia o which a ound 61% we e
in child en below 5yea s o age [1]. Child en in mala ia-
endemic a eas become mos ulne able o mala ial in ec-
ion a 4 o 6mon hs o age as ma e nally ans e ed
an ibodies wane [2] and hey hen begin o acqui e hei
own an ibodies in esponse o epea ed in ec ion. Na u-
ally acqui ed immuni y o mala ia de elops o e ime
wi h con inuous exposu e o in ec ion [3].
An ibodies play a c ucial ole in media ing acqui ed
immuni y o mala ia. Blood s age me ozoi e an igens
and a ian su ace an igens (VSA) exp essed on in ec ed
e y h ocy es (IE) a e impo an a ge s o his p o ec i e
immuni y. An ibodies o me ozoi e an igens inhibi in a-
sion o ed blood cells (RBCs), p e en in a-e y h ocy ic
g ow h [4], and p omo e opsoniza ion o phagocy ic
clea ance [5] and complemen ixa ion [6]. An ibodies o
me ozoi e an igens o su icien magni ude and unc ion
appea o con ibu e o immuni y [7]. In young child en
o hose wi h limi ed mala ia exposu e, hey may ins ead
ac as bioma ke s o mala ia exposu e [8, 9], wi h po en-
ial o in o m su eillance and con ol ac i i ies [10].
Among he es ed an igens, me ozoi e su ace p o ein 1
(MSP1) is he mos copious p o ein ound on he su ace
o he me ozoi e [11]. C ucial o he p ima y in e ac ion
be ween me ozoi es and RBCs in pa asi e in asion [12],
MSP1 is a majo a ge o opsonizing ollowing na u al
exposu e [13]. MSP2 is ano he abundan , GPI-ancho ed
su ace p o ein necessa y o me ozoi e in asion.
Inc eased IgG le el agains MSP2 was associa ed wi h
inc easing age, highe haemoglobin le el and educed
pa asi aemia sugges ing i s p o ec i e e ec [14]. E y h-
ocy e binding an igen 175 (EBA175) is eleased om
mic onemes [15] and agg ega es a he apical egion o
he me ozoi e su ace. An ibodies o he RIII-V egion o
EBA175 ha e also been associa ed wi h p o ec ion om
mala ia [16–18]. Rhop y-de i ed Rh2A9 help binding o
he RBC ecep o s a e he p ima y in e ac ion be ween
he RBC and me ozoi e su ace p o eins is comple ed.
The le el o IgG agains Rh2A9 in child en (5–14yea s)
was associa ed wi h lowe isk o mala ia [19].
An ibodies o VSA diminish mala ia isk by obs uc -
ing cy oadhesion o di e en hos ecep o s [20, 21] and
ini ia ing phagocy ic clea ance o IE [22]. Se e al s udies
ha e epo ed associa ions be ween le els o an i-VSA
an ibodies and p o ec ion agains symp oma ic mala ia
[23–26] bu ew s udies ha e examined he dynamics o
na u ally occu ing an i-VSA IgG in in an s in a mala ia-
endemic se ing [27, 28]. In one s udy [27], child en up
o 24mon hs o age did no acqui e an ibodies o VSA
bu in a high- ansmission a ea o Tanzania [29], chil-
d en had d ama ic inc eases in an ibodies o VSA om
1 o 2yea s o age. Recen s udies in Papua New Guinea
sugges ha acqui ed an ibodies o VSA play an ea lie
ole in immuni y o mala ia han an ibodies o me ozo-
i e an igens [30]. These con adic o y indings indica e
he need o u he s udies o in es iga e he dynamics
o na u ally acqui ed immuni y a ge ing bo h me ozoi e
an igens and VSA in e y young child en.
This s udy examines he dynamics o an ibody acqui-
si ion o mul iple me ozoi e an igens, schizon ex ac
and VSA in young child en in esponse o ongoing
exposu e o mala ia. The s udy was pa o he In e na-
ional Lipid-based Nu ien Supplemen (iLiNS) P ojec
DYAD-Malawi andomized con olled ial (clinical i-
als.go egis a ion numbe NCT01239693). The o igi-
nal s udy epo ed ha nu ien supplemen a ion did no
ha e any signi ican impac on an h opome ic indices
in 18mon hs old child en [31]. Ano he sub-s udy om
he same coho [32] epo ed ha mala ia an ibody
acquisi ion (agains he same an igens epo ed he e) in
ea ly in ancy was no imp o ed by addi ional lipid-based
nu ien supplemen a ion. Fo his epo , mala ia an i-
body measu emen a 18mon hs o age was ela ed o
concu en mala ia in ec ion and o documen ed epi-
sodes o clinical mala ia du ing ea ly childhood o unde -
s and he impac o exposu e on an ibody acquisi ion.
