Effect of nutrient supplementation on the acquisition of humoral immunity to Plasmodium falciparum in young Malawian children

Ba ua e al. Mala J (2018) 17:74 h ps://doi.o g/10.1186/s12936-018-2224-6 RESEARCH E ec o nu ien supplemen a ion on he acquisi ion o humo al immuni y o Plasmodium alcipa um in young Malawian child en P iyanka Ba ua1, Upeksha P. Chand asi i1, James G. Beeson1,2,3, Ka h yn G. Dewey4, Kenne h Male a5, Pe Asho n6 and S ephen J. Roge son1* Abs ac Backg ound: The e is e idence ha sugges s ha unde nu i ion has a de imen al e ec on mala ial immuni y in child en. The aim o he s udy was o disco e whe he nu ien supplemen a ion imp o ed de elopmen o mala ial an ibody immuni y in child en up o 18 mon hs o age. Me hods: The s udy was conduc ed wi h a subse o 432 Malawian child en om a andomized con olled ial o nu i ional supplemen s. The a ms included p e- and pos na al small-quan i y lipid-based nu ien supplemen s o bo h mo he and child; p ena al supplemen a ion wi h i on and olic acid; and p e- and pos na al supplemen a ion wi h mul iple mic onu ien s. Pai ed plasma samples we e collec ed a 6 and 18 mon hs o age. The le els o an ibod- ies agains me ozoi e su ace p o ein 1 (MSP1 19kD) and MSP2, e y h ocy e binding an igen 175 (EBA175), e icu- locy e binding p o ein homologue 2A (Rh2A9), schizon ex ac and a ian an igens exp essed on he su ace o in ec ed e y h ocy es we e measu ed. Resul s: A 18 mon hs o age, 5.4% o child en we e pa asi aemic by mic oscopy and 49.1% we e anaemic. An ibod- ies o he es ed me ozoi e an igens and schizon ex ac inc eased be ween 6 and 18 mon hs and his inc ease was s a is ically signi ican o MSP1, MSP2 and EBA175 (p < 0.0001) whe eas IgG o a ian su ace an igens dec eased wi h inc easing age (p < 0.0001). Howe e , he supplemen a ion ype did no ha e any impac on he p e alence o le els o an ibodies a ei he 6 o 18 mon hs o age o any o he es ed mala ia an igens in ei he uni a ia e analysis o mul i a ia e analysis a e adjus ing o co a ia es. Conclusions: P e- and pos na al lipid-based nu ien supplemen a ion did no al e mala ia an ibody acquisi ion du - ing in ancy, compa ed o p ena al supplemen a ion wi h i on and olic acid o p e- and pos na al supplemen a ion wi h mul iple mic onu ien s. T ail egis e a ion Clinical ials.go egis a ion numbe NCT01239693 Keywo ds: Mala ial immuni y in child en, 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) 2018. 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 ecommons.o g/licenses/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 ecommons.o g/ publicdomain/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 Depa men o Medicine (RMH), Pe e Dohe y Ins i u e o In ec ion and Immuni y, Uni e si y o Melbou ne, Melbou ne, VIC, Aus alia Full lis o au ho in o ma ion is a ailable a he end o he a icle Page 2 o 13 Ba ua e al. Mala J (2018) 17:74 Backg ound Mala ia is one o he leading causes o dea h in child en and p egnan women wi h an es ima ed 214 million new cases and 438,000 dea hs wo ldwide in 2015. The disease can be caused by i e di e en species o he genus Plas- modium, o which Plasmodium alcipa um causes he highes a es o mo ali y and mo bidi y and is pa icu- la ly p ominen in young child en o sub-Saha an A ica, wi h an es ima ed 292,000 dea hs in 2015 [1]. In sub-Saha an A ica, mala ia and malnu i ion o en co-exis , and bo h con ibu e signi ican ly o dea hs in young child en. Howe e , s udies o possible syne gis ic clinical e ec s o mala ia and malnu i ion ha e gi en con lic ing esul s, indica ing he need o u he s ud- ies in his a ea. Fo example, in a c oss-sec ional s udy among p e-school Kenyan child en [2] and a longi udi- nal s udy in Gambian child en unde 5yea s o age [3], s un ing was associa ed wi h inc eased mala ial isk, bu in Papua New Guinea i was epo ed ha s un ing migh p o ec child en agains clinical mala ia episodes [4]. Some o he s udies no ed no signi ican associa- ion be ween an h opome ic measu emen s [5], s un - ing [6] o unde nu i ion [7] and al e ed suscep ibili y o mala ia. A limi ed numbe o s udies ha e examined he impac o nu ien supplemen a ion on mala ia suscep ibili y in child en. Zinc and i amin