A n t ib o d y r e s p o n s e s t o P l a s m o d iu m f a l c ip a r u m s p o r o z o it e -, LIVER- AND BLOOD-STAGE SYNTHETIC PEPTIDES IN MIGRANT AND AUTOCHTHONOUS POPULATIONS IN MALARIA ENDEMIC AREAS.
F E R R E IR A - D A - C R U Z M .F .* , D E S L A N D E S D .C .* , O L IV E IR A - F E R R E I R A J.*, M O N T E N E G R O - J A M E S S.**, T A R T A R A.***, D R U I L H E P .**** & D A N IE L - R I B E IR O C .T .*
S u m m a r y :
This study evaluates the differences in host immune responses to defined plasmodial antigens in four geographically different regions in which malaria is endemic. Sera from 5 2 7 individuals were tested for the presence of antibodies specific for three types of plasmodial antigen : liver-stage antigen (LSA-1), blood-stage antigen (SPF 70) and circumsporozoite (CS) antigen (NANP)4.
The individuals taking part in the study comprised : patients with transfusional malaria due to Plasmodium falciparum or P. vivax;
non-immune migrants residing in an endemic area in Rondônia;
Amazonian Indians from the states of Para (Xingu PA) and Mato Grosso (Xingu MT); people living in a hyperendemic area in Africa (Burkina-Faso); and controls that had never been to a mala
ria endemic area. None of the transfusional sera displayed antibo
dies against sporozoite or to liver stage antigen, although 80% of the P. falciparum transfusional malaria sera contained IgG antibo
dies against the blood-stage peptide. A low percentage of Indians from Xingu PA and of non-immune migrants displayed antibodies against liver-stage (27% and 17%) and sporozoite (11% and 12%) peptides, although a greater frequency of antibodies against blood-stage peptide (50% and 49%) was observed in both cases.
Indians from Xingu MT exhibited a greater frequency of antibodies against liver, sporozoite and blood-stage peptides (45%, 50%
and 58%). Only hyperimmune African individuals exhibited higher percentages of antibodies against liver- (64%) and blood-stage antigens (87%), contrasting with a low frequency of antibodies against the CS repeat (33%). Taken together, the present data confirm that Rondonian migrants and Indians from Xingu PA consti
tute populations with limited exposure and immunity to P. falcipa
rum malaria infection and conversely, Xingu MT Indians and Africans have been more exposed to malaria infection. In conclu
sion this study indicates that the immune response to these malaria parasite peptides can be used to assess malaria transmission in epidemiological surveys.
* W orld H ealth O rgan ization C ollaborating C en ter fo r R esearch and T raining in th e Im m u nology o f Parasitic D iseases; D epartm ent o f Im m u nology , Institu to O sw ald o Cruz, FIOCRUZ, R io de Ja n e iro , Brazil.
** T u lan e U niversity M edical C enter, Louisiana, USA.
*** Institut Pasteur, Lille, France.
**** Institut P asteur de Paris, France.
C orresp on d en ce : Maria de Fatim a Ferreira da Cruz. D epartm ent o f Im m u n ology , Institu to O sw ald o Cruz, FIO CRU Z, Av Brasil 43 6 5 , C E P 2 1 0 4 5 .9 0 0 , R io d e J a n e i r o , B r a z il. F a x : ( 0 2 1 ) 2 8 0 - 1 5 8 9 ; (0 2 1 )5 9 0 -3 5 4 5 ; P h o n e : (0 2 1 )2 8 0 -1 4 8 6 .
Financial support : th e w ork w as sup ported by th e C om m ission o f th e E u ro p e an C o m m u n ities and th e B razilian N ation al R esearch C ouncil.
