INFECTION AND IMMUNITY, Mar. 1993, p. 988-9930019-9567/93/030988-06$02.00/0Copyright © 1993, American Society for Microbiology

Plasmodium falciparum-Specific Immunoglobulin G (IgG),IgM, and IgE Antibodies in Paired Maternal-Cord Sera from

East Sepik Province, Papua New GuineaROBERT S. DESOWITZ,* JOSEPH ELM, AND MICHAEL P. ALPERS

Department of Tropical Medicine and Medical Microbiology, John A. Bums School ofMedicine,University ofHawaii, Honolulu, Hawaii 96186, and Papua New Guinea Institute ofMedical Research,

Goroka, Papua New Guinea

Received 26 May 1992/Accepted 31 December 1992

Enzyme-linked immunosorbent assay (ELISA) and Western blot (immunoblot) serological analyses forimmunoglobulin G (IgG), IgM, and IgE antibodies to Plasmodium falciparum were made from 46 maternal-cord serum pairs obtained from parturient East Sepik (Papua New Guinea) women and their newborn.Concurrent study of these women had shown that placental parasitemia rates were related to parity with thehighest rate (41%) in the primiparous group and the lowest rate (3%) in the women who had given birth morethan three times (>3 parity group). Overall ELISA positivity rates for antimalarial IgG, IgM, and IgEantibodies in the maternal sera were 54.3, 28.2, and 8.3%, respectively, while those for the cord sera were 36.9,0, and 16.6% respectively. Seropositivity rates were not related to maternal parity group, except for maternalIgE, in which there was a higher rate, of borderline significance, in the >3 parity group than in the primiparousgroup. Cord IgE positivity was largely independent of maternal positivity and vice versa. Cord and maternalIgG immunoblot pairs showed near homology. IgG antibodies to the P. falciparum antigens of sizes <36 kDawere either weak or absent in parity group 1 and 2 maternal-cord serum pairs. Neither ELISA or immunoblotrevealed IgM antibody in the cord serum samples. Maternal IgM antibodies showed a heterogeneity ofresponses both between paired IgG immunoblots and between different serum samples. The IgE immunoblotsexhibited a similar diversity, albeit of less complexity. The presence of P. falciparum-specific IgE in the cordsera would indicate that prenatal immune hypersensitization of the fetus to malaria had occurred.

Maternal-fetal immunological interactions in malaria are

still poorly understood. The vulnerability of the pregnantwoman, particularly the primigravida, to severe falciparummalaria is thought to be, in part, the result of the generalimmune "dampening" during pregnancy (reviewed in refer-ences 2 and 7). However, the degree of susceptibility and theclinical severity during pregnancy are not uniform, varyingfrom one region where falciparum malaria is endemic toanother (10, 20). It has been hypothesized that this variabil-ity may be due to differences in natural immune factors, suchas blood polymorphisms, and acquired immune factors be-tween populations (10).Plasmodium falciparum-specific immunoglobulin M

(IgM), IgGl to IgG4 isotypes, and IgE antibodies have beendemonstrated in sera of malaria patients and inhabitants ofregions where malaria is endemic (9, 25, 26). It has beenknown for over 20 years that of these, only IgG maternalantibody crosses the placenta to passively establish a nearlyequal titer in cord sera (28, 29). IgM antibodies have beendemonstrated in cord and neonatal sera in cases of congen-ital malaria (12, 22). However, there have been few investi-gations of P. falciparum-specific antibodies in paired mater-nal and cord sera comparatively analyzed for Ig class andantigen recognition as they may be related to the parasito-logical status and parity of the mother. This paper charac-terizes, by enzyme-linked immunosorbent assay (ELISA)and immunoblot, the P. falciparum-specific IgG, IgM, andIgE antibodies in 46 paired maternal-cord serum samples

* Corresponding author.

988

obtained in the East Sepik Province of Papua New Guinea,a region of meso-to-hyperendemic malaria.

MATERIALS AND METHODS

Subjects and samples. The source of the serum samplesused in this study has been described elsewhere (10). Syn-optically, placental crush smears and paired maternal-cordserum samples were obtained from women immediately aftergiving birth at the Wewak General Hospital, East SepikProvince, Papua New Guinea. Parasitological findings fromthese subjects noted that 41% of the primiparas, 23 and 25%of women who had given birth two and three times (parity 2and 3 subjects), respectively, and 3% of the >3 parity grouphad a P. falciparum placental parasitemia. However, in allinstances of placental parasitemia, the density was relativelylow, the average being 1.6%. No fetal (cord blood) para-sitemias were detected. None of the subjects exhibited signsor complaints of clinical malaria, nor were the birth weightor maternal and cord blood hematocrits related to thepresence of a placental parasitemia. Forty-six paired mater-nal-cord serum samples were obtained and used in thisstudy.