Me hods
S udy loca ion andpa icipan s
The s udy pa icipan s we e 601 in an s o 18 mon hs
o age om u al Malawi, pa icipan s in he iLiNS P o-
jec DYAD-Malawi ial. De ailed desc ip ion o he ial
design and supplemen s has been published elsewhe e
[33]. B ie ly, p egnan women we e andomly alloca ed
o supplemen a ion g oups ha ecei ed ei he i on and
olic acid (IFA), mul iple mic onu ien s (MMN) o 20g
o lipid based nu ien supplemen s (LNS) daily. A e
deli e y, women in he IFA g oup ecei ed placebo,
whe eas MMN and LNS supplemen a ion was sus ained
o 6mon hs pos -pa um. F om 6 o 18mon hs o age,
child en in he LNS g oup ecei ed 10g LNS wice daily.
De ec ion o mala ia
Child en we e ollowed up weekly a home and eb ile
episodes we e in es iga ed o mala ia. Clinical mala ia
was de ined as e e wi h axilla y empe a u e abo e
37.5°C and pa asi aemia was con i med by mic oscopy
o apid diagnos ic es (RDT). Fo mic oscopy, slides
we e examined unde 100× magni ica ion and pa asi es
we e coun ed agains 200 leucocy es. The RDT was he
Clea iew® Mala ia Combo (B i ish Biocell In e na ional
Page 3 o 12
Ba uae al. Mala J (2019) 18:11
L d, Dundee, UK) which de ec s he p o eins P. alcipa-
um lac a e dehyd ogenase and his idine- ich p o ein 2.
P epa a ion o plasma samples
Blood om pa icipan s was collec ed a he 18-mon h
s udy isi . Plasma was sepa a ed by cen i uga ion and
s o ed a − 80°C p io o shipping on d y ice o Aus-
alia. Samples we e hawed, hea inac i a ed a 57°C o
45min, and we e s o ed a − 80°C un il assayed.
Main enance o pa asi e cul u e
Th ee P. alcipa um lines we e cul u ed as p e iously
desc ibed [34]. The E8B-ICAM line binds o ICAM-1
and CD36 [35], and exp esses g oup B/C a genes [36].
Rose ing line R29 exp esses a genes om g oup A
[37]. A 3D7-de i ed line had a g oup A a gene as i s
dominan ansc ip , bu i s binding pheno ype was
no de ined [30]. Cul u es we e synch onized by hypo-
onic lysis using 5% so bi ol, and by egula gela in lo-
a ion [38]. To selec R29 o high le els o ose ing, i
was subjec o wo ounds o gela in lo a ion. A e he
i s ound, he pelle was collec ed and esuspended
in gela in wi h hepa in li hium sal , (0.05mg/ml Sigma
Ald ich), added o dis up ose es, and he supe na an
was collec ed.
Measu emen o IgG ome ozoi e an igens andschizon
ex ac
Me ozoi e an igens MSP119kD, MSP2 (FC27 clone), EBA
175, and P. alcipa um e iculocy e binding p o ein hom-
ologue 2 (Rh2A9) we e exp essed and pu i ied as p e i-
ously desc ibed [16, 19, 32, 39, 40], and schizon ex ac
was p epa ed as p e iously desc ibed [41]. ELISAs we e
pe o med as p e iously desc ibed [32].
Measu emen o IgG le els agains VSA
IgG an ibody le els agains VSA we e measu ed by low
cy ome y as p e iously desc ibed [32], and low cy om-
e y da a we e analysed as desc ibed [42].
Da a analysis
Da a we e analysed using S a a e sion 13.0 (S a aCo p,
Texas, USA) and g aphed using G aphPad P ism e sion
5 (La Jolla, CA, USA). An ibody le els we e measu ed as
op ical densi y (OD) o schizon and me ozoi e an igens,
o as geome ic mean luo escence in ensi y (MFI) o
VSA. They we e exp essed ela i e o he posi i e con ol
which was a pooled plasma sample om mala ia exposed
indi iduals om A ica.
Se op e alence was speci ied as he pe cen age o
child en whose ela i e an ibody le el was g ea e han
he mean plus h ee s anda d de ia ions o he nega-
i e con ols’ an ibody le els. Nega i e con ols we e a
panel o mala ia-naï e Melbou ne blood dono s. Socio-
economic s a us (SES) was de i ed om an in en o y
o key household asse s (HHA) adap ed om [43]. Chi
squa ed es s we e pe o med o es he di e ences in
se oposi i i y. Mann–Whi ney es s we e pe o med
o compa ing an ibody le els be ween wo g oups and
K uskal–Wallis es s we e pe o med o compa e he
an ibody le els among mo e han wo g oups.