R ésum é
: RÉPONSE ANTICORPS À DES PEPTIDES SYNTHÉTIQUES DES STADES SANGUINS HÉPATIQUE ET SPOROZOITE DE P . FALCIPARUM DANS DIFFÉRENTES RÉGIONS ENDÉMIQUESCette étude a évalué les différences de la réponse immune de sujets vivant dans quatre régions géographiques ou le paludisme est endé
mique. Les sérums de 5 2 5 individus ont été testes vis-à-vis de trois peptides de Plasmodium falciparum, correspondants aux stades : hépatique (LSA-1), sanguin (SPF70) et sporozoite (NANP)4. Les indi
vidus étudiés comprenaient des malades avec paludisme post-trans- fusionnel à P. falciparum ou à P.vivax, des migrants non immuns vivant à Rondonia (Brésil), des indiens brésiliens vivant dans les états du Para (Xingu-PA) et du M ato Grosso (Xingu-MT), des africains rési
dant dans une région holoendémique de G aretanga (Burkina-Faso) et des sujets sains n'ayant jamais séjourné dans une zone endé
mique. Aucun des sérums de paludisme post-transfusionnel n 'a réagi avec les peptides LSA-1 ou (N A N P )4 bien que 80% présentaient des anticorps pour le peptide SPF-70. Un faible pourcentage d'indiens du Xingu-PA et de migrants possédaient des anticorps contre les stades hépatique (27% et 17%) et sporozoite ( 11% et
12%) contrastant avec un pourcentage plus élevé d'anticorps contre le stade sanguin (50% et 49%). Les indiens du Xingu-MT portaient plus fréquemment des anticorps contre les stades hépatique, sporo
zoite et sanguin (45%, 50% et 58%). Les africains ont montré des taux élevés d'anticorps contre les stades hépatique (64%) et sanguin (87%) contrastant avec la faible fréquence d'anticorps contre le (NANP)4 (33%). Ces résultats suggèrent que l'étude de la réponse immune contre ces peptides peut être utilisée comme indicateur du degré d'exposition des populations.
KEY W ORDS :
Plasmodium falciparum.
peptide. vaccine, malaria.MOTS
CLÉS :
Plasmodium falciparum.peptide. vaccin, paludisme.
INTRODUCTION
T
he im portance o f the hum oral immune response in the d efense against P. fa lc ip a r u m in fe c tio n s w as clearly d em o n strated th ree decades ago by Cohen et al., 1961. They show ed that the transfer o f immunoglobulins from immune adults
to in fected ch ild ren ind u ced a rapid redu ction in parasitemia levels. These results w ere recently corro
b o ra te d by an a n a lo g o u s stu d y by B o u h a ro u n - Tayoun et al., 1990. O ne clear implication o f these
Parasite, 1995, 2, 23-29
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2 3Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1995021023
FERREIRA-DA-CRUZ M.F., DESLANDES D.C., OLIVEIRA-FERRF.IRA J. et al.
studies is that the humoral immune status o f a popu
lation should be characterized prior to vaccination field trials in order to facilitate both the choice o f an approp riate target po p u latio n and the su bsequ en t analysis o f vaccination results that are often difficult to interpret.
In Brazil, th ere has so far b e e n little research on humoral immune responses to P. fa lc ip a r u m synthe
tic peptides, and existing studies have focused their attention on the prevalence o f antisporozoite antibo
d ies (T o sta and M oura, 1986; Arruda e t a l., 1989;
Oliveira-Ferreira et al., 1992). W e therefore decided to study P. fa lc ip a r u m - specific humoral immune res
p o n se in au to ch th o n o u s and m igrant pop u lation s from endem ic areas in four geographically different regions in w hich malaria is endem ic, as w ell as in individuals with transfusion induced malaria, in order to evaluate the applicability o f using this approach to study the degree o f exposure to malaria. Immune res
ponses against w hole parasite antigen (PA) w ere also evaluated for comparative purposes.
MATERIAL AND METHODS
Po p u l a t io n s t u d ie d
T
h e su b jects investigated co rresp o n d ed to a representative sample o f the populations studied. The enrolm ent criteria used w ere : to be
a permanent resident o f the tribe since birth (indians and africans) or to be a migrant from non endem ic area and residing in the municipality o f Ariquemes (Rondonia). All individuals gave informed consent to participate in the study.
1) The population o f non-im m une migrants lives in the state o f Rondonia in the southwestern part o f the Brazilian Amazon. According to the Brazilian Ministry o f Health, malaria is endem ic in the area, without any marked seasonal variation. Rondonia, which accounts for 50% o f the nation’s registered malaria cases, saw the im plementation o f federal governm ent agricultural settlement programs and a surge in mining activity in the 1970s. As a result, immigrants w ere attracted from other states (in southern and northeastern Brazil) that w ere m ostly m alaria free. Many o f th ese m igrants have been living in the state for as little as 2-4 years (41% ) and the great majority (80% ) for less than 10 years.