A supplemen a ion educed clinical mala ia episodes caused by P. alcipa um in young child en [8–10]. In a high mala ia ansmis- sion se ing, i on supplemen a ion was associa ed wi h inc eased pa asi aemia [11] and inc eased mo ali y [12] in i on-su icien child en, whe eas he p o ision o i on wi h mic onu ien s was associa ed wi h educed isk o mala ia in i on-de icien child en [13]. O he s ud- ies ha e ound e idence o associa ions be ween acu e mala ia and de iciency o hiamine [14] and an ioxidan s including i amin E [15], which sugges s hey ha e oles in p o ec ion agains mala ia. While he e is limi ed e i- dence ha supplemen a ion wi h mic onu ien s such as zinc o i amin B12 can imp o e an ibody esponse o accina ion [16, 17], he abili y o mic o- o mac onu i- en supplemen a ion o a ec he acquisi ion o an ibody o pa hogens ollowing na u al exposu e is unknown. The aim o his s udy was o iden i y whe he p e- and pos na al nu i ional supplemen s could imp o e mala - ial immuni y in young child en. The s udy was pa o a nu ien supplemen a ion clinical ial, he In e na- ional Lipid-based Nu ien Supplemen (iLiNS) P ojec DYAD-Malawi ial (clinical ials.go egis a ion num- be NCT01239693). Fo his epo , he le el and p e a- lence o an ibody o me ozoi e an igens, schizon ex ac and a ian su ace an igens (VSA) exp essed by P. alci- pa um-in ec ed e y h ocy es (IEs) we e de e mined in in an s aged 6 and 18mon hs as an ibodies o me ozoi e an igens and VSAs a e belie ed o play impo an oles in media ing acqui ed immuni y agains mala ia [18, 19]. Me hods S udy loca ion and pa icipan s The s udy pa icipan s we e a coho o 432 in an s esid- ing in Lungwena, Malindi and Mangochi om u al Malawi who pa icipa ed in he iLiNS P ojec DYAD- Malawi nu ien supplemen a ion ial, pa o he iLiNS P ojec [20]. The de ails o he ial design and supple- men s ha e been published elsewhe e [21]. In b ie , pa - icipa ing p egnan women we e andomly alloca ed o ecei e i on and olic acid (IFA), mul iple mic onu ien s (MMN) o a small-quan i y (20g) o lipid based nu i- en supplemen (LNS) daily. A e deli e y, women in he IFA g oup ecei ed placebo able s, while MMN and LNS supplemen a ion was con inued du ing he i s 6mon hs o lac a ion. Child en o mo he s in he LNS g oup also ecei ed LNS 10g wice daily om 6 o 18mon hs o age. A 18mon hs o age, an h opome ic assessmen s e ealed no signi ican di e ences in he child en’s mean leng h, mean weigh , he p e alence o s un ing, o head o mid-uppe a m ci cum e ence be ween he in e en- ion g oups o all pa icipan s in he iLiNS p ojec [22]. Plasma samples we e collec ed om in an s a 6 and 18mon hs o age. A hese ime poin s blood haemo- globin concen a ion was measu ed wi h a Hemo-Cue® haemoglobinome e om enous blood samples. Mala ia pa asi aemia was sough by mic oscopic examina ion o hick blood ilm and by apid diagnos ic es (RDT) using Clea iew® Mala ia Combo (B i ish Biocell In e na ional L d., Dundee, UK). Plasma samples p epa a ion Blood om pa icipan s was sepa a ed by cen i uga ion sho ly a e collec ion and plasma was s o ed a −80°C be o e shipmen on d y ice o Aus alia. Plasma samples we e hea -inac i a ed o 45min a 57°C o inac i a e complemen p o eins. The hea -inac i a ed samples we e hen s o ed a −80°C. Cul u ing and main aining pa asi es The P. alcipa um lines used we e E8B-ICAM, R29 and 3D7 a A o e -exp essing pa asi e line. E8B-ICAM adhe es o ICAM-1 and CD36 [23], and exp esses g oup B/C a genes whe eas R29 exp esses g oup A a genes and o ms ose es [24]. The 3D7 line spon aneously exp essed a g oup A a gene as i s dominan ansc ip [25]; i s binding ligands ha e no been cha ac e ized. The pa asi es we e g own and main ained in cul u e as desc ibed p e iously [26]. IEs we e synch onized wi h 5% so bi ol and subjec o gela in lo a ion egula ly [27]. Page 3 o 13 Ba ua e al. Mala J (2018) 17:74 To selec R29 o ose ing, gela in lo a ion wi hou hen wi h hepa in li hium sal , 0.05 mg/ml Sigma Ald ich), was pe o med. Measu ing IgG o mala ia me ozoi e an igens and schizon ex ac Recombinan me ozoi e p o ein 1 (MSP-1 19 kD, 3D7 clone), egion III-V o e y h ocy e binding an igen 175 (EBA 175), and P. alcipa um e iculocy e binding