The associa ion be ween mala ia episodes and an i-
body se op e alence in 18 mon hs old child en was
in es iga ed using logis ic eg ession, and linea eg es-
sion was pe o med o s udy he associa ion o mala ia
episodes wi h an ibody le els. Linea eg ession was
done by ans o ming an ibody le els o hei na u al
loga i hm; hese we e back ans o med o epo ing
desc ip i e esul s. Mul i a ia e eg ession adjus ed
o he co a ia es du a ion o ges a ion, HIV in ec ion,
gende o he child and ma e nal anaemia.
Resul s
S udy popula ion cha ac e is ics
A o al o 601 samples om 18mon hs old child en
we e es ed (48.8% male and 51.2% emale). The nu i-
en supplemen s ecei ed by hei mo he s we e IFA
in 33.6%, MMN in 33.4%, and 32.9% ecei ed LNS.
The mean haemoglobin le el was 10.8 ± 1.5g/dl wi h
a p e alence o anaemia (haemoglobin le el ≤ 10.9g/
dl) o 46.3%. The pe cen age o child en ha ing P. alci-
pa um pa asi aemia a he 18-mon h isi was 6.5% by
mic oscopy and 9.9% by RDT (Table1).
Table 1 S udy popula ion cha ac e is ics
a Anaemia de ined as Hb ≤ 10.9g/dl
b Rapid diagnos ic es
Cha ac e is ics o  hepa icipan s Numbe
(%)
[n = 601]
Female 308 (51.2)
Blood haemoglobin concen a ion, mean ± SD, g/dl 10.8 ± 1.5
Anaemiaa278 (46.3)
Pa asi aemia by mic oscopy 39 (6.5)
Pa asi aemia by RDTb60 (9.9)
Numbe o child en wi h mala ial episodes 144 (23.9)
Single episode 119 (19.8)
Mul iple episodes 25 (4.2)
Low socio-economic s a us 357 (59.4)
Mo he ’s educa ion below median 321 (53.4)
Page 4 o 12
Ba uae al. Mala J (2019) 18:11
Magni ude andp e alence o an ibodies andIgG
esponses inpa asi aemic andapa asi aemic child en
a 18mon hs
An ibody se op e alence agains me ozoi e an igens,
schizon ex ac and VSA was measu ed o all he
es ed child en (Table2). The se oposi i i y was highes
o MSP1 (54.4%), ollowed by schizon ex ac (54.1%).
Howe e , IgG agains VSA o all he es ed pa asi e lines
we e e y low and ew child en we e se oposi i e.
Child en who we e pa asi aemic (n = 60) by RDT
(9.9%) a he ime o sample collec ion a 18mon hs had
highe an ibody le els han apa asi aemic child en, and
di e ences we e s a is ically signi ican o all he es ed
an igens excep Rh2A9. The se op e alence da a we e
also in acco dance wi h he an ibody le el da a and pa a-
si aemic child en we e mo e equen ly se oposi i e o
all he an igens han he apa asi aemic child en (Table2).
Child en who we e pa asi aemic a 6 mon hs (n = 57)
we e somewha mo e likely o expe ience mala ial epi-
sodes om 6 o 18mon hs o age; 14 ou o 57 (24.6%)
pa asi aemic child en a 6mon hs had episodes be ween
6 o 18mon hs compa ed o 99 ou o 544 (18.2%) apa a-
si aemic child en (p = 0.24, Chi squa e).
Associa ion o p e ious mala ia episodes wi han ibody
se op e alence in18mon hs old child en
One-hund ed and o y- ou child en (23.9%) had one o
mo e mala ia episodes be o e 18mon hs o age. An ibody
se op e alence was highe in he child en wi h p e ious
clinical mala ia episodes han hose wi hou mala ia o
all he es ed an igens, and di e ences we e s a is ically
signi ican o MSP1, MSP2 and schizon ex ac and IgG
agains E8B VSA (Table3).