2) The two Indian populations com prise individuals living a) in the state o f Para (near the Tucurui dam) and b) in the state o f Mato G rosso (in the Amazon Basin in northern Brazil). These two tribes are widely separated and offer the possibility o f studying P. f a l c ip a ru m immune responses in isolated localities. The characteristics o f each tribe have already b een repor
ted elsew here (Arruda et al., 1989). Blood sample co l
lections w ere perform ed at the end o f the rainy sea
son, a period o f high transmission.
3) The African population is chronically exposed to the risk o f malaria infection. It resides in Garetanga- Burkina Faso, a village located near the village o f D onse (Druilhe et al., 1986, Druilhe and Khusmith, 1987).
4) The transfusional malaria cases corresponded to individuals that have never b een in endem ic areas.
The P. fa lc ip a r u m cases w ere from individuals living in Rio de Jan eiro and those o f P. v iv a x from indivi
duals living in B elo Horizonte (Minas Gerais state) - malaria free areas - and w ere therefore prim einfected individuals.
D e t e r m i n a t i o n o f Pla sm o d iu m s p e c ie s
Malaria diagnosis was perform ed by m icroscopic ex a
m ination o f 500 fields o f G iem sa-stained thick and thin blood smears. Parasitemia was quantified by ex a
m ination o f at least 200 leu k ocy tes in thick b loo d films.
Se r u m c o l l e c t io n
Serum sam ples w ere o btain ed from a total o f 527 individuals, as follow s : 187 from non-im m une sub
jects; 236 from Indians living in Para (1 1 7 ) or the Mato G rosso (1 1 9 ); 78 from hyperim m une African individuals; 11 from patients with post-transfusional malaria (6 infected with P. v iv a x and 5 with P. f a l c i p a r u m ); and 15 from control individuals w ho had
n e v e r b e e n to an e n d e m ic a re a . All th e seru m sam ples w ere co llected w ithout any p referen ce in term s o f age, race or sex. For the analysis o f the results according to age, the age distribution o f each group w as d one in order to have a hom ogen eous num ber o f individuals in each one (Table 1).
P.
FALCIPARUM SYNTHETIC PEPTIDES- (NANP)4, this synthetic peptide corresponds to four repeats o f the repetitive epitope o f the CS protein o f P. fa lc ip a r u m ( Zavala et al., 1986).
- LSA-1, w hich corresponds to a repetitive epitope, part o f a 200kDa m olecule that is specific to the liver stage o f P. fa lc ip a r u m (Druilhe et a l , 1984; Gué rin- Marchand et al., 1987). Its sequence is : LAKEKLQG- QQSDLEQERLAKEKLQEQQSDLEQERLAKEKLQ and purity higher than 90%.
- SPF70, which corresponds to a schizont protein, is a 70kDa antigen degraded from a 120kDa precursor membrane protein in mature schizonts and increases in am ount at the time o f schizont rupture (m erozoite release/reinvasion). Although this m olecu le derives
A n tib o d y re sp o n s e s t o P. f a l c ipa r u m pep tides
AGE GROUPS - (years)
(NANP)4 LSA-1
RO p/n
XPA p/n
XMT p/n
AF p/n
RO p/n
XPA p/n
XMT p/n
AF p/n
1-10 13/39 8/50 22/37 12/32 5/39 14/50 24/37 78/32
11-20 12/50 9/21 50/26 32/22 22/50 14/21 46/26 95/22
21-30 9/44 14/21 74/23 40/10 11/44 33/21 69/23 0/10
>30 13/54 16/25 67/33 78/14 26/54 60/25 51/33 29/14
Total 12/187 11/117 50/119 33/78 17/187 36/117 45/119 64/78
T a b le 1. - Freq u en cy o f an ti-circum sporozoite (NANP)4 and anti-liver-stage (LSA-1) an tibo d ies acco r
ding to ag e in different populations.
p : percent positives; n : total num ber o f individuals; RO : Rondonia; XPA : Xingu Para; XMT : Xingu Mato Grosso;
AF : Africa.
from P. f a l c i p a r u m , it also cross-reacts with anti-P . v ivax antibodies. The synthesis o f SPf70 was done by copolym erization o f equal amounts o f four different se q u e n ces o f the 70 kDa m o lecu le term ed : C2 - G Q D E G E E N E G ; C3 - LV FLV Q Q PFLFV LW D Q NEKFPVFMGVYDP; C5 DQFFDANPNLFQLLEPVEFDED and C 10 - GRNGLGANTDQDDQLEDE (Jam es et al.,
1993; T oebe et al., 1993).