hom- ologue 2 (P Rh2, cons uc P Rh2-2030) we e exp essed in Esche ichia coli as p e iously epo ed [28–30]. Full- leng h MSP-2 (FC27 clone) exp essed in E. coli was kindly p o ided by Robin Ande s (La T obe Uni e si y, Aus alia). The schizon ex ac was p epa ed acco ding o a p e iously published me hod [31]. B ie ly, magne ic- ac i a ed cell so ing (MACS) pu i ied schizon pelle was mixed wi h h ee imes he olume o he pelle wi h PBS. Cell-lysis was done by eeze- hawing o 6 imes, hen i was spun down o cla i y he supe na an and his ex ac was used a e op imizing he coa ing concen a ion. Each me ozoi e an igen was coa ed a 0.5–2 µg/ml and schizon ex ac a 1:8000 dilu ion on o 384 well NUNC MaxiSo p™ pla es (The mo Fishe Scien i ic Inc, MA, USA) and le o e nigh a 4°C. The pla es we e washed wi h PBS/Tween 20 and non-speci ic binding was blocked wi h 0.1% casein (The mo Fishe Scien i ic) on he ollowing day. The pla es we e hen incuba ed wi h pa icipan se a dilu ed a 1:250 in 0.1% casein in ipli- ca es o 1h and washed. Ho se adish pe oxidase-conju- ga ed goa an i-human IgG (Li e Technologies, Aus alia) was added a 1:2500 dilu ion o 1h and pla es we e again washed. ABTS [2,2′-azino-bis(3-e hylbenz hiazoline- 6-sul onic acid)] was added as enzyme–subs a e o 15min and abso bance a 405nm was measu ed using a BMG POLARs a Omega luo ime e (BMG Lab ech, Ge many). Measu emen o o al IgG le els agains VSA To al IgG an ibody le els agains VSAs exp essed on he su ace o IEs we e measu ed by low cy ome y as p e- iously desc ibed [32] wi h sligh modi ica ions. These an ibodies a e belie ed o p ima ily a ge P. alcipa um e y h ocy e memb ane an igen 1 (P EMP1) [33]. In b ie , 2.5μl o pa ien se a (1:20 dilu ion) we e co-incuba ed wi h IEs a 0.2% haema oc i and a app oxima ely 7–8% pa asi aemia, dilu ed in PBS solu ion wi h 1% HI-FBS (Hea inac i a ed e al bo ine se um) o 30 min. Fol- lowing incuba ion he cells we e washed 3 imes wi h PBS/1% HI-FBS and incuba ed wi h 25μl o 1:100 ab- bi an i-human IgG (Dako, Aus alia) dilu ed in PBS/1% HI-FBS. The cells we e washed again in PBS/1% HI-FBS and incuba ed wi h Alexa Fluo 647 donkey an i- abbi IgG in 1:500 dilu ion (Li e Technologies, Aus alia) and 10 μg/ml e hidium b omide (E B ) in PBS/1% HI-FBS o 30min. Following incuba ion he cells we e washed in PBS/1% HI-FBS and esuspended in ice-cold 2% pa a- o maldehyde ixa i e solu ion (p epa ed in PBS). The ixed IEs we e hen un h ough a Hype Cy ® sys em wi h a pla e eade adap e (In ellicy ®, NM, USA) con- nec ed o a Cyan low cy ome e (Beckman Coul e Inc., CA, USA) whe e he cells we e acqui ed. The low cy ome y da a was analysed acco ding o a p e iously published me hod [32]. Da a analysis S a is ical analyses we e pe o med using S a a e sion 13.0 (S a aCo p, Texas, USA). S a is ical analyses we e pe o med acco ding o a p e-planned and app o ed analysis plan a ailable a [34]. Measu ed an ibody le els (in op ical densi y [OD] o schizon and me ozoi e an i- gens and geome ic mean luo escence in ensi y [MFI] o VSA) we e p esen ed as a pe cen age o he posi i e con ol. The posi i e con ol came om a pool o plasma om mala ia immune A ican adul s whe eas he nega- i e con ol came om plasma samples om 3 Mel- bou ne dono s o he ELISA and 8 Melbou ne dono s o he low cy ome y assay. Se op e alence was de ined as he pe cen age o he coho ha ing an ibody esponses g ea e han he mean an ibody esponse plus h ee s anda d de ia ions o neg- a i e con ols, which we e mala ia naï e samples om Melbou ne blood dono s. Socioeconomic s a us (SES) was calcula ed on he basis o a sco ing sys em o house- hold asse s (HHA) adap ed om [35]. Pa icipan cha ac e is ics including demog aphic and basic clinical cha ac e is ics we e ca ego ized by in e - en ion g oups and he median and in e qua ile ange o each cha ac e is ic we e abula ed. Di e ences in cha ac e is ics ac oss he g oups we e de e mined by Mann–Whi ney es (non-pa ame ic con inuous a i- ables wi h wo g oups), K uskal–Wallis (non-pa ame ic con inuous a iables wi h mo e han wo g oups) o Chi2 es ( o ca ego ical a iables) whe e applicable. Di e - ences in he an ibody le el and he se oposi i i y be ween 6 and 18mon hs old child en