To de e mine whe he he pe cen age o se oposi i -
i y a 18mon hs o age a ies acco ding o he numbe o
mala ia episodes, child en we e di ided in o g oups ha -
ing ei he a single episode o mala ia o mo e han one
episode o mala ia (de ec ed by RDT). One-hund ed and
nine een child en had single episodes o mala ia whe eas
25 child en had mul iple episodes eco ded. An ibody
Table 2 Magni ude and p e alence o  an ibodies and IgG esponses in pa asi aemic and apa asi aemic child en
a 18mon hs
Signi ican p aluein i alic o ma
a An ibody le els exp essed as uni s ela i e o posi i e con ol
b In e qua ile ange
c Samples de ined as an ibody posi i e i le els g ea e han mean + 3SD o nega i e con ols
d p alue be ween pa asi aemic and apa asi aemic child en calcula ed by Mann–Whi ney es o Chi squa ed es o an ibody le el o se oposi i i y, espec i ely
e Me ozoi e su ace p o ein 1
Me ozoi e su ace p o ein 2
g E y h ocy e binding an igen 175
h Re iculocy e binding p o ein homologue 2A
i Va ian su ace an igen an ibody esponses
An igen es ed/pa asi e
isola e Median an ibody le ela (IQRb)
Numbe o  se oposi i ec child en (%) p alued
All child en; (n = 601) Pa asi aemic child en;
(n = 60) Apa asi aemic child en;
(n = 541)
MSP1 19kDe2.80 (0.65, 7.09) 12.31 (4.38, 28.79) 2.37 (0.56, 6.08) < 0.0001
327 (54.4) 53 (88.3) 274 (50.7) < 0.0001
MSP2 2.91 (0.87, 9.04) 15.14 (3.47, 35.16) 2.62 (0.79, 7.28) < 0.0001
162 (26.9) 36 (60.0) 126 (23.3) < 0.0001
EBA175g3.38 (1.95, 16.09) 5.89 (2.89, 21.37) 3.24 (1.92, 15.89) 0.0228
69 (11.5) 15 (25.0) 54 (9.9) 0.001
Rh2A9h7.17 (2.1, 25.3) 8.59 (3.28, 19.24) 7.11 (4.05, 13.27) 0.5630
151 (25.1) 23 (38.3) 128 (23.7) 0.013
Schizon 3.30 (1.27, 8.09) 17.31 (5.22, 48.66) 2.95 (1.09, 6.98) < 0.0001
325 (54.1) 58 (96.7) 267 (49.4) < 0.0001
E8Bi0 (0, 0.20) 0.15 (0, 0.86) 0 (0, 0.16) < 0.0001
43 (7.2) 9 (15.0) 34 (6.3) 0.013
R29i0 (0, 0.07) 0 (0, 1.12) 0 (0, 0.002) < 0.0001
21 (3.5) 9 (15.0) 12 (2.2) < 0.0001
3D7i0 (0, 0.35) 0.30 (0, 2.08) 0 (0, 0.29) < 0.0001
50 (8.3) 17 (28.3) 33 (6.1) < 0.0001
Page 5 o 12
Ba uae al. Mala J (2019) 18:11
Table 3 Associa ion be weenmala ia episodes andan ibody se op e alence in18mon hs old child en
Signi ican p aluein i alic o ma
a Samples de ined as an ibody posi i e i le els g ea e han mean + 3SD o nega i e con ols
b p alue compa ed o child en wi h no episode calcula ed by Chi squa ed es
c p- alue de i ed om logis ic eg ession wi h Odds Ra ios (OR) and 95% con idence in e als (CI)
d p- alue de i ed om mul i a ia e logis ic eg ession wi h Adjus ed Odds Ra ios (OR) a e con olling o du a ion o ges a ion, HIV in ec ion, gende o he child, ma e nal anaemia
e Me ozoi e su ace p o ein 1
Me ozoi e su ace p o ein 2
g E y h ocy e binding an igen 175
h Re iculocy e binding p o ein homologue 2A
i Va ian su ace an igen an ibody esponses
An igen
es ed/
pa asi e
isola e
Se oposi i esa
(%) No episode
(n = 457) Any episode
wi hin18mon hs
% (n = 144)
p aluebSingle
episode
% (n = 119)
p aluebMul iple
episodes %
(n = 25)
p aluebUnadjus ed
OR(95% CI) p aluecAdjus ed
OR(95% CI) p alued
MSP1 19 kDe327 (54.4) 224 (49.0) 103 (71.5) < 0.0001 83 (69.8) < 0.0001 20 (80.0) 0.003 2.24 (1.59, 3.17) < 0.0001 2.21 (1.56, 3.12) < 0.0001
MSP2 162 (26.9) 113 (24.7) 49 (34.0) 0.028 40 (33.6) 0.051 9 (36.0) 0.207 1.41 (1.02, 1.94) 0.034 1.38 (1.00, 1.90) 0.049
EBA175g69 (11.5) 50 (10.9) 19 (13.2) 0.459 13 (10.9) 0.996 6 (24.0) 0.047 1.33 (0.87, 2.05) 0.182 1.31 (0.85, 2.01) 0.216
Rh2A9h151 (25.1) 108 (23.6) 43 (29.9) 0.133 34 (28.6) 0.265 9 (36.0) 0.160 1.32 (0.95, 1.83) 0.095 1.30 (0.93,1.80) 0.117
