epi.1986.wahlgren.ajtmh.anti-plasmodium falciparum antibodies acquired by residents in a holoendemic...

8/14/2019 Epi.1986.Wahlgren.ajtmH.anti-Plasmodium Falciparum Antibodies Acquired by Residents in a Holoendemic Area of

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Am . J. Trop. M ed. H yg.. 35(1), 1986, pp. 2229Copy ri gh t 1986 b y Th e Amer ic an Soc ie ty o f T r op ic al Me di ci ne a nd Hygi en e

ANTI-PLASMODIUM FALCIPARUM ANTIBODIES ACQUIREDBY RESIDENTS IN A HOLOENDEMIC AREA OF LIBERIADURING DEVELOPM ENT OF CLINICAL IMMUNITYMAT S WAHLGREN ,*t A ND ER S B JO RKMAN,66t H EDVIG P ER LMANN,8KLAVS BERZIN S,* ANDPETER PERLMANN@

*De pa rtm en t o f Immu no lo gy, U niv ers ity o f S to ckh olm , S -10 6 9 1 S to ck ho lm , S we den ,Y ek ep a C lin ic al R es ea rc h a nd T ra in in g Unit o f t he L ib er ia n I ns ti tu tef or B iome di ca l R es ea rc h, L ib er ia , a nd tDep ar tm en t o f I nf ec tio us D is ea se s,Karol inska Ins ti tu te, Roslagtu ll s Hospi ta l,S -1 148 9 S to ckh olm , S wed enA bstract. Sera from 48 children and adolescents (215years of age), residing in a m alariaholoendemic area of Liberia were investigated for specificities and isotypes of anti-P .falciparum antibodies. N o clear-cut relationship to the developm ent of clinical im munitywas found when the overall antibody activities to total parasite antigens were determ inedby enzyme-linkedimmunosorbent assay(ELISA).Although therewas a certainriseof

1gM , total IgG- and IgG2 antibody activities, this was most pronounced at ages when aclinical but nonsterile immunity is already present. W hen the sera were investigated byim munoprecipitation of 35S-m ethionine labeled parasite polypeptides, the total num ber ofparasite antigens precipitated w as sim ilar at all ages. A nalysis by indirect im munofluorescence (IFA), registering antibodies to intracellular parasite antigens, revealed no agedependent changes in antibody titers. In contrast, when the sera were assayed by a novelIFA, specific for a restricted number of parasite antigens in the membrane of infectederythrocytes, the frequency of positive sera as well as the anti-P. fakiparum titers rose inparallelith the development of clinicalmmunity. Thus, theseantigensappearedto beimportant inducers of protective immune responses and may be suitable candidates for avaccineagainstthe asexualblood stagesof P.fakipa rum.

In highly endem ic malarious areas, a relativeclinical immunity is normally acquired by theage of 5 years and severe malaria becomes lessfrequent. A fully developed but nonsterile immunity is generally present by 10 years of ageand Plasmodium fakiparum malariaisusuallykept at subpatent levels in adults. Serum antibodies isolated from immune donors living inThe Gambia have a protective capacity wheninjected into children acutely infected with P.fakiparum .2 Sim ilarly, passive transfer of antibody via the placenta has been suggested toprotect new borns against the disease.3D issection by recently developed techniquesof the humoral response in clinically immuneindividualss compared tothatinnonimmunesmay be expected to provide further insights inthe protective capacities of different anti-P. falciparum antibodies. In this study we have attem pted to correlate different param eters of din

A cc ep ted 3 Ju ly 1 98 5.

ica l imm un ity (ag e, sp le en size , p aras ite d ens itie s)w ith the occurrence of antibodies of different isotypes or P. fakiparum specificities. Sera from 48children or adolescents, aged 215 years andliving in a holoendemic area of Liberia, wereassayed for anti -P.fakiparum antibodies by several different methods. Using a novel imm unofluorescence assay,@ '7 we found a positive correlation between the development of clinicalimmunity and the appearance of antibodies directed against P . fakiparum antigens in them em bran e o f in fec te d e ry th roc yte s.