we e de e mined by Wil- coxon ma ched-pai s signed- anks es and McNema ’s es , espec i ely. S a is ical di e ences be ween he g oups we e epo ed as p<0.05 and 95% con idence in e als we e also epo ed o he analyses. An ibody le els a 6 and 18 mon hs o age we e epo ed as he median pe cen age o he posi i e con- ol and he in e qua ile ange (IQR). The K uskal–Wal- lis es was pe o med o compa e an ibody le els ac oss supplemen a ion g oups. Reg ession analyses we e ca - ied ou using na u al loga i hmically ans o med an i- body le els which we e back ans o med o epo ing Page 4 o 13 Ba ua e al. Mala J (2018) 17:74 desc ip i e esul s. Linea eg ession uni a ia e analysis was pe o med be ween LNS e sus IFA, LNS e sus MMN and MMN e sus IFA o de e mine he an ibody le el di e ences be ween supplemen a ion g oups. Mul- i a ia e eg ession was also pe o med adjus ing o he ollowing co a ia es: ma e nal BMI a en olmen , du a- ion o ges a ion ( om en olmen o deli e y), numbe o p egnancies, sex o he child, ma e nal educa ion, p oxy o SES, s udy si e, ma e nal anaemic s a us a en ol- men , ma e nal HIV s a us and bed ne use by child en and hese co a ia es we e uni o mly included in all he adjus ed analyses acco ding o he p e-speci ied analysis plan. Fo bo h uni a ia e and mul i a ia e analyses, coe - icien s and 95% con idence in e als (CI) we e epo ed. The numbe and he pe cen age o child en who we e se oposi i e o each mala ia an igen we e epo ed by supplemen a ion g oups, and chi2 es was pe o med o de e mine he di e ences ac oss he supplemen a ion g oups. Uni a ia e logis ic eg ession was pe o med be ween LNS e sus IFA, LNS e sus MMN and MMN e sus IFA o de e mine he di e ences in se oposi i i y be ween di e en supplemen a ion g oups. Mul i a i- a e logis ic eg ession was pe o med adjus ing o he abo e-men ioned co a ia es, epo ing odds a ios (OR) and 95% CI. Resul s S udy popula ion cha ac e is ics F om a o al o 1391 en olled p egnan women ec ui ed o he ial, 869 comple ed he in e en ion and ollow- up o 18mon hs a e deli e y. Fou hund ed and hi y- wo single on child en om hese women wi h sample a ailabili y a bo h 6 and 18mon hs we e es ed o his s udy (Fig.1). O he 432 child en (47.9% male and 52.1% emale) es ed a 6 and 18mon hs o age, 33.6% we e om he IFA g oup, 32.4% om he MMN g oup and 34.0% in he LNS g oup. Table1 summa izes he pa icipan cha ac e is ics Fig. 1 Pa icipan low in Consolida ed S anda ds o Repo ing T ials ecommended o ma ; adap ed and modi ied om [22]. ges ges a ion, IFA i on and olic acid, LNS lipid-based nu ien supplemen , MMN mul iple mic onu ien s Page 5 o 13 Ba ua e al. Mala J (2018) 17:74 acco ding o di e en supplemen a ion g oups. The cha - ac e is ics did no di e subs an ially be ween he h ee in e en ion g oups. The pe cen ages o child en who we e pa asi aemic a 6mon hs we e highe in child en om he IFA g oup (8.3% by mic oscopy and 7.6% by RDT) compa ed o MMN (3.6% by mic oscopy and 7.1% by RDT) o LNS (3.4% by mic oscopy and 6.8% by RDT) bu he di e ences we e non-signi ican . The cha ac e is ics o he included and excluded chil- d en a 6mon hs a e illus a ed in Table2. Signi ican ly mo e included child en had pa asi aemia by mic oscopy (P=0.01), bu esul s o RDT showed no such di e ence. The pe cen age o anaemia a 6mon hs was signi ican ly highe (P<0.0001) in he excluded pa icipan s (71.7%) han he included g oup (58.1%). The magni ude and p e alence o an ibodies in di e en age g oups The le els o an ibodies and se op e alence o he an i- bodies agains me ozoi e an igens, schizon ex ac and VSA o h ee di e en pa asi e lines we e measu ed a 6mon hs and 18mon hs o age (Table3). The an ibody le els o he me ozoi e an igens and schizon ex ac we e signi ican ly highe a 18 mon hs compa ed o he le els a 6mon hs and i was s a is ically signi ican (P<0.0001) o MSP1, MSP2 and EBA175. Howe e , he le els o na u ally acqui ed IgG agains VSAs we e signi ican ly lowe in child en a 18mon hs o age compa ed o he same child en a 6mon hs. This di - e ence was signi ican (<0.0001) o all he es ed pa a- si e lines. As wi h he an ibody le el da a, se op e alence o an i- bodies agains he es ed me ozoi e an igens and schizon ex ac