Serological methods. The ELISAs were done according tothe methods of Quakyi (17) for IgG and IgM and Desowitz(9) for IgE. Antigen was prepared by I. Quakyi from aculture of clone NFS4 of P. falciparum and was a kind giftfrom her. The immunologic reagents were alkaline phos-phatase-conjugated monoclonal (mouse ascites) antibody tohuman IgG and IgM and affinity-isolated goat anti-humanIgE antibody (Sigma Chemical Co., St. Louis, Mo.). Thesubstrate was p-nitrophenyl phosphate disodium. Twelve

Vol. 61, No. 3

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/i

ai o

n 15

Jan

uary

202

2 by

155

.133

.51.

245.

ANTIBODIES TO P. FALCIPARUM IN MOTHER AND NEWBORN 989

normal serum samples from Hawaiian residents who hadnever been exposed to malaria and four cord serum samplesfrom neonates born in Hawaii served as controls. All serumsamples were assayed for IgG and IgM at a dilution of 1:100in phosphate-buffered saline (PBS) but were undiluted forthe IgE assay. These dilutions were selected as givingmaximum sensitivity from box titrations of known positivePapua New Guinea sera and normal control sera. Thethreshold for positivity for all ELISA assays was an absor-bance >3 standard deviations above the mean of the con-trols.The methods of immunoblot analyses followed, with some

modifications, those described by Towbin et al. (23) andChang et al. (5). The parasite antigen was an extract,prepared by saponin lysis, of the FUP (Uganda-Palo Alto)strain of P. falciparum maintained in culture. The antigenwas mixed with an equal volume of Laemmli's reducedsample buffer and electrophoresed in 10% polyacrylamidegels containing 0.1% sodium dodecyl sulfate. Prior to use,the antigen was heated to 90°C for 90 s and sonicated.Antigen was then electrophoresed, by means of a mini-gelapparatus (Bio-Rad Laboratories, Richmond, Calif.), in 10%polyacrylamide gels and then electrophoretically blottedonto nitrocellulose transfer membranes, which were then cutinto strips. The strips were blocked with 5% nonfat pow-dered milk in PBS for 1 h. Three strips were incubated for 2h at room temperature with each serum sample: two samplesdiluted in PBS-5% nonfat powdered milk to 1:100 for IgGand IgM and the third sample diluted to 1:10 for IgE.Alkaline phosphatase-labelled monoclonal mouse IgG hybri-doma antibodies to human IgG and IgM were used assecondary antibodies to develop Ig class-specific immuno-blots. IgE immunoblots were made by use of mouse anti-human IgE monoclonal antibody followed by incubation inalkaline phosphatase-conjugated goat anti-mouse IgG anti-body (all antibody reagents were from Sigma Chemical Co.).The strips, after incubation in the secondary antibodies for 1h, were developed with Nitro Blue Tetrazolium-5-bromo-4-chloro-3-indolyl phosphate substrate (Kirkegaard and PerryLaboratories, Gaithersburg, Md.). The strips were washedsix times with PBS-0.05% Tween 20 between each step inthe procedure. A sample of a set of high-molecular-weightmarkers (Sigma Chemical Co.) was included in each run, andthe marker strip was stained with Coomassie blue. Antigenreferences were obtained by staining the gel with silver stain(Bio-Rad) and staining a blotted but unprocessed membranestrip with Coomassie blue.

RESULTS

ELISA. The results of the ELISAs categorized for sero-logical positivity according to antibody class, maternal par-ity, and placental parasitemia are shown in Table 1. MaternalIgG positivity rates did not significantly differ between paritygroups nor was there, overall, a significant difference be-tween those with and without a placental parasitemia. Fourof the 12 subjects with a placental parasitemia were ELISAIgG negative, although 3 of those serum samples gave anabsorbance value only slightly below the threshold forpositivity. The cord blood IgG ELISA positivity rates for allmaternal parity groups were lower than those for maternalsera, but again many negative cord sera from newborns ofseropositive mothers had an absorbance value only margin-ally below the threshold for positivity.Maternal P. falciparum-specific IgM ELISA seropositiv-

ity rates did not differ significantly between parity groups or

TABLE 1. ELISA seropositivity rates of paired maternal-cordserum samples by Ig class, maternal parity,

and placental parasitemiaSerum sample % Positive (maternal/cord) for:group (no. ofsamples)a IgG IgM IgE