Schizon 325 (54.1) 222 (48.6) 103 (71.5) < 0.0001 85 (71.4) < 0.0001 18 (72.0) 0.023 2.14 (1.52, 3.00) < 0.0001 2.20 (1.55, 3.13) < 0.0001
E8Bi43 (7.2) 24 (5.3) 19 (13.2) 0.001 17 (14.3) 0.001 2 (8.0) 0.554 1.85 (1.15, 2.98) 0.010 1.87 (1.15, 3.04) 0.011
R29i21 (3.5) 13 (2.8) 8 (5.6) 0.122 7 (5.9) 0.107 1 (4.0) 0.738 1.55 (0.78, 3.09) 0.204 1.51 (0.75, 3.02) 0.245
3D7i50 (8.3) 36 (7.9) 14 (9.7) 0.485 10 (8.4) 0.850 4 (16.0) 0.152 1.31 (0.80, 2.14) 0.278 1.30 (0.79, 2.14) 0.291

Page 6 o 12
Ba uae al. Mala J (2019) 18:11
se op e alence was highe in child en ha ing a single epi-
sode o mul iple episodes han hose wi hou any episode
de ec ed. Fo a single episode, di e ences in p e alence
we e s a is ically signi ican o MSP1, schizon ex ac
(p ≤ 0.051) and IgG agains VSA exp essed by he E8B
pa asi e line (p = 0.001). In he smalle numbe o chil-
d en ha ing mul iple episodes, p e alence o an ibody
o MSP1 (0.003), EBA175 (0.047) and schizon ex ac
(0.023) was signi ican ly highe han in child en wi hou
mala ia. The e we e no signi ican di e ences in an ibody
se op e alence be ween hose wi h single o mul iple
episodes.
Logis ic eg ession e ealed ha child en wi h any epi-
sode o mala ia om bi h o 18mon hs o age had sig-
ni ican ly highe odds o being se oposi i e o MSP1,
MSP2, schizon ex ac and VSA agains E8B pa asi e
line in bo h unadjus ed (p ≤ 0.034) and adjus ed analysis
(p ≤ 0.049) han child en wi hou any episode.
Associa ion o p e ious mala ia episodes wi hle els
o an ibodies in18mon hs old child en
The le els o an ibody o mala ia an igens we e com-
pa ed be ween in an s who did and did no ha e p e ious
mala ia episodes (Figs.1, 2). As obse ed wi h an ibody
se oposi i i y, child en ha ing mala ia episodes had
highe le els o an ibodies o se e al an igens when com-
pa ed o child en wi hou mala ia his o y, and his was
signi ican o MSP1, MSP2 and schizon ex ac . How-
e e , he e we e no signi ican di e ences be ween he
MSP1
Ab le els (% o he posi i es)
wi hou episode
single episode
mul iple episodes
0
20
40
60
80
100
a
b
cd
<0.0001
0.0007
0.6885
MSP2
Ab le els (% o he posi i es)
wi hou episode
single episode
mul iple episodes
0
20
40
60
80
100
0.0015
0.0418
0.7627
EBA 175
Ab le els (% o he posi i es)
wi hou episod
e
single episode
mul iple episodes
0
20
40
60
80
100
0.2338
0.8680
0.4167
Rh2A9
Ab le els (% o he posi i es)
wi hou episode
single episod
e
mul ipl
e episodes
0
20
40
60
80
100
0.4630 0.4635
0.3024
Fig. 1 Le els o an ibody a 18 mon hs o a MSP1, b MSP2, c EBA175 and d Rh2A9, by his o y o episodes o mala ia; (N; Wi hou episode = 457,
Single episode = 119, Mul iple episodes = 25). Da a p esen ed as box plo s wi h he Y axis ep esen ing an ibody le els as a pe cen age o posi i e
con ol (pooled plasma o mala ia immune adul s). The boxes deno e he 25 h o 75 h pe cen ile while he whiske s deno e he 10 h and he 90 h
pe cen ile wi h ou lie s. p alue calcula ed using Mann–Whi ney es be ween he wo g oups indica ed by he line
Page 7 o 12
Ba uae al. Mala J (2019) 18:11
an ibody le els o child en wi h single o mul iple epi-
sodes o any an igen.
Le els o IgG agains VSA we e signi ican ly highe
o all he es ed pa asi e lines in child en ha ing single
mala ia episode han hose who had none (p alue 0.038
o E8B, 0.028 o R29 and < 0.0001 o 3D7).
Linea eg ession (Table4) indica ed ha ha ing single
o mul iple episodes o mala ia om bi h o 18mon hs
o age was associa ed wi h signi ican ly highe an ibody
le els o MSP1, MSP2 and schizon ex ac in unad-
jus ed analysis (p ≤ 0.047) and o MSP1 and schizon
ex ac in adjus ed analysis (p < 0.0001) o 18mon hs old
child en.