ParasitesM ATERIALS AND M ETHODS

T he T anzanian strain F 32 ofP lasm odium falciparum, isolated in 1978, was used for all thetests. The parasites were cultivated in red bloodcells (RBC), blood group 0, at a hematocrit of5% .

22


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23NTI-P. FALCIPARUMANTIBODIESAND CLINICALIMMUNITYStudy area

Blood specimens were collected near YekepaTown in northern Liberia in a rural area wherem alaria epidem iology has been studied in detail.8In the 2 villages included in this study, malariais holoendem ic and perennial. The sporozoiteinoculation rate is 0.2/person-night in the dryseason. P. fakiparum is the main malaria parasite but P. malariae and P. ovale are also comm only found.

Sample collectionThe study included a survey of 40 children,aged 212years, from L ugbeh V illage and 8 adolescent boys (aged 15) from Bonah Village. No

subject had taken any antimalarial drug for thelast three months. Age and sex were recordedand spleen size was estimated with the childstanding using Hackett's classification. Bloodspecimens were collected by finger prick in heparinized m icrohem atocrit tubes and centrifuged.T he plasm as w ere initially kept at 20 C ,atera t 70 C.Thick and thin film s were made form i cr os co pi c o bs er va ti on .

Paras it ol og ic al e xaminat io nsBlood films were stained with the standardG iem sa m ethod. P arasite species w ere identifiedand parasite counts were calculated from thenumber observed per 100400 leukocytes, assum ing a constant leukocyte count of 10 x 10@ /l.

Ant i-P.fal cipa rumELI SAA trophozoite-schizont-enriched antigen fraction, obtained by centrifugation on a Percoll gradient, was used as antigen as described previously.9 For post-coating, plates w ere incubated

5 h r at r oom t emp er atu rew it h 0 .2 5% ge la tininstead of 2% bovine serum album in as this further decreased the background. All sera weretested at dilutions 1:1,000, 1:5,000 and 1:25,000.1gM antibodies were assayed with a goat antihum an 1gM preparation conjugated w ith alkalinephosphatase (ALP) (Sigma Chem ical Co., St.Louis, M issouri). IgG antibodies were assayedw ith A LP -conjugated rabbit antibodies specificfor the Fc fragment of hum an im munoglobulin

gam ma chains. Specificity of the conjugates waschecked in an ELISA where the plates were coated with purified hum an myeloma proteins of differen t i so types. 'IgG subclasses of the anti-P. fakiparum antibodies were determined in an antigen ELISAwith subclass-specific monoclonal mouse antibodies as previously described.'0 The monoclonals were from the same clones as used previously except that the antibodies from cloneJL5 12, earlier used as IgG1 reagent, were substituted by antibodies from clone N L16 (Sew ardLabs, London, England). Human sera were absorbed twice with human erythrocyte ghosts andtested at dilutions 1:200, 1:800 and 1:3,200. Subclass specificity of the reactions w as ascertainedas be fo re. '

Indirect im munofluorescence assay (IF A)For the assay of antibodies to parasite antigensin the membrane of infected erythrocytes, different dilutions of test sera were added to m onolayers of glutaraldehyde-fixed and air driede ry th ro cy te s (5% l0% p ar as itemi a) a s d es cri be dby Perlm ann et al.5 Slides were stained with biotinylated goat antibodies to human immunoglobulin and F IT C-conjugated avidin.A ntibodies to intraerythrocytic parasites w ereassayed sim ilarly using monolayers of air driedb ut u nf ix ed e ry th ro cy te s. T es ts w er e c on sid ere d

positive w hen late stage parasites gave a distincta nd b ri gh t immuno fl uo re sc en ce .

Immunopr ecipitat ionfparasiteantigensRing stage dominated P. fakiparum cultureswere labeled until late schizont stage with 355Wm ethionine, solubilized and precipitated w ith 10@ dundiluted test serum and a polyvalent rabbitanti-hum an im munoglobulin serum . The precipitated antigen antibody com plexes w ere analyzedby sodium dodecyl sulphate-polyacrylam ide gelelectrophoresis (SDS-PAGE) under reducingc on dit io ns a nd f lu or og ra ph ed .'2

S ta ti st ic al ana ly si sSpearman's rank correlation test was used. Pvalues > 0.05 w ere considered insignificant.