Im m u n o r a d io m e tr ic a s s a y
(IRMA)
FOR d e t e c t i o n OF ANTI-P. FALCIPARUM SPOROZOITE-STAGE ANTIBODIES
T h e IRMA w as p e rfo rm e d as a lre a d y d e s c rib e d (O liveira-Ferreira et a l., 1992; Zavala e t a l., 1986) using m icro p lates (D y n atech A lexand ria, Virginia, USA) coated with (NANP) 4 conjugate to bovine serum albumin (BSA; Sigma). Control wells w ere prepared by omitting the peptide from the coat procedure. The counts per minute (CPM) in control w ells w ere sub
tracted from the CPM in corresponding peptides co a
ted plates. A serum sample was considered positive w hen the cpm value was higher than the average CPM o f the normal sera plus two standard deviations (314 CPM).
En z y m e l in k e d i m m u n o s o r b e n t a s sa y ( E L I S A ) FOR THE DETECTION OF ANTI-P. FALCIPARUM LIVER- STAGE ANTIBODIES
The ELISA was perform ed in polystyrene microplates (NUNC M A XISO RP) c o a te d w ith 1 0 0 μ l/well o f a 2μg/ml antigen solution in phosphate-buffered saline (PB S). T he plates w ere incubated overnight at 4°C and the antigen solution removed. The plates w ere
w ashed with PBS-0.05% Tw een20 (Sigma; PBS-T20) and saturated w ith PBS co n tain ing 2.5% skim m ed m ilk o v e rn ig h t at ro o m te m p e ra tu re (R T ). A fter washing with PBS-0.02% Tw een20, test sera diluted 1:100 in PBS solution containing 1.25% skimmed milk and 0.5% Tw een20 w ere added to the wells in dupli
cate, in volum es o f 100μl/well, and incubated at RT for 1 hour. The plates w ere then w ashed as described a b o v e and 10 0 μ l o f p e ro x id a se -c o n ju g a te d a n ti
human IgG (Sigma), diluted 1:1000 in the same buffer as the one used for sera dilution, was added to each w ell. A fter in cu b a tio n ( 1h - R T), th e p lates w ere washed again and 100(μl/well o f the substrate solution w as added (3% o f O PD - O -p h e n y len e d iam in e - Sigma - in 0.05M citrate buffer, pH 5.1, containing 0.07% H2O 2 - Merck). The reaction was stopped by addition o f 50μ l o f 2N HC1 to ea ch w ell and the absorbance was m easured at 492nm with a Titertek Multiskan ELISA reader. The average optical density (O D ) o f a one- hundred-fold dilution o f normal sera plus 2 standard deviations was defined as the cut-off limit (0.184 OD).
En z y m e l in k e d im m u n o s o r b e n t a s s a y (E L ISA ) FOR THE DETECTION OF ANTI-P . FALCIPARUM PEP
TIDE BLOOD-STAGE ANTIBODIES
W e used polystyrene microplates (CORNING) coated with 100μl/well antigen solution (5μg/ml) in tris-buf- fered saline pH 8.0 (TBS). The plates w ere incubated for 16h at 4°C and the antigen solution removed. The plates w ere rinsed three times (for 10 min each time, with vigorous agitation) and then once for 30 min at 37°C w ith 2 0 0 μ l/w ell T B S -0 .5 % T w e e n 2 0 (T 2 0 ) (Sigm a), to block n onsp ecific bindings. After b lo c
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king, 100μl o f test sera (diluted 1:100 in TBS-0.05% - T 2 0 ) w e r e a d d e d to th e w e lls in d u p lic a te , in volu m es o f 100μl/well, for 2h at 37°C. T h e plates w ere then w ashed 3 times, for 10 min each time, in TBS-0.1% -T20. The remaining steps in this assay were similar to those used for the detection o f liver-stage antigen. The cut-off value was 0.132 OD.