Associa ion o age a  he ime o mala ia episode
onan ibody le els andse op e alence in18mon hs old
child en
To in es iga e whe he age a he ime o clinical
mala ia a ec ed an ibody le els o se op e alence a
18 mon hs, an ibody le els and se op e alence we e
compa ed be ween child en who had mala ial episodes
p io o 6mon hs and hose who had hem be ween 6
and 18mon hs o age (Table5). Bo h child en who had
mala ia episodes be o e 6mon hs o age and hose who
had mala ia episodes om 6 o 18mon hs had highe
an ibody le els and se oposi i i y o mul iple an igens
compa ed o hose wi hou any his o y o clinical mala ia.
schizon
wi hou episode
single episode
mul iple episodes
0
20
40
60
80
100
<0.0001
0.0025
0.6126
Ab le els (% o he posi i es)
wi hou episode
single episode
mul iple episodes
0
1
2
3
4
50.0378
E8B
Ab le els (% o he posi i es)
wi hou episod
e
single episode
mul iple episode
s
0
1
2
3
4
50.0277
R29
Ab le els (% o he posi i es)
wi hou ep
isode
single episode
mul iple ep
isodes
0
1
2
3
4
5< 0.0001
3D7
ab
cd
Fig. 2 Le els o an ibody a 18 mon hs o a schizon ex ac , b E8B, c R29 and d 3D7 pa asi e line, by his o y o episodes o mala ia; (N; Wi hou
episode = 457, Single episode = 119, Mul iple episodes = 25). Da a p esen ed as box plo s wi h he Y axis ep esen ing an ibody le els as a
pe cen age o posi i e con ol (pooled plasma o mala ia immune adul s). The boxes deno e he 25 h o 75 h pe cen ile while he whiske s deno e
he 10 h and he 90 h pe cen ile wi h ou lie s. p alue calcula ed using Mann–Whi ney es be ween he wo g oups indica ed by he line
Page 8 o 12
Ba uae al. Mala J (2019) 18:11
Howe e , an ibody le el o se op e alence o all o he
es ed an igens did no di e be ween child en who had
mala ia be o e 6mon hs o om 6 o 18mon hs.
Discussion
Na u ally acqui ed immuni y o mala ia is achie ed wi h
ongoing exposu e o in ec ions and subsequen acquisi-
ion o an i-mala ial an ibodies. An ibodies agains me -
ozoi e an igens and VSA a e hough o play key oles in
con e ing immuni y agains mala ia [26, 44]. The aim o
his s udy was o examine how asymp oma ic pa asi ae-
mia a he ime o blood sampling and episodes o clinical
mala ia in young child en a ec he ea ly de elopmen
o an ibodies agains me ozoi e an igens and VSA. This
s udy in es iga ed whe he he numbe o episodes in
ea ly li e, o he age a which hey occu ed, in luenced
he de elopmen o an ibody. The s udy ound ha a
18mon hs o age child en who we e pa asi aemic had
signi ican ly highe le els o an ibodies and se op e a-
lence o all he es ed VSA and me ozoi e an igens han
he apa asi aemic child en, wi h he excep ion o an i-
body o Rh2A9. Child en who had expe ienced clinical
mala ia episodes be o e 18 mon hs o age had highe
an ibody le els and se op e alence o he es ed an igens
Table 4 Associa ion be weennumbe o mala ia episodes
andan ibody le el in18mon hs old child en
Signi ican p aluein i alic o ma
a p- alue de i ed om linea eg ession o an ibody le els and epo ed as
coe icien and 95% con idence in e als (CI)
b p- alue calcula ed using mul i a ia e linea eg ession o an ibody le els
epo ing coe icien and 95% con idence in e als (CI) while adjus ing o
du a ion o ges a ion, HIV in ec ion, gende o he child, ma e nal anaemia
c Me ozoi e su ace p o ein 1
d Me ozoi e su ace p o ein 2
e E y h ocy e binding an igen 175
Re iculocy e binding p o ein homologue 2A
g Va ian su ace an igen an ibody esponses
An igen
es ed/
pa asi e
isola e
Unadjus ed
coe icien
(95% CI)
p alueaAdjus ed
coe icien
(95% CI)
p alueb
MSP1 19 kDc1.85 (1.43, 2.41) < 0.0001 1.83 (1.41, 2.37) < 0.0001
MSP2d1.28 (1.00, 1.63) 0.047 1.26 (0.99, 1.61) 0.057
EBA175e0.92 (0.75, 1.11) 0.399 0.91 (0.76, 1.10) 0.378
Rh2A9 1.14 (0.98, 1.33) 0.074 1.14 (0.98, 1.33) 0.082