we e also signi ican ly highe a 18mon hs com- pa ed o he se op e alence a 6mon hs. The highes pe - cen age o se oposi i i y was obse ed agains MSP1 in Table 1 Pa icipan cha ac e is ics acco ding o supplemen a ion g oups a I on and olic acid supplemen a ion b Mul iple mic onu ien supplemen a ion c Lipid-based nu ien supplemen s d P alue ob ained by K uskal–Wallis es (con inuous a iables) o Chi squa e es e Rapid diagnos ic es Anaemia de ined as haemoglobin le el < 110 g/l (Wo ld Heal h O ganiza ion [1]) Cha ac e is ics IFAa n (%) MMNb n (%) LNSc n (%) P alued Numbe o child en 145 (33.6) 140 (32.4) 147 (34.0) 0.87 Male 69 (47.6) 61 (43.6) 77 (52.4) 0.32 Pa asi aemia by mic oscopy a 6 mon hs 12 (8.3) 5 (3.6) 5 (3.4) 0.10 Pa asi aemia by RDTe a 6 mon hs 11 (7.6) 10 (7.1) 10 (6.8) 0.97 Haemoglobin le el a 6 mon hs, mean ± SD, g/l 102.7 ± 16.7 103.6 ± 16.0 103.7 ± 14.7 0.74 Anaemia a 6 mon hs 86 (59.3) 81 (57.9) 84 (57.1) 0.93 Low socioeconomic s a us 95 (65.5) 75 (53.6) 81 (55.1) 0.08 Mo he ’s educa ion below median 79 (54.5) 66 (47.1) 80 (54.4) 0.34 Table 2 Compa a i e cha ac e is ics o he included and excluded pa icipan s a 6 mon hs o age Values a e numbe (%) o mean ± SD a P alue ob ained by Chi squa e es o Mann–Whi ney es (con inuous a iables) b Rapid diagnos ic es c Anaemia de ined as haemoglobin le el < 110 g/l (Wo ld Heal h O ganiza ion [1]) Cha ac e is ics Included (432) Excluded (349) P aluea Pa asi aemia by mic os- copy 22 (5.1) 6 (1.7) 0.01 Pa asi aemia by RDTb31 (7.17) 35 (10.0) 0.15 Haemoglobin le el, mean ± SD, g/l 103.4 ± 15.8 101.4 ± 15.9 0.09 Anaemiac251 (58.1) 250 (71.7) < 0.0001 Low socioeconomic s a us 251 (58.1) 215 (61.6) 0.32 Mo he ’s educa ion below median 225 (52.1) 193 (55.3) 0.37 Page 6 o 13 Ba ua e al. Mala J (2018) 17:74 18mon h old child en; 54.6% compa ed o 29.6% se oposi- i es a 6mon hs (P<0.0001). Signi ican inc eases in se o- posi i i y wi h inc easing age we e also obse ed o Rh2A9 (P<0.0001) and schizon ex ac (P=0.0314). Howe e , he se op e alence o IgG agains VSAs o di e en pa asi e lines declined signi ican ly be ween 6 and 18mon hs o age o he h ee es ed pa asi e lines (P<0.0001 o IgG agains E8B and 3D7 and P=0.0016 o R29). Associa ion be ween nu ien supplemen a ion and an ibody se op e alence in 6 mon hs old child en Se op e alence o he mala ia an igens in 6mon hs old child en was no signi ican ly di e en be ween any o he ea men a ms o any o he es ed an igens excep Rh2A9 (P=0.044) (Table4). Adjus men o he analysis o he selec ed co a ia es did no signi ican ly change he esul s o he analysis, and he only signi ican di e ence was obse ed o he odds a io o Rh2A9 be ween LNS and IFA g oup in he uni a ia e analysis (P=0.047), wi h he odds o being se oposi i e a 6mon hs o age in LNS g oup being 54% less han in he IFA g oup. This associa- ion did no emain signi ican in he adjus ed analysis. Associa ion be ween nu ien supplemen a ion and an ibody se op e alence in 18 mon hs old child en Se op e alence o he es ed mala ia an igens in 18 mon hs old child en was no signi ican ly di e en be ween any o he ea men a ms o any o he an i- gens es ed (Table 5). Adjus men o he analysis o he selec ed co a ia es did no signi ican ly change he esul s o he analysis, and no signi ican di e ences in an ibody se op e alence we e obse ed be ween he di - e en supplemen a ion g oups o any o es ed an igens. Associa ion be ween nu ien supplemen a ion and an ibody le els in 6 mon h old child en The le el o an ibodies did no di e signi ican ly acco d- ing o di e en nu ien supplemen a ion g oups o any o he es ed an igens a 6mon hs o age. Mo eo e , mul- i a ia e linea eg ession showed no signi ican di e - ences in he le els o an ibodies agains any o he es ed an igens when hey we e ca ego ized by di e en supple- men a ion g oups (Table6). Associa ion be ween nu ien supplemen a ion and an ibody le els in 18 mon h old child en The le el o an ibodies did no di e signi ican ly be ween di e en nu ien supplemen a ion g oups o any o he es ed an igens excep o IgG agains E8B pa asi e line (P=0.043) in 18mon hs old child en. Mul i a ia e linea eg ession showed no signi ican di e ences in he le els o an ibodies agains any