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A low.Thereassignificantncreasen1gMantiP.fakiparum activityith increasingge (r=0.66, P = 8.9 x l06). IgG antibodies to P. falciparum were present in all 48 sera. There wasalso an age-dependent increase in antibody aco tivity but the statistical significance of this correlation was less pronounced (r = 0.38, P = 1.1 x1 0_ 2; F ig. lB ).8 C@@O IgG1and IgG3antibodies'erepresentin a lo most all sera (46/48 and 47/48, respectively) ando showed a similar rise w ith age as the total IgGantibodies. H ow ever, this age correlation w as notlo 15 statistically ignificantr = 0.27,P = 7.5 x 10-2YEARS for IgG, and r = 0.28, P = 6.0 x 10-2 for IgG3,respectively). In contrast, IgG 2 antibodies w hichwere detectable in 75% of the samples (29/38;10 samples excluded because of lack of serum)rose with age similarly to what was seen for 1gM(r= 0.69,P = 3.6 x l0@).IgG4antibodieserepresent in 50% of the samples available for test

O ing(18/38). E levated activities were seen in someO children younger than 4 years as well as in some

O 8 adolescents, and there was no increase with ageO (r=0.l3,P=4.6 x 10').In individual donors, there w as no correlationbetw een parasite densities and the 0D 405 for totalIgG , IgG ,, IgG 3 and IgG 4 antibodies, respectively. However, a weak negative correlation wasfound for 1gM and IgG2 (r = 0.243; r = 0.246,respectively).

Immunoprecipitationf355-methionine-labeledP . f alc ip arum p oly pe pt id esTo establish whether or not there was a majordifference in the antigens recognized by the anti

P. fakiparum antibodiesoccurringat differentages, serum samples were reacted with 35S-meth io n.in e-la beled p ara site ex tra cts, p rec ip ita te dw ith rabbit anti-human immunoglobulin andsubjected to S DS -P AG E and autofluorography.The results of a representative experiment areshown in Figure 2. In general, all sera precipitated some 20 parasite derived polypeptides ofdifferent apparent m olecular w eights. T he m ajorantigens seen were of M @ 195,000 d, 150,000 d,140,000 d, 129,000 d, 118,000 d, 90,000 d,80,000 d and 50,000 d, respectively. A ntibodiesto these antigens were present at all agesbetween 212years and there was no significant age-dependent difference in the intensity ofthe signals seen on the fluorographs. Identicalbanding patterns but slightly stronger signals w ere

24 WAHLGRENET AL

0 00c&@@

5

0 o 0O 0o @o8o8 0

2.01. 5

1.00.5

>2.02.01. 5

1.00.5

00 0008 000O 00005 10 15YEARS

FIGURE 1. ELISA values of sera (diluted 1/1,000;o rd in at e 0D@5 ) o f c hil dr en a nd a do le sc en ts ( ab sc is sa ,age in years) in the P .fakiparum E LIS A. A . 1gM react iv it y; B . I gG r ea ct iv it y.

Allvalu esareexpresse das thet estval ueminusbackground (i.e., incubation buffer). T he 0D@5for sera of healthy Swedish blood donors at thed ilu tio ns p re se nte d v aried b etw een 0 0 .0 5(1gM,IgG ), 00.075 (IgG ,, IgG 3, IgG 4) and 00.1 forIgG2.

RESULTSELISA

Antibodyisotypes1gM antibodies9 were present in 42/48 seratested (0D405 > 0.1). Figure 1 shows the agedependent changes of these antibodies at serumdilutions of 1:1,000. Similar results were obtained when the sera were tested at dilutions of1:5,000 or 1:25,000 although the readings obmined at these dilutions were frequently rather


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25NTI-P. FALCIPARUMANTIBODIESAND CLINICALIMMUNITY

12 IC

195@@,,29,,...@ 118

5

FIGURE 2. Fluorograph of SDS-PAGE after separation of-S methionine-labeled P.fakiparum polypeptidesimmun op re cip ita te d w it h u nd ilu te d s era ( 10 1 d) of c hi ld re n o f d if fe re nt a ge s o r o f 3 h ea lt hy Swed is h b lo od d on or s(C ). Molecu la r we igh ts o f some of t he ma jo r po lypep ti de s a re i nd icat ed x l0@.