Schizon 1.52 (1.21, 1.91) < 0.0001 1.50 (1.19, 1.87) < 0.0001
E8Bg1.06 (0.75, 1.50) 0.719 1.03 (0.73, 1.46) 0.826
R29h1.33 (0.79, 2.24) 0.267 1.39 (0.81, 2.40) 0.222
3D7h1.16 (0.79, 1.72) 0.434 1.13 (0.77, 1.67) 0.508
Table 5 Associa ion be weenage a  he ime o mala ia episode andan ibody le els andse op e alence in18mon hs
old child en
Signi ican p aluein i alic o ma
a Fo each an igen ep esen ed, ow 1: median an ibody le el (in e qua ile ange); ow 2: numbe o se oposi i e child en (%)
b p alue calcula ed by Mann–Whi ney es o Chi squa ed es o an ibody le el o se oposi i i y, espec i ely
c Me ozoi e su ace p o ein 1
d Me ozoi e su ace p o ein 2
e E y h ocy e binding an igen 175
Re iculocy e binding p o ein homologue 2A
g Va ian su ace an igen an ibody esponses
An igen es ed/
pa asi e isola e No episodea (n = 457) ≥ 1 episode
be o e6mon hs o  agea
(n = 31)
p alueb≥ 1 episode be ween6
and18mon hs o  agea (n = 113) p alueb
MSP1 19 kDc2.36 (0.51, 5.73) 6.16 (1.92, 11.16) 0.0071 4.71 (1.43, 21.25) < 0.0001
224 (49.0) 24 (77.5) 0.002 79 (69.9) < 0.0001
MSP2d2.76 (0.79, 8.23) 5.76 (1.01, 10.55) 0.1043 3.06 (1.06, 13.97) 0.3165
113 (24.7) 12 (38.8) 0.084 37 (32.7) 0.083
EBA175e3.49 (1.96, 17.05) 3.25 (1.89, 8.79) 0.5785 3.19 (1.93, 9.91) 0.3753
50 (10.9) 3 (9.7) 0.827 16 (14.2) 0.338
Rh2A9 7.03 (3.97, 13.13) 8.24 (4.41, 17.32) 0.3790 7.92 (4.12, 15.87) 0.2223
108 (23.6) 10 (32.3) 0.278 33 (29.2) 0.219
Schizon 2.95 (1.02, 7.24) 7.40 (2.24, 13.97) 0.0211 4.70 (1.77, 14.87) 0.0018
222 (48.6) 23 (74.2) 0.006 80 (70.8) < 0.0001
E8Bg0 (0, 0.15) 0.03 (0, 1.11) 0.0177 0 (0, 0.26) 0.0967
24 (5.3) 10 (32.3) < 0.0001 9 (7.9) 0.269
R29g0 (0, 0.002) 0 (0, 0.43) 0.0424 0 (0, 0.24) 0.2341
13 (2.8) 5 (16.2) < 0.0001 3 (2.6) 0.913
3D7g0 (0, 0.33) 0.01 (0, 0.37) 0.4718 0 (0, 0.42) 0.8673
36 (7.9) 4 (12.9) 0.324 10 (8.9) 0.734
Page 9 o 12
Ba uae al. Mala J (2019) 18:11
han child en who did no ha e any episode, and he le -
els and se op e alence o an ibodies a 18mon hs o age
did no di e depending on he age o he child a he
ime o mala ia episodes. This knowledge is ele an o
in o ming accine de elopmen and s a egies o se o-
su eillance o mala ia [10].
P e ious s udies ha e shown ha newbo n babies and
young in an s a e ela i ely p o ec ed om symp oma ic
mala ia [45, 46], and his has been a ibu ed mainly o
ma e nally ans e ed an ibodies p esen in he i s
ew mon hs o li e [2, 47]. Young child en become mos
suscep ible o in ec ion as ma e nally de i ed an ibod-
ies wane, and hen g adually begin o acqui e an ibody
in esponse o in ec ions, hus de eloping na u ally
acqui ed immuni y o mala ia [2, 3].
The p esen s udy examined he e ec o clinical
mala ia episodes and pa asi aemia on acquisi ion o
an ibodies by 18mon hs o age. A his ime, li le o no
placen ally ans e ed an ibody emains, so an ibodies
elici ed a e likely o e lec na u ally acqui ed immuni y
[47]. An ibody le els agains all he es ed an igens we e
ela i ely low, in ag eemen wi h ea lie s udies showing
he age dependen acquisi ion o an ibody [48, 49]. The
p opo ion o child en wi h de ec able an ibodies was
highes o MSP1 (54.4%) and schizon ex ac (54.1%)
pe haps because schizon ex ac ac s as a c ude ma ke
o blood s age mala ia in ec ion whe eas MSP1 is he
mos abundan me ozoi e su ace p o ein [11]. An ibod-
ies o VSA exp essed by IE p edominan ly consis o an i-
bodies o P EMP1, he main an igen on he IE su ace
[50]. An ibodies o VSA a e la gely s ain-speci ic and
epea ed exposu e leads o he acquisi ion o a epe oi e
o an ibodies o di e en a ian s [51]. Slow acquisi ion
o a ian -speci ic an ibody, and possible lack o in ec ion
wi h a ian s simila o hose es ed, could explain he
e y low p e alence o de ec ed an ibodies o VSA.