o he es ed an igens be ween he di e en supplemen a ion g oups (Table7). Discussion This s udy in es iga ed he impac o nu i ional sup- plemen a ion on mala ial immuni y in a subse o young child en om a andomized con olled ial o p e-na al Table 3 Magni ude and p e alence o an ibodies in 432 child en a 6 and 18 mon hs a Plasmodium alcipa um b An ibody le el p esen ed as a pe cen age o he posi i e con ol showing he median and in e qua ile ange c Se oposi i i y de ined as sample mean op ical densi y o luo escence in ensi y > mean + 3 s anda d de ia ions o he nega i e con ols d P alue calcula ed using Wilcoxon ma ched-pai s signed- anks es e P alue calcula ed using McNema ’s es Me ozoi e su ace p o ein 1 g Me ozoi e su ace p o ein 2 h E y h ocy e binding an igen 175 i Re iculocy e binding p o ein homologue 2A Signi ican P alues <0.05 a e indica ed in i alics An igen es ed/P a isola e An ibody le elbP aluedAn ibody se oposi i i yc (%) P aluee 6 mon hs 18 mon hs 6 mon hs 18 mon hs MSP1 0.97 [0.38, 2.89] 2.80 [0.83, 6.35] < 0.0001 128 (29.6) 236 (54.6) < 0.0001 MSP2g2.14 [1.31, 3.59] 2.95 [0.92, 9.12] < 0.0001 114 (26.3) 117 (27.0) 0.1004 EBA175h2.50 [1.44, 4.13] 3.73 [2.19, 18.73] < 0.0001 48 (11.1) 51 (11.8) 0.5764 Rh2A9i5.49 [2.01, 33.78] 8.5 [4.68, 14.29] 0.2829 14 (3.2) 122 (28.2) < 0.0001 Schizon 1.92 [0.84, 6.99] 3.32 [1.44, 6.89] 0.2522 202 (46.7) 233 (53.9) 0.0314 E8B 0.20 [0.03, 0.45] 0 [0, 0.21] < 0.0001 105 (25.2) 34 (7.8) < 0.0001 R29 0.18 [0, 0.63] 0 [0, 0.11] < 0.0001 37 (9.1) 16 (3.7) 0.0016 3D7 0.23 [0.01, 0.53] 0 [0, 0.29] < 0.0001 94 (22.2) 36 (8.3) < 0.0001 Page 7 o 13 Ba ua e al. Mala J (2018) 17:74 nu ien supplemen a ion wi h IFA o p e- and pos na al MMN o LNS. An ibodies o se e al impo an me ozoi e an igens (MSP1, MSP2, EBA175, Rh2A9), schizon ex ac and VSA o h ee di e en pa asi e lines (E8B-ICAM, R29 and 3D7 o e exp essing a A) we e measu ed in o de o de e mine whe he nu ien supplemen a ion imp o es he acquisi ion o mala ia an ibody in young child en. In mala ia endemic a eas, an ibodies o mala ia an i- gens inc ease wi h age, being highe in adul s han in child en, bu he dynamics o an ibody p oduc ion in in an s a e less well s udied [36]. The obse a ions ha he le els and se op e alence o an ibody o mos o he me ozoi e an igens es ed inc eased be ween 6 and 18mon hs o age a e in ag eemen wi h obse a ions in a case con ol s udy conduc ed in Kenyan child en, in which he le el o an ibodies agains MSP1 and P Rh2 inc eased s eadily wi h inc easing age om bi h o 2yea s [37], and a coho s udy om Benin [38], in which MSP1 and MSP2 an ibody le els showed a cons an inc ease un il 18mon hs o age. In a highe ansmission Table 4 Associa ion be ween nu ien supplemen a ion and se op e alence in 6 mon hs old child en a I on and olic acid b Mul iple mic onu ien s c lipid based nu ien supplemen s d P alue calcula ed using he Chi2 es e P alue calcula ed using logis ic eg ession epo ing Odds Ra ios (OR) and 95% Con idence in e als (CI) Me ozoi e su ace p o ein 1 g P alue calcula ed using mul i a ia e logis ic eg ession epo ing odds a ios (OR) while adjus ing o ma e nal BMI a en olmen , du a ion o ges a ion ( om en olmen o deli e y), numbe o p egnancies, sex o he child, ma e nal educa ion, socioeconomic s a us, s udy si e, ma e nal anaemic s a us a en olmen , ma e nal HIV s a us and bed ne use by child en h Me ozoi e su ace p o ein 2 i E y h ocy e binding an igen 175 j Re iculocy e binding p o ein homologue 2A k Va ian su ace an igens Signi ican P alues <0.05 a e indica ed in i alics Ou come Numbe o child en se oposi i e/ o al numbe o child en Compa ison be ween LNS and IFA g oup Compa ison be ween LNS and MMN g oup Compa ison be ween MMN and IFA g oup IFAaMMNbLNScP aluedOR (95% CI) P alueeOR (95% CI) P alueeOR (95% CI) P aluee MSP-1 19kD 43/145 (29.7%) 44/140 (31.4%) 41/147 (27.9%) 0.806 0.96 (0.74, 1.23) 0.739 0.84 (0.51, 1.40) 0.512 1.07 (0.64, 1.77) 0.804 Adjus ed modelg0.97 (0.73, 1.28) 0.808 0.86 (0.49, 1.49) 0.583 1.01 (0.57, 1.76) 0.981 MSP-2h38/145 (26.2%) 40/140 (28.6%) 36/147 (24.5%) 0.734 0.96 (0.73, 1.24) 0.736 0.81 (0.48, 1.37) 0.434 1.07 (0.63, 1.80) 0.807 Adjus ed modelg0.98 (0.74, 1.31) 0.893 