V

obtained w ith the sera from the 8 adolescent boys,aged 15 years (not shown). However, as seenfrom Figure 2, the frequency of sera having ag en era lly re du ce d a ctiv ity in th is te st w as h ig he rin th e y oun ger a ge g ro up s th an in th e 8 1 2-y earo ld c hi ld re n.Antibodies to P. falciparum antigens in them em brane of inftcted erythrocytes

W ith sera diluted 1:20, this test detects antibodies against parasite antigens in the m emb ran e o f in fec ted erythro cy tes, p rim arily th osec on ta in ing r in g s ta ge s a nd e ar ly t ro phozoi te s. T hepredom inant antibody recognizes an antigen ofMr 155,000 d. Intraerythrocytic parasites or nonin fe cte d e ry th ro cy te s a re not s ta in ed .4 '5T he frequency of sera giving positive immunof luore sc en ce in th is te st r os e s ig nif ic an tl y w ithage. T hus, for the 43 sera available, the proportion of positive sera (at dilutions 1:20) was29% , 50% and 88% in the age groups 25,69an d 10 -1 5 yea rs, resp ectiv ely. T he freq uen cy o fpo sitiv e sera from L iberia n ad ults from th e sam eareas is also approxim ately 90% .@ T he endpointtiters of the test sera also show ed a significantage-dependent increase (43 sera available fortesting, r = 0.56, P = 2.2 x l0@). In some of

the adolescent sera these titers were very high(Fig. 3). Furthermore, for the 43 donors investig ate d, th ere w as a sig nific an t n eg ativ e c orre lation betw een parasitem ia and antibody titers inthis assay (r = 0.43, P = 6.9 x l0@). As seenfrom Figure 4, parasite densities > 100/mm3blood were rarely found in donors w hose antibody titers were >1:80 and none or very fewparasites were found in donors of high titeredsera. In addition, children with elevated antibody titers to the parasite antigens in the erythro cy te s urfa ce ra re ly h ad e nla rg ed s ple en s. R athe r, sp lee n size ap pea red to b e n ega tiv ely co rrelatedto a ntib od y titer. T he p rop ortio n of p ositiv e sera( 1:20) as a function of spleen size according toHackett is shown in Figure 5.A ntib od ie s to in tr ac ellula r p ara sites

T he se w ere a ssa ye d in a n IFA u sin g p ara sitiz ederythrocytes as above but w ithout fixation. All43 sera tested contained parasite staining antibodies with titers ranging from 1:320 to >1:1 0,2 40. H ow ev er, th ere w as no ag e-d epe nd en tc ha ng e in a ntib od y le ve ls (F ig ure 3 ). Mo re ov er,th ere w as n o a ge -d ep en de nt c orre la tio n b etw ee nparasitem ia and antibody titers, or betw een thelatter an d sp leen size (d ata n ot shown ).