Child en who we e pa asi aemic a he ime o sample
collec ion a 18mon hs had signi ican ly highe le els o
an ibodies and se op e alence o all he es ed VSA and
me ozoi e an igens (excep Rh2A9) han he apa asi ae-
mic child en. This inding is consis en wi h s udies om
Kenya in which an ibodies o VSA [52] and o me ozo-
i e an igens [53] we e highe in cu en ly pa asi aemic
indi iduals. In he la e s udy, he e we e mo e d ama ic
di e ences in IgG le els be ween pa asi aemic and apa a-
si aemic child en, han be ween pa asi aemic and apa a-
si aemic adul s. O e wo ime pe iods o highe and
lowe ansmission, pa asi aemia was s ongly associa ed
wi h highe an ibody le els o MSP2 and Rh2A9 (and o
o he an igens including AMA1 and MSP4), and weakly
wi h an ibody o MSP1, EBA175 and schizon ex ac
[53]. In Malawi, by con as , an ibodies o all es ed
an igens excep Rh2A9 we e s ongly associa ed wi h
pa asi aemia. Howe e , i is possible ha some in ec ed
child en may ha e been missed because pa asi aemia
was de ec ed using RDT, which may miss low densi y
in ec ions. Selec ing an ibody a ge s o su eillance o
mala ia exposu e [10] will equi e e alua ion in mul iple
popula ions and age g oups.
The ela ionship be ween an ibody le els o se op e a-
lence and his o y o clinical mala ia in ec ions was also
in es iga ed. Bo h measu es we e highe in child en
who had expe ienced clinical mala ia episodes be o e
18mon hs o age han child en who did no , and he di -
e ences we e signi ican o MSP1 19 kd, MSP2, schizon
ex ac , and ( o an ibody le els, bu no se op e alence)
o IgG agains all es ed pa asi e lines. This is in acco d-
ance wi h o he s udies ha epo an ibody boos ing
ollowing exposu e o clinical mala ia [9, 27, 53–55], and
sugges s ha me ozoi e (and VSA) an ibodies ac p ima -
ily as bioma ke s o exposu e in e y young child en [8,
9]. Some child en wi h no mala ia episodes de ec ed did
ha e an ibodies; his likely e lec s he occu ence o low-
densi y asymp oma ic in ec ions ha we e no de ec ed
du ing ollow up and we e su icien o gene a e an ibod-
ies o some an igens.
When he coho was di ided in o child en wi h single
o mul iple mala ia episodes, hose wi h single mala ia
episodes had signi ican ly highe le els o an ibody
agains MSP1, MSP2, schizon ex ac (≤ 0.0015) and IgG
agains VSA o all h ee es ed pa asi e lines (≤ 0.028)
compa ed o hose wi hou episodes. Fewe child en had
mul iple episodes, limi ing s a is ical powe , bu hese
child en also had signi ican ly highe an ibody le els
agains MSP1, MSP2 and schizon ex ac (≤ 0.0418) han
hose who did no ha e mala ia. The e was no ela ion-
ship be ween he numbe o clinical episodes and ei he
an ibody le els o p e alence. These indings sugges ed
ha an ibodies o MSP1, MSP2 o schizon ex ac we e
use ul ma ke s o his o y o clinical mala ia, bu an ibod-
ies o EBA175 and Rh2A9 we e no . In Kenyan child en
1–8yea s old, MSP1, MSP2, Rh2A9 and schizon ex ac
also showed mode a e induc ion ollowing mala ia epi-
sodes in he p e ious yea , while EBA175 did no [53].
In sum, an ibodies o MSP1, MSP2 and schizon p o ein
ex ac may se e as good bioma ke s o se o-su eil-
lance o mala ia [10, 56], al hough o he an igens such as
AMA1 and MSP4 [53] wa an u he e alua ion.
In an s’ immune sys ems a e apidly de eloping, and
esponses o in ec ion may di e be ween younge and
olde child en in magni ude o longe i y. To in es i-
ga e his, an ibody le els and p e alence a 18mon hs
we e compa ed be ween child en ha ing mala ia be o e
6mon hs o age, o om 6 o 18mon hs. Symp oma ic
mala ia is uncommon in e y young in an s [3], and
only 31 pa icipan s (5.15%) had clinical mala ia be o e