Im m u n o f l u o r e s c e n t a n t i b o d y t e s t (IFA T ) f o r
DETECTION OF ANTI-P . FALCIPARUM WHOLE BLOOD- STAGE ANTIBODIES
W e used P. fa l c i p a r u m - parasitized red blood cells cultured asynchronously in vitro (Trager and Jensen, 1976). T h e IFAT w as perform ed acco rd in g to the m ethod d esc rib e d by Ferreira and S an ch es, 1988 using b lo o d sm ears co n tain in g 20 parasitized red blood cells per field and fluorescein isothiocyanate conjugated goat anti-human IgG (Sigma). A reciprocal titer o f 20 or greater was considered positive.
Ep id e m io l o g ic a l s t u d y
Data were analyzed according to sex and age in all the population studied. In Rondonian subjects, other para
meters, such as the presence o f circulating parasites at the time o f serum collection, the number o f previous malaria attacks, and the date o f the last attack, were also consid ered . Statistical analysis w as perform ed using the EPISTAT softw are for the Chi-Square test with Y ates’ correction with 95% confidence intervals.
RESULTS
Ma l a r ia p r e v a l e n c e
A
t the time o f blood collection in the state o f R ondonia the p rev alen ce o f positive thick blood smears am ong the 187 individuals studies was 11%. Eighty percent o f these positives were due to P. f a lc ip a r u m and 20% to P. vivax. No indivi
dual carried both species.
T h e p rev alen ce o f p ositive thick b lo o d sm ears in Indians from Xingu MT (119) was 4.2% , with three individuals carrying P. fa lc ip a r u m and two cariying P. vivax. T he level o f parasitemia w as very low in positive individuals (usually below 0.02% ). In Indians from Xingu PA (117), the prevalence o f positive thick blood smear was also low (3.4% ). P. v iv a x was pre
sent in all the positive individuals, and none o f them w as positive for P. fa lc ip a r u m . Among the African subjects, 65% carried blood-stage parasites, and the positivity was higher in children than in adults. P. f a l c ip a r u m w as the m ost frequent species accounting for 95% o f the positives.
A n t i b o d i e s a g a i n s t
P.
f a l c i p a r u m l i v e r - s t a g e p e p tid e (L SA -1)As was the case for antibodies against the sporozoite- stage peptide, none o f the transfusional malaria sera presented IgG antibodies against the LSA-1 peptide.
The frequency o f antibody responses to this antigen in Indians from Xingu PA and non-im m une migrants in Rondonia w as low (27% and 17%, respectively).
Indians from Xingu MT and hyperim m une African individuals displayed higher percentages (45% and 64%, respectively) (Fig. 1). No relationship was obser
ved betw een anti-LSA-1 IgG antibodies and sex in any o f the populations studied. Previous research, like the present study, has show n that the d evelopm ent o f antibod ies to (NANP)4 is ag e-d ep en d en t (O liveira- Ferreira et al., 1992). To determ ine w hether a similar correlation exists for the LSA-1 peptide, w e stratified antibody-positive rates by age, since all our selected populations had a h o m ogeneou s distribution in all the age groups studied (1-10; 11-20; 21-30; and >30 years). T he frequ ency o f anti-LSA-1 antibodies w as significantly higher among individuals above 20 years old in the group from Xingu PA (p<0.001), above 10 years old in the group from Xingu MT (p<0.01) and less than 20 years old in the African group (p<0.001) (Table 1). Among Rondonian individuals, no correla
tion w as o bserv ed b etw een the frequ en cy o f anti- LSA-1 IgG antibodies on the one hand, and either the p resen ce o f circulating parasites or the date o f the last malaria attack o f malaria or age, on the other.
A n t i b o d i e s a g a i n s t
P.
f a l c i p a r u m s p o r o z o i t es t a g e-p e p t i d e (N A N P) 4
None o f transfusional sera displayed IgG antibodies against the circum sporozoite protein (CSP). The fre
quency o f IgG antibodies against CSP in migrants from Rondonia and Indians from Xingu PA was low (12%
and 11%, respectively). Indians from Xingu MT exhibi
ted a higher frequency (50%), while African individuals showed an unexpectedly low frequency (33% ) (Fig. 1).
No significant correlation was observed betw een anti- CSP IgG antibody and sex in any o f the populations studied. H ow ever, as befo re, these antibod ies was significantly higher in individuals above 20 years o f age from Xingu MT and Africa (p <0.001) (Table 1).