YEARS

t2:i!'-@t3,@5@;;@6r@ ;4@9@


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27NTI-P. FALCIPARUMANTIBODIESAND CLINICALIMMUNITYreflects an antigen-dependent restriction of theIgG 2 a ntib Od ies p re sen tly is n ot k nown. N o d istinct age pattern of this type w as found for IgG 4antibodies present in a more limited number ofsera. A lth oug h a w eak n eg ativ e co rrelation w asfound betw een parasite densities and IgG 2 antibody activities, this was not the case for theo th er IgG su bc la sse s. T he re fo re , th e re le va nc e o fthis finding, for a possible causal relationshipb etw een elev ated antib od y lev els as d etected b yELISA a nd p ara site d en sitie s, is u nc erta in .No age -d ep endent d if fe re nc es in t he f orma tio no f a n tib od ie s to in div id ua l p ara site a ntig en s w ererevealed by analyzing the sera by immunoprec ip ita tio n a fte r SDS -PAGE o f b io sy nth etic allylab ele d P .fa kiparum ex tracts. T he to tal n um bero f p ar as ite polypepti de s p re cip ita te d wa s s im ila rat all ages and not significantly different fromw hat is seen w ith ad ult sera from th e sam e area.'8A ntibodies against the m ajor polypeptides includ in g th e m ajo r sch izo nt-d eriv ed su rfac e glycoprotein of M r 195,000 d,'2 w ere present at allag es an d sometim es at elev ated c on cen tration seven in very young children. The frequency ofnegative or w eakly reacting sera appeared to behigher in the younger children than in the olderones. T his w as probably due to a generally poorantibody response in som e donors rather thandue to a selective lack of antibodies against ind iv id ua l a ntig en s. It s ho uld b e empha siz ed , h owev er, th at th is a ssa y is o f q ualitative rath er tha nquantitative nature. M oreover, as the parasitee xtra cts h ad b ee n la be le d w ith 3 5S -m eth io nin e,a ntib od ie s to m eth io nin e-p oo r a ntig en s5 '6 w ereno t de tect ed .W hen the sera w ere analyzed by indirect IF Ao f u nfixed p arasites, n o ag e-dep en den t ch an ge sin antibody titers were seen and there was noco rrelatio n to p arasite d en sities o r sp leen size.T his confirm s previous results obtained w ith ad ifferen t series of d ono rs from th e sam e area,'3u sin g th e IFA d esc rib ed b y Volle r a nd O 'N eill.'9Similar results also have been reported fromT an zan ia b y D rap er C t al.2 n con trast, othe rsh av e sh ow n ag e-relate d increases in a nti-P . falciparum antibody titers in IFA studies of serumsam ples from other W est African areas.21'22However, in those studies, the blood donorsw ere from areas in w hich m alaria tra nsm issio nw as seaso na lly re stricted an d o ccu rred at low errates. In addition, the test groups w ere dividedd ifferen tly an d in clu de d sera from c hild ren lessthan 2 years of age and from adults older than

w>IC00a

0 I IISPLEEN SIZE

FIGURE 5. Percentage of sera positive ( 1/20) inthe IFA w ith GA-fixed an d a ir dried in fected RBC(ordinate);nd spleensizeestimatedccordingoH ack ett: a bs cissa , 0 = no rm al sp lee n (n = 6 ); I = sp le enpa lp ab le b elo w co stal m arg in on d ee p in sp ira tio n (n =7); II = spleen palpable below costal m argin, but notpro ject ed beyond a hori zon ta l l ine ha lf way be tweenthe costa! margin and the umbilicus (n = 18); III =s ple en w ith l ow es t p alp ab le p oi nt p ro je cte d mor e th anhalf w ay to the um bilicus but not below a line draw nho rizo nta lly th ro ug h it (n = 5).2 0 y ears. O th er exp la natio ns for th e d ifferen cesin result could be the relatively sm all num ber ofs era te ste d b y u s a nd d iffe re nc e in th e se nsitiv ityo f th e assay s used .In earlier w ork no correlation has been foundb etw een titers o btain ed usin g co nv en tion al IFAor E LIS A and those found w ith the corresponding sera in the modified IFA.'8 In contrast toth e E LISA an d th e IFA o f u nfix ed p arasites, th atof glutaraldehyde-fixed and air dried erythrocytes detects antibodies against only a few para site a ntig en s in th e membr an e o f i nf ec te d c ell s. 5'6In serum from infants and adult donors, the m ajority o f the an tibo dies g iv in g p ositiv e stain in gin this assay are directed against a soluble andheat stable polypeptide of M r 155,000 d (Pf 155)deposited in the erythrocyte membrane by theb urstin g s ch iz on t o r in va din g m ero zo ite s. P f 1 55app ears to b e im po rtant fo r th e in vasio n pro cessas it binds to glycophorin A , a putative P. falc ipa rum recep to r i n t he e ry th rocy te membrane6 '23and hum an antibodies against Pf 155 very efficien tly in hib it m ero zo ite rein vasio n in vitro.'8