Maternal parity1 (13) 61.5/38.4 38.4/0 0/15.42 and 3 (18) 44.4/27.7 11.1/0 16.6/17.7>3 (15) 60.0/46.6 40.0/0 26.6/21.4All (46) 54.3/36.9 28.2/0 15.2/13.3

Placenta' (12) 66.6/41.6 33.3/0 8.3/16.6

Placenta- (34) 50.0/35.2 26.4/0 17.6/11.8a +, parasitemia; -, no parasitemia.

between the placental parasitemia-positive and -negativegroups. IgM seropositivity rates for all groups were approx-imately half the IgG rates. There were no samples whichwere positive for IgM antibody alone; all samples that wereIgM positive were also ELISA positive for IgG antibody. Nocord samples were ELISA positive for IgM antibody.The maternal IgE ELISA positivity rate progressively

increased with parity: 0% for parity 1, 15.8% for parity 2 and3, and 26.6% for parity >3. The number of samples in eachparity group was relatively small, but there was a differenceof borderline significance in the P. falciparum-specific IgEseropositivity rate between the parity 1 and >3 parity groups(P = 0.06, Fisher's exact test). The serum samples, as notedearlier, represent a subset of all women whose blood andplacental tissues were examined. Placental P. falciparumparasitemia rates were related to parity, with the highest rate(41%) in the primiparas. Table 1 shows the inverse associa-tion between the placental parasitemia rates, parity, and theP. falciparum-specific IgE ELISA maternal seropositivityrates. Six of the seven maternal serum samples positive forIgE antibody also had IgG antibody (three of those also hadIgM antibody). There was one IgE-positive maternal serumsample giving a high absorbance value that had no ELISA-detectable IgG or IgM anti-P. falciparum antibody.Of particular interest is the presence of P. falciparum-

specific IgE antibody in six cord serum samples. Moreover,the presence of IgE antibody to P. falciparum in the motherseemed to bear little or no relationship to that in hernewborn. There were four samples in which the cord serumonly was IgE positive, six cases in which the maternal serumonly was positive, and only two cases in which the maternal-cord pair were copositive.The average absorbance values of maternal and cord sera,

variously categorized by maternal parity group, ELISApositivity only, placental parasitemia positivity only, andplacental parasitemia negativity only, were calculated foreach Ig class ELISA. No significant differences (Student's ttest) were found between maternal and cord samples, be-tween maternal samples alone and cord samples alone, orbetween parity groups and their subgroups described above.There was a trend toward higher antibody concentration(absorbance values) of all three Igs with increasing parity inthe placenta parasitemia-negative women, but, again, thosetrends were not statistically significant.Immunoblot. Normal maternal and cord serum controls

showed no immunoblot reactions of any Ig class antibody tothe malaria antigen.

VOL. 61, 1993

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/i

ai o

n 15

Jan

uary

202

2 by

155

.133

.51.

245.

990 DESOWITZ ET AL.

liI

1

-195

-14- ~~~~~~484'i}v:*l4 *4i 36

-25

Et 5Fm?u-I gtm

2 3 45S 64F*R

;..

i,

qW.._...~ _

la .;

?.-Wii~~~~~~~,.j ~ .J

-195

-83

,i-

......

:.

-48

-42

-36

FIG. 1. IgG antibodies to P. falciparum, detected by Westernblotting, in representative maternal-cord serum pairs from parturi-ent women and their newborns of East Sepik Province, Papua NewGuinea. The immunoblot of the maternal serum is at the left side ofeach pair. The pair at lane 1 are sera that were seronegative for IgGantibody by ELISA; lanes 2 through 6 are serum samples that wereIgG ELISA seropositive. The immunoblots of lanes 2 through 6 arearranged according to ascending maternal parity as follows: lane 2,parity 1; lane 3, parity 2; lane 4, parity 3; lane 5, parity 4; and lane6, parity 5. Numbers are masses in kilodaltons.

IgG. Representative IgG immunoblots of maternal-cordserum pairs, categorized according to ELISA positivity andmaternal parity, are shown in Fig. 1. It will be seen that,although minor differences occur, the cord and maternalserum immunoblots of any one pair are essentially identical.Almost all of the ELISA-negative East Sepik sera tested

produced a positive IgG immunoblot, although they showedfewer and fainter bands than immunoblots of ELISA-posi-tive sera. The banding patterns of the ELISA-negative serawere highly variable, with 2 to 15 bands present. An exampleof the latter is shown in lane 1, Fig. 1; sera from a parity 1woman and her newborn had immunoblots with 15 distinctbands, the most prominent being at the 195-, 96-, and 44-kDalevels.Lanes 2 through 6 of Fig. 1 show the IgG immunoblots of