D etection o f these antibodies was not related to the presence o f parasites in the peripheral blood or to the level of parasitemia in Rondonian subjects.
A n t i b o d i e s a g a i n s t
P.
f a l c i p a r u m b l o o d - s t a g e PEPTIDE (S P F 7 0 ) AND AGAINST THE WHOLE PARASITE ANTIGEN (P A )
Four out o f five individuals with P. f a lc ip a r u m trans
fusional malaria displayed IgG antibodies against the
A n t i b o d y r e s p o n s e s t o P . f a l c ip a r u m p e p tid e s
Fig. 1. - P rev alen ce o f IgG an tib od ies against liver-stage (LSA-1), sp orozoite (NANP)4 and blood -stage (S P F 70) and to bloo d -stage parasite an tigen (PA ) in different populations.
TM : transfusional malaria; RO : Rondonia; XPA : Xingu Para; XMT : Xingu Mato Grosso; AF ; Africa; Pf : P. fa lcip a ru m ; Pv : P. vivax.
SPF70 peptide and against PA, as com pared with only o n e (17% ) P. v iv a x transfusional m alaria case with antibodies against the SPF70 peptide. The frequencies o f anti-SPF70 and anti-PA IgG antibodies w ere higher in individuals from Africa and Xingu MT (87%, 58%
an d 8 8 % , 8 1 % , r e s p e c tiv e ly ) . In d iv id u a ls fro m Rondonia and Xingu PA exhibited similar frequencies o f anti-SPF70 IgG antibodies (50% and 49%, respecti
vely). In Rondonians, the frequency o f anti-PA IgG antibodies w as also similar (52% ) but in individuals from Xingu PA it was lower (36% ) (Fig. 1). No rela
tionship was observed betw een anti-blood stage anti
bodies and sex in any o f the populations studied. The prevalence o f IgG antibodies against PA was higher in the group aged over 10 years in Xingu PA. Among Indians from Xingu MT, the frequency o f these anti
bodies increased with age, reaching 100% in indivi
duals over 20 years old (Table 1). In the group from Rondonia, the frequency o f anti-SPF70 IgG antibodies w as significantly higher in individuals w hose m ost recent malaria attack occurred within the last year (1 to 12 m onths) (p < 0.001). No significant correlation was observed betw een these antibodies and age in Rondonian individuals.
W hen antibody responses to the peptides and to PA w ere analyzed according to degree o f malaria exp o sure, two main observations w ere made : a) the anti- LSA-1 a n tib o d y re sp o n se is th e o n ly s e r o lo g ic a l
m arker that appears to indicate that the Xingu PA population has clearly b een more exposed to P. f a l c i p a r u m th a n th e R o n d o n ia n m ig ra n ts; and b ) co n versely, the anti-(NANP) 4 resp o n se is the only antibody response w hich does not reflect the greater e x p o su re o f A frican individuals to P. f a l c i p a r u m malaria, as com pared with other populations (Fig. 1).
DISCUSSION
I
n our study, none o f the transfusional malaria (TM) patients had antibodies against (NANP)4 or LSA-1, notwithstanding the high (80%) percentage o f P. fa lc ip a r u m TM-infected individuals with antibodies against SPF70 and PA, and despite the data o f Hope et al., 1994, showing cross-reactivities between sporozoite and bloodstage antigens. This may be due to the relatively small num ber o f TM cases in our study. Similarly, although antigen sharing betw een blood-and liver-stage schizonts has also been reported (Szarfman et al., 1988a, 1988b; Suhrbier et al., 1989), our data confirm the stage specificity o f the liver-stage P. fa lc ip a r u m peptide used in the present study.
In Rondonian individuals, no correlation was found betw een the frequ en cy o f anti-(NANP) 4 antibodies and parasitemia. Similar observations w ere made by Oliveira-Ferreira et al., ( 1992) in Brazil and Esposito
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et al., (1988) in Africa, with both studies show ing that the frequency o f anti-sporozoite antibodies was not related to anti-blood-stage antibody titers or to reduc
tions in parasitemia induced by chem oprophylaxis.
Similarly, the ab sen ce o f any relationship betw een the presence o f anti-LSA-1 antibodies, the presence of circulating parasites and the time elapsed since the previous malaria attack suggest that these antibodies can n o t b e u sed as in d icators o f cu rrent or recen t natural malaria infection.