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28 WAHLGRENF@ALT his an tig en is po or in m eth io nin e an d therefo renot detected in im munoprecipitation experim en ts p erfo rm ed w ith 3 5S -m eth io nin e-la be le dp ara site e xtra cts.5 '6 In th is re sp ec t it re semb le sth e hea t sta ble S -a ntige ns d escrib ed b y o th er inv estig ato rs .2 4 H ow ev er, it a pp ea rs to la ck th e m ajo r se ro lo gic al v aria tio n c ha ra cte ris tic fo r th es eparas ite components .6T he relev an ce o f th ese p arasite an tig en (s) forin v iv o p ro tectio n against P . f akipa rum m ala riais s up po rte d b y th e p re se nt fin din gs s howin g b otha m ark ed in crease in th e freq ue ncy o f an tib od ycontaining sera and antibody titers in parallelw ith the acquisition of clinical immunity. D on ors w ho se sera co ntain ed elev ated leve ls o f antibodies to these antigens consistently had lowparasite counts and spleens of sm all or m odera te ly e nl ar ge d s iz e, s ugge st in g t ha t th eir immunes ys tem suc ce ss fu lly h andl ed t he ma la ria p re ss ur eto which they w ere exposed.'3 Taken togetherw it h p re vious f in dings@ 8th es e r es ults s ugge stth at P 1' 1 55 an d p ossib ly othe r p arasite an tig en sin the m em brane of infected erythrocytes m aybe co nsidered as prim e cand id ates for a v accineaga in st th e a se xual b lood s ta ge s o f P .f ak ip ar um .

REFERENCES1. Coh en,S.,McGr ego r,I.A.,and Carr ingt on,.P.,1 96 1. G amma-g lo bu lin a nd a cq uire d immun ity to h um an m ala ria . N ature , 1 92 : 7 33 7 37 .2 . McGregor, I . A . ,Ca r ri ng ton ,S . P ., and Cohen , S .,196 3.Tr eatm entofEa stAfri can.fak ipar umwith West A f ri ca n- gl ob ul in . T r an s. R . So c. T r op .Med . Hyg ., 5 7: 1 70 1 79 .3. Edozien, J. C ., Gilles, H . M ., and Udeozo, I. 0.K ., 1 96 2. A du lt a nd c ord b lo od g amma-g lo bulin and imm unity to malaria in Nigerians.L an ce t, 2 : 9 5 1 9 55 .4 . Per lmann , H ., a nd Wahlg re n, M ., 1 983. I gG -ant ib od ie s s pe cif ic f or th e a lte re d s urf ac e o f P /a smod iu m fa kip ar um mala ria infec ted er yth rocytes. S cand. J. Immunol.. 18: 71.5. Per lmann, H. ,Berz ins ,K., WahlgrenM.,Car lsson,J ., B j rkman,A ., P atarro yo , M . E ., an d P erlmann , P ., 1 984. An tib od ie s i n ma la ria l s er a toa nt ig en s in t he memb rane o f e ry th ro cy te s inf ec ted wi th ear ly a sexua l b lood s tages o f P /a s

m odium falciparum . J. E xp. M ed., 159: 1686-1704.6. Wahlgre n,.,Berzins,.,Perlman n,.,Wh!in ,B., Ca rl sson , J ., B j rkman,A ., P er lmann , P .,McN ic ols, L -A ., D am e, J. B ., an d McC utch an ,T . F., 1985. Pf 155, a vaccine candidate forp ro te ct io n a ga in st a se xu al s ta ge s o f P la smodiumfalciparum m alaria. Pages 5 1 -56 in R . Lernerand R . C hanock, E ds., M odern A pproaches to

Vaccine s. Co ld Spr ing Harbor Laborator y, Co ldSpring Harbo r.7. F ranzn,L ., and W ahlgren, M ., 1985. M alariad ia gnos is : Par as it ol og ic al a nd s er ol og ic al t es ts .Postgradua teDoctor, 8 : 386392.8. B jorkm an, A ., H edm an, P ., B rohult, J., W illcox,M ., D iam an t, I., P eh rsso n, P . 0 ., R om bo, L .,a nd B en gts so n, E ., 1 98 5. D if fe re nt m ala ria c ont ro l a ct iv itie s in a n a re a o f L ib er ia . E ff ec ts onm alariom etric param eters. A nn. Trop. M ed.Paras it ., 79 : 239246.9. W ahlgren, M ., Berzins, K ., Perlm ann, P., andB j rkman , A ., 1 983 . C hara cteriza tio n o f th ehumoral immune r esponse i n P la smod ium fa/c ip ar um mal ar ia . I . E st imat io n o f a nt ib od ie s to