ELISA-positive sera arranged in order of maternal parity.All of these sera produced highly complex immunoblotpatterns with more than 30 bands ranging in antigen molec-ular mass from 220 to 22 kDa. In all of these paired maternal-cord samples, the most prominent bands were consistentlyat the 195-, 48-, 44- through 42-, and 36-kDa antigen levels.Although there was a general similarity in pattern betweenall serum samples, there appeared to be one parity-relateddifference. The IgG antibody(s) of parity 1 and 2 women andtheir newborns had a weak recognition of antigens less than36 kDa in size, particularly the 25- and 22-kDa antigens (Fig.1, lanes 2 and 3). With increasing maternal parity (Fig. 1,lanes 4 through 6), there was an intense recognition of thesetwo antigens. No obvious differences could be distinguishedbetween the IgG immunoblots from women of equal or nearequal parity with a placental parasitemia and those without aplacental parasitemia. This similarity is shown by comparing

MA'" -25

1 2 3 4 5 6 7FIG. 2. IgM antibodies to P. falciparum in representative IgM

ELISA-negative (lane 1) and -positive (lanes 2 through 7) maternalsera. The parity of the mothers from whom the positive sera wereobtained are as follows: lanes 2 and 3, parity 1; lane 4, parity 3; lane5, parity 4; and lanes 6 and 7, parity 5.

the immunoblot in lane 4, from a parity 3 woman with a 1%placental parasitemia, with that of lane 5, from a parity 4woman without a placental parasitemia.

IgM. Figure 2 shows representative IgM immunoblots ofmaternal sera arranged according to IgM ELISA positivityand parity order. Immunoblots of ELISA-negative sera wereeither devoid of bands, or when present, the bands were fewand very faint (Fig. 1, lane 1). None of the cord sera hadELISA- or immunoblot-detectable P. falciparum-specificIgM antibody when the enzyme-conjugated monoclonalmouse anti-human IgM was used as the second antibody.The most striking feature of the 1gM immunoblots of the

ELISA-positive maternal sera (Fig. 2, lanes 2 through 6) isthe heterogeneity of antigen-antibody responses. The IgGimmunoblot patterns from all sera tested essentially line up.In contrast, the IgM immunoblots exhibit relatively littleuniformity in recognition patterns from one serum sample toanother. However, similar to the parity-related IgG re-sponse, the IgM immunoblots from parity 1 women showweak recognition of P. falciparum antigens less than 36 kDain size (Fig. 2, lanes 2 and 3), whereas the IgM antibodiesfrom parity 4 and 5 women (lanes 4 through 6) have a strongreaction to the 25- and/or 22-kDa antigens.Another striking difference between the anti-P. falciparum

IgG and IgM antibodies is their variation in antigen specific-ities. This is revealed by comparing the paired IgG and IgMimmunoblots from maternal sera that are ELISA positive forboth Ig classes (Fig. 3). One such example is that of a parity5 woman whose IgM immunoblot is shown in lane 6, whichis compared with the IgG immunoblot at lane 7. In this serum

INFECT. IMMUN.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/i

ai o

n 15

Jan

uary

202

2 by

155

.133

.51.

245.

ANTIBODIES TO P. FALCIPARUM IN MOTHER AND NEWBORN

I

'95-

83-

48-i

36-i

25- ON

GM GM G M GM G MFIG. 3. Representative paired IgG (G) and IgM (M) immunoblots

of five maternal serum samples positive for antibodies of both Igs toP. falciparum by ELISA.

sample, the prominent IgG antibody(s) to the 195-, 83-, and42-kDa antigens is either absent or at low concentrations inthe IgM immunoblot. Conversely, the IgM immunoblot hasstrong bands at 127-, 46-, and 34 kDa, levels that are weak orabsent in the IgG blot.

IgE. Representative IgE immunoblots of maternal, cord,and paired maternal-cord sera that were IgE ELISA positiveare shown in Fig. 4. The immunoblots reveal a diversity ofantigen-IgE antibody specificities, consisting of 2 to 20bands, between the individual serum samples. There is aband at the 48-kDa level present in all sera of both maternaland cord origins. Bands at the 195-, 97-, 71-, 43-, 38-, and22-kDa levels were present in many sera, but not uniformlyso. Compared with the IgG and IgM immunoblots, the bandswere faint, as would be expected considering the low con-centration of IgE in serum. However, one sample, a mater-nal serum sample from a parity 5 woman, produced arelatively strong immunoblot of 17 bands, ranging from 195to 22 kDa, the most prominent being 109, 97, 48, 47, and 38kDa (Fig. 4, lane 1).The IgE immunoblots of the cord sera that were IgE