In Indians from Xingu PA, the frequency o f anti-CS protein antibodies was similar to that observed in the m ig ra n t p o p u la tio n o f R o n d ô n ia . A m o n g th e s e Indians, exposure to P. v iv a x is higher than to P. f a l
cip a ru m , in contrast to Indians from Xingu MT, w ho are more exposed to P. fa lc ip a r u m . This may explain w hy w e fo u n d h ig h er fr e q u e n c ie s o f a n tib o d ie s against (NANP)4, LSA-1 and PA in Indians from Xingu MT. Both the Xingu PA and Xingu MT Indian popula
tions w ere visited at the end o f the rainy season (D e cem b er-A p ril), a perio d o f high tran sm issio n . Therefore, the difference in the frequencies o f antibo
dies against (NANP)4, a P. fa lc ip a r u m - specific anti
g e n , in th e tw o In d ia n p o p u la tio n s c o u ld b e explained by different degrees o f exposure to P. f a l c ip a ru m and P. v iv a x rather than by seasonal varia
tions as observed in other endem ic areas (Esposito et al., 1988; Druilhe et al., 1986; Hoffman et al., 1987;
D eloron and Cot, 1990). Conversely, the similarity in the frequencies o f anti-SPF70 antibodies observed in the two Indian populations probably reflects the fact that this peptide is recognized by antibodies from both P. vivax- and P. fa lc ip a r u m infected individuals (as d em onstrated w ith the sera from transfusional malaria patients).
W e observed a low frequency o f anti-(NANP) 4 antibo
dies in the African population, in contrast to a high prevalence o f anti-LSA-1 antibodies. This could indi
cate that in holoendem ic areas, the immune response to the liver-stage peptide is a better indicator o f mala
ria transmission than the response to the CS protein.
Am ong th e p o p u latio n s in the p re sen t study, the African show ed the highest frequency o f anti-LSA-1 antibodies. In addition to the fact that this population was the most exposed to malaria, one other possible explanation for these data could be the existence o f variant epitopes in the Brazilian strains o f P. f a l c i p a
rum. This seem s unlikely, however, since sequence data from tw o isolates have show n that the LSA-1 g e n e is w ell co n se rv e d (P. D ru ilh e, u n p u b lish ed data). It seem s that the low frequency o f anti-LSA-1 an tib o d ies am ong ag ed 21 -3 0 or o v er A fricans is inversely related to anti-sporozoite immunity, which in crea ses p ro g ressiv ely w ith age, b ein g fou n d in
around 80% o f individuals over 30. This latter immu
nity may protect the population from the establish
m ent o f hepatic forms o f the parasite. T he African population also displayed the highest frequ ency o f anti-SPF70 and anti-PA IgG antibodies. No significant correlation w as observed betw een the frequency o f antibodies against blood -stage antigens and age, in contrast to som e previou s stud ies on pop u lation s from endem ic areas, (D eloron and Cot, 1990; Baird et al., 1991), but in agreem ent with the study o f Druilhe et al. (1986).
The present data confirm that R ondonian migrants and Indians from Xingu PA are characterized by limi
ted exp osu re and immunity to P. f a l c i p a r u m anti
gens, and therefore should be considered a priority target for control measures in the future. Conversely, Xingu MT Indians seem to be m ore exposed to mala
ria in fectio n and have p ro bably reach ed a higher d eg ree o f im m unity to P. f a l c i p a r u m antigens. In con clu sion this study indicate that malaria parasite peptides can be used to assess malaria transmission in epidem iological surveys.
ACKNOWLEDGEMENTS
W
e a re g ra te fu l to D r C lo v is N a k a ie ( D e p a r tm e n t o f B io p h y s ic s , E s c o la Paulista de M edicina, Brazil) for providing the (NANP)4 peptide and to Dr Hoom an M omen (Departm ent o f Biochem istry and M olecular Biology, Instituto O sw ald o Cruz) fo r critical review o f the m an u scrip t. W e are also in d eb ted to D rs R om eu R od rigu es F ia lh o and Sandra F erra cio li (N ation al Health Foundation) for all the facilities provided for the field work.
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Accepté le 2 janvier 1995
Parasite, 1995, 2, 2 3-29