P. fakiparum or human erythrocytes by meansofmic ro ELISA. C /i n.Exp . ImmunoL ,53: 127-134.10.Wahlgr en,M., Berzins ,., Perlman n,P.,andP ersso n, M ., 1 98 3. C ha rac te riz atio n o f th e h umor al immun e r es po ns e i n P /o smod iumf ak ipa rum mala ria . II. Ig G su bc la ss le ve ls o f a nti P .fa/ciparum antibodies in different sera. C /in.E xp . Immu no L, 54 : 1 35 14 2.1 1. L un dg ren , K ., W ah lg ren , M ., B erzins , K ., T ro yeBlomberg,M. , Pe rlmann , H ., and Pe rlmann , P .,1 983. Monoclona l, p ar as ite a nd ant i RBC ant ib od ie s p ro du ce d b y s ta bl e EBV- tr an sf orme d Bcell lines from m alaria patients. /. Im munol,,131: 20002003 .1 2. H old er, A . A ., an d F reem an , R . R ., 1 98 2. B iosyn thesi s and proces si ng of a P /a smod ium fa/ciparum schizont antigen recognized by immunese rum anda monoc lonal ant ibody . J . Exp .Med ., 1 56 : 1 5281538 .1 3. B jo rkma n, A . H olo en demic m ala ria a nd th e e ffectsof chemotherapy, 1985.Thesis, KarolinskaInsti tute, Stockholm, Sweden.14. J ay awar dena ,A . N ., 1 981. Immune r es pons es inma la ri a. Pages 8516 in J . M. Mansf ie ld , ed . ,P ara sitic D isea se s, V olu me 1 . M arc el D ek ke rI nc ., New York .15 . Sp iege lbe rg ,H. L . , 1974 . B io log ical ac ti vi ti es o fimm uno glob ulin s o f d iffere nt clas ses an d su bclasses. Ad v. Im muno/., 19: 259 294.16. M orell, A ., S kvaril, F., H itzig, W . H ., and B arandun , S ., 1972 . I gG subc la ss es :Deve lopmen to f th e s er um concentr at io ns i n norma l in fa ntsand ch il dr en . J . Pedi at r. , 80 : 960964 .1 7. Y ou nt, W . J ., D on ne r, M . M ., K un ke l, H . G ., a ndKabat, E. A ., 1968. Studies on human antibodies. VI. Selectivevariat ion in subgroupcomposit ionand genet icmarkers. J . Exp. Med. ,127:633646.18. Whl in , . , Wahlgren,M. , Perlmann, H. , Berz ins ,K. , Bjrkman,A ., P ata rro yo , M . E ., a nd P erlm ann, P., 1984. Hum an antibodies to a M@155 ,000 P /a smodi um f ak ip ar um anti ge n e ff ic ien tl y i nh ib it merozoi te i nvasi on . Proc . Na tLA ca d. S ci. USA , 8 1: 7 91 2 79 16 .19. Vo lle r, A ., a nd O 'Ne ill, P ., 1 975. Immuno fluorescence method suitable for large-scale applic atio n to ma la ria . Bu lL W.H.O ., 4 5: 524529 .20. D raper, C . C ., L eiijveld, J. L. M ., M atola, Y . G .,and W hite, 0. B ., 1972. M alaria in the P are


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m une response to m alaria before, during anda ft er t he a pp li ca tio n o f c on tr ol m ea su re s: A l ong itu din al stu dy in th e We st A fric an s av an na .Bul L W .H .O ., 5 6: 5 79 6 00 .23. Per kin s, M ., 1 984. Sur fa ce p ro te in s o f P la .smod iumf al ci pa rum mer oz oit e b in di ng to t he e ry thr oc yte r ec ep to r, g ly co ph or in . J . E xp . Me d., 1 59 :788789.2 4. W ilso n, R . J . M ., McGre go r, I. A ., H all, P ., W illiam s, K ., and B artholom ew , R., 1969. A ntig en s a sso ciated w ith P la sm od iu m fa kip aru min fe ction s in m an . L an cet, 1 : 2 0 1 2 05 .

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