0.80 (0.45, 1.42) 0.447 1.09 (0.62, 1.94) 0.764 EBA-175i15/145 (10.3%) 20/140 (14.3%) 13/147 (8.9%) 0.320 0.92 (0.62, 1.35) 0.663 0.58 (0.28, 1.22) 0.152 1.34 (0.65, 2.76) 0.426 Adjus ed modelg0.96 (0.63, 1.47) 0.857 0.54 (0.25, 1.18) 0.120 1.53 (0.70, 3.37) 0.289 Rh2A9j9/145 (6.21%) 3/140 (2.14%) 2/147 (1.4%) 0.044 0.46 (0.21, 0.99) 0.047 0.63 (0.10, 3.83) 0.616 0.33 (0.09, 1.23) 0.098 Adjus ed modelg0.48 (0.21, 1.06) 0.070 0.79 (0.12, 5.42) 0.814 0.34 (0.09, 1.30) 0.114 Schizon ex ac 68/145 (46.9%) 67/140 (47.86%) 67/147 (45.6%) 0.927 0.97 (0.77, 1.23) 0.821 0.91 (0.57, 1.45) 0.699 1.07 (0.67, 1.71) 0.774 Adjus ed modelg0.91 (0.70, 1.18) 0.468 0.89 (0.53, 1.51) 0.677 0.97 (0.58, 1.63) 0.923 VSAk o E8B pa asi e line 31/145 (21.4%) 39/140 (27.9%) 35/147 (23.8%) 0.437 1.09 (0.83, 1.43) 0.552 0.80 (0.47, 1.36) 0.402 1.52 (0.88, 2.64) 0.134 Adjus ed modelg1.01 (0.74, 1.37) 0.957 0.70 (0.38, 1.26) 0.233 1.55 (0.86, 2.80) 0.146 VSA o R29 pa asi e line 14/145 (9.7%) 12/140 (8.6%) 11/147 (7.5%) 0.803 0.86 (0.57, 1.30) 0.470 0.83 (0.35, 1.94) 0.659 0.88 (0.39, 1.98) 0.756 Adjus ed modelg0.76 (0.48, 1.20) 0.234 0.69 (0.27, 1.74) 0.427 0.91 (0.38, 2.14) 0.822 VSA o 3D7 pa asi e line 27/145 (18.6%) 33/140 (23.6%) 34/147 (23.1%) 0.530 1.16 (0.87, 1.54) 0.313 0.97 (0.56, 1.69) 0.924 1.35 (0.76, 2.41) 0.301 Adjus ed modelg1.04 (0.75, 1.43) 0.829 0.89 (0.48, 1.65) 0.721 1.24 (0.66, 2.31) 0.501 Page 8 o 13 Ba ua e al. Mala J (2018) 17:74 se ing, he se op e alence o an ibody o MSP1 eached a peak a e 6mon hs o age, whe eas p e alence o an i- body o MSP2 peaked a 9mon hs o age [39]. In con as o he inc ease in an ibodies o me ozoi e an igens, he le els o an ibodies agains VSA dec eased om 6 o 18 mon hs, sugges ing an ongoing loss o ma e nal an i-VSA an ibodies wi h li le o no de elop- men o he child’s own VSA an ibody esponses. Con- sis en wi h his, in a ecen s udy an i-VSA an ibodies waned by 6–9mon hs o age and did no eappea du - ing in ancy and ea ly childhood [40]. I is possible ha he an i-VSA an ibodies equi e mo e in ec ions han he me ozoi e an igens o de elop, and an i-VSA esponses a e known o o en be sho -li ed ollowing in ec ion [41]. This may be ela ed o he highly a ian na u e o VSA [42], equi ing epea ed exposu e o de elop c oss- eac i e an ibodies o mul iple VSA a ian s, whe eas me ozoi e an igens a e mo e conse ed. The s udy ound no e idence ha nu ien supplemen- a ion al e ed he de elopmen o an ibody esponses o ei he me ozoi e an igens o VSA in young child en, in acco dance wi h a s udy conduc ed on Beninese child en Table 5 Associa ion be ween nu ien supplemen a ion and se op e alence in 18 mon hs old child en a I on and olic acid b Mul iple mic onu ien s c lipid based nu ien supplemen s d P- alue calcula ed using he Chi2 es e P- alue calcula ed using logis ic eg ession epo ing Odds Ra ios (OR) and 95% Con idence in e als (CI) Me ozoi e su ace p o ein 1 g P- alue calcula ed using mul i a ia e logis ic eg ession epo ing odds a ios (OR) while adjus ing o ma e nal BMI a en olmen , du a ion o ges a ion ( om en olmen o deli e y), numbe o p egnancies, sex o he child, ma e nal educa ion, socioeconomic s a us, s udy si e, ma e nal anaemic s a us a en olmen , ma e nal HIV s a us and bed ne use by child en h Me ozoi e su ace p o ein 2 i E y h ocy e binding an igen 175 j Re iculocy e binding p o ein homologue 2A k Va ian su ace an igens Ou come Numbe o child en se oposi i e/ o al numbe o child en Compa ison be ween LNS and IFA g oup Compa ison be ween LNS and MMN g oup Compa ison be ween MMN and IFA g oup IFAaMMNbLNScP aluedOR (95% CI) P alueeOR (95% CI) P alueeOR (95% CI) P aluee MSP-1 19kD 82/145 (56.5%) 72/140 (51.4%) 82/147 (55.7%) 0.646 0.98 (0.78, 1.24) 0.895 1.19 (0.75, 1.90) 0.460 0.83 (0.52, 1.33) 0.444 Adjus ed modelg0.98 (0.77, 1.25) 0.865 1.10 (0.67, 1.82) 0.708 0.80 (0.48, 1.34) 0.398 MSP-2h42/145 (28.9%) 39/140 (27.8%) 36/147 (24.4%) 0.669 0.88 (0.66, 1.16) 0.359 0.79 (0.45, 1.40) 0.428 0.95 (0.54, 1.66) 0.851 Adjus ed modelg0.82 (0.60, 1.13) 0.226 0.63 (0.34, 1.18) 0.149 0.96 (0.53, 1.76) 0.906 EBA-175i19/145 (13.1%) 14/140 (10.0%) 18/147 (12.2%) 0.705 0.99 (0.67, 1.43) 0.967 1.59 (0.69, 3.63) 0.273 0.61 (0.27, 1.40) 0.242 Adjus ed modelg0.91 (0.60, 1.37) 0.642 1.19 (0.48, 2.95) 0.710 0.65 (0.27, 1.58) 0.342 Rh2A9j42/145 (28.9%) 40/140 (28.5%) 40/147 (27.2%) 0.941 0.96 (0.74, 1.24) 0.739 0.93 (0.56, 1.57) 0.797 0.96 (0.57, 1.61) 0.880 Adjus ed modelg0.96 (0.73, 1.26) 0.763 0.84 (0.48, 1.47) 0.540 0.98 (0.56, 1.71) 0.931 Schizon ex ac 73/145 (50.3%) 78/140 (55.7%) 82/147 (55.7%) 0.568 1.12 (0.89, 1.40) 0.352 1.003 (0.63, 1.60) 0.991 1.24 (0.78, 1.99) 0.365 Adjus ed modelg1.18 (0.90, 1.54) 0.223 0.87 (0.52, 1.46) 0.600 1.40 (0.83, 2.35) 0.208 VSAk o E8B pa asi e line 14/145 (9.6%) 11/140 (7.9%) 9/147 (6.1%) 0.534 0.78 (0.51, 1.21) 0.266 0.76 (0.30, 1.89) 0.554 0.78 (0.33, 1.83) 0.562 Adjus ed modelg0.80 (0.50, 1.29) 0.360 0.72 (0.27, 1.89) 0.504 0.75 (0.29, 1.93) 0.557 VSA o R29 pa asi e line 7/145 (4.8%) 5/140 (3.5%) 4/147 (2.7%) 0.632 0.74 (0.40, 1.39) 0.351 0.76 (0.20, 2.87) 0.680 0.72 (0.22, 2.32) 0.582 Adjus ed modelg0.79 (0.39, 1.60) 0.506 0.95 (0.22, 4.07) 0.946 0.85 (0.23, 3.14) 0.808 VSA o 3D7 pa asi e line 15/145 (10.3%) 11/140 (7.8%) 10/147 (6.8%) 0.532 0.80 (0.52, 1.21) 0.283 0.86 (0.35, 2.08) 0.732 0.85 (0.37, 1.97) 0.710 Adjus ed modelg0.74 (0.46, 1.22) 0.241 0.55 (0.20, 1.52) 0.250 1.11(0.43, 2.83) 0.830 Page 9 o 13 Ba ua e al. Mala J (2018) 17:74 Table 6 Associa ion be ween nu ien supplemen a ion and an ibody le els in 6 mon hs old child en a I on and olic acid b Mul iple mic onu ien s c lipid based nu ien supplemen s d P alue calcula ed using K uskal–Wallis es e P alue calcula ed using linea eg ession o an ibody le els be ween supplemen a ion g oups epo ing coe icien and 95% Con idence in e als (CI) Me ozoi e su ace p o ein 1 g P alue calcula ed using mul i a ia e eg ession adjus ing o ma e nal BMI a en olmen , du a ion o ges a ion ( om en olmen o deli e y), numbe o p egnancies, sex o he child, ma e nal educa ion, socioeconomic s a us, s udy si e, ma e nal anaemic s a us a en olmen , ma e nal HIV s a us and bed ne use by child en h Me ozoi e su ace p o ein 2 i E y h ocy e binding an igen 175 j Re iculocy e binding p o ein homologue 2A k Va ian su ace an igens Ou come An ibody le els by s udy g oup, median (IQR) LNS and IFA LNS and MMN MMN and IFA IFAaMMNbLNScP aluedCoe (95% CI) P alueeCoe (95% CI) P alueeCoe (95% CI) P aluee Numbe o pa icipan s N = 145 N = 140 N = 147 MSP-1 19kD 0.81 (0.38, 2.45) 1.13 (0.37, 3.02) 1.03 (0.40, 0.72) 0.430 1.03 (0.85, 1.25) 0.773 0.88 (0.60, 1.28) 0.498 1.15 (0.78, 1.70) 0.467 Adjus ed modelg1.06 (0.87, 1.30) 0.542 0.94 (0.63, 1.39) 0.755 1.11 (0.75, 1.64) 0.614 MSP-2h2.36 (1.43, 3.57) 2.02 (1.33, 4.13) 1.98 (1.19, 3.27) 0.313 0.99 (0.87, 1.12) 0.833 0.87 (0.66, 1.15) 0.334 1.08 (0.82, 1.42) 0.592 Adjus ed modelg0.99 (0.86, 1.13) 0.834 0.88 (0.66, 1.19) 0.417 1.06 (0.80, 1.40) 0.703 EBA-175i2.37 (1.34, 3.85) 2.60 (1.50, 4.50) 2.50 (1.42, 4.02) 0.335 1.03 (0.92, 1.14) 0.610 0.91 (0.74, 1.13) 0.390 1.14 (0.91, 1.43) 0.240 Adjus ed modelg1.04 (0.93, 1.16) 0.502 0.86 (0.69, 1.08) 0.195 1.24 (0.99, 1.54) 0.061 Rh2A9j4.93 (1.53,18.55) 5.97 (2.12, 39.95) 5.86 (2.45, 33.70) 0.245 1.11 (0.89, 1.38) 0.364 1.00 (0.66, 1.53) 0.986 1.23 (0.77, 1.95) 0.382 Adjus ed modelg1.08 (0.85, 1.37) 0.532 0.99 (0.63, 1.57) 0.990 1.16 (0.70, 1.93) 0.559 Schizon ex ac 1.85 (0.80, 4.93) 2.11 (0.82, 8.08) 2.06 (0.89, 7.23) 0.569 1.07 (0.89, 1.28) 0.465 0.97 (0.68, 1.40) 0.875 1.21 (0.85, 1.73) 0.282 Adjus ed modelg1.06 (0.89, 1.27) 0.481 0.88 (0.61, 1.26) 0.475 1.30 (0.91, 1.85) 0.155 VSAk o E8B pa asi e line 0.17 (0.03, 0.46) 0.26 (0.06, 0.47) 0.19 (0.02, 0.44) 0.453 1.04 (0.87, 1.24) 0.688 1.00 (0.70, 1.42) 0.995 1.07 (0.76, 1.51) 0.709 Adjus ed modelg1.00 (0.82, 1.21) 0.996 0.99 (0.68, 1.47) 0.987 1.02 (0.71, 1.47) 0.925 VSA o R29 pa asi e line 0.16 (0, 0.59) 0.25 (0, 0.81) 0.12 (0, 0.55) 0.452 0.93 (0.75, 1.15) 0.496 0.70 (0.46, 1.07) 0.098 1.21 (0.80, 1.82) 0.358 Adjus ed modelg0.91 (0.73, 1.13) 0.407 0.63 (0.41, 0.97) 0.063 1.30 (0.88, 1.91) 0.187 VSA o 3D7 pa asi e line 0.19 (0.01, 0.48) 0.24 (0.02, 0.64) 0.26 (0.01,0.59) 0.544 1.19 (0.98, 1.46) 0.081 1.06 (0.72, 1.55) 0.774 1.34 (0.91, 1.99) 0.138 Adjus edg1.13 (0.92, 1.39) 0.245 1.05 (0.69, 1.61) 0.810 1.18 (0.81, 1.73) 0.382