ELISA positive without copositivity of the mother's serumalso showed a diversity of antigen recognition (Fig. 4, lanes3 and 4). However, the cord serum immunoblots generallyhad fewer and lighter bands than the IgE immunoblots of thematernal serum specimens.The IgE antibodies in the maternal and cord ELISA-

copositive pairs had essentially the same specificities, asevidenced by the similarity of their immunoblots (Fig. 4,lanes 5 and 6). However, it will be seen from the twoexamples illustrated in Fig. 4 that considerable variationbetween the sera of different maternal newborn subjects canoccur. The immunoblots of the lane 5 pair had only one

195 ...;

109-i83-

71-

48-,

43.-

22-

1 2 3 4 5 6FIG. 4. Representative IgE immunoblots of maternal and cord

sera positive for P. falciparum-specific IgE antibody by ELISA.Lanes are as follows: 1 and 2, maternal sera whose paired cordsample was negative; 3 and 4, cord sera whose paired maternalsample was negative; 5 and 6, cord-maternal pairs in which bothserum samples were positive (the cord sample is the left immunoblotof each pair).

strong band, that to the 48-kDa antigen, while the blots of thelane 6 pair had 18 bands, 8 of which, ranging in molecularmass from 195 to 38 kDa, were prominent.

DISCUSSION

In this study, we have attempted to elucidate an immuno-logical rationale, by characterization of antigen recognitionby antibodies of different Ig classes, for the phenomenaassociated with falciparum malaria and pregnancy. It shouldbe noted that the women of our study differed from those inother immunological investigations on malaria in humanpregnancy in other malaria-endemic areas, notably fromtropical Africa, in that in the East Sepik falciparum malariaof pregnancy presented as a relatively benign infection withhigh placental parasitemia rates of low density in the primip-arous group. However, similar to the case in Africa, therewere indications of increasing immunity (or retention ofimmunity) in the higher-parity groups. There was no corre-lation between parity and either IgG ELISA positivity ratesor antibody concentration. It is not known whether this ispeculiar to our study population, because other serologicalinvestigators on malaria in pregnancy have not analyzedtheir data on the basis of maternal parity (4, 6, 28). The IgGimmunoblots, however, revealed a parity-associated patternof antigen recognition of a more frequent and higher concen-tration of antibody to the 25- to 22-kDa antigens in thewomen (and their newborns) of higher (>3) parity. The P.

j

991VOL. 61, 1993

... ....

.,I

I4

tk.

ri.

VI'

..f.f.

;"i

-7

iii

W.I 1....

,.OP,--

04 wi

11 I"

..4'. *114:1 --.

,.,z 11" .-i-ow.,

4-,;, -%,,

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/i

ai o

n 15

Jan

uary

202

2 by

155

.133

.51.

245.

992 DESOWITZ ET AL.

falciparum antigen of this molecular size group has beendescribed (11) as being protective in that antibody to itinhibited in vitro merozoite reinvasion of erythrocytes. Thevirtual absence of placental parasitemia in the >3 paritygroup would presumably signify them to be more immunethan women of lower parity, particularly the primiparas.Otherwise, there was a similarly multitudinous complex ofimmunoblot bands noted by other investigators (6, 21).Among those bands, we noted the 48- to 51-kDa complex ofantigens to be most strongly and consistently present in theIgG immunoblots of sera positive by IgG ELISA. In con-trast, Thelu et al. (21) found that the two major antigensassociated with high antibody levels in sera from Africa wereof molecular masses 115 and 103 kDa.The cord serum IgG ELISA rates for neonates born of

mothers of all parity groups were lower than the maternalserum rates. However, many of the negative ELISA valueswere only marginally below the threshold of positivity, and itmay be that the threshold was set unrealistically high inpursuit of unequivocally clean results. That those marginallyIgG ELISA-negative cord sera did have IgG antibody wasevidenced by the presence of antigen-IgG bands, albeit oflower intensity and fewer in number, on the immunoblots.The possibility that humoral antibody levels toP. falciparumare significantly decreased in a significant proportion ofprimigravidas with a placental parasitemia deserves furtherinvestigation. Transplacental passage of IgG would accountfor the near IgG immunoblot homology between pairedmaternal and cord sera. The minor differences in IgG immu-noblot pattern between a maternal and cord serum pair maybe due to the presence of isotypes present only in thematernal serum, because both IgG2 and IgG3 are poorlytransported to the fetal circulation (6, 14). Alternatively, orin addition, the cord IgG may differ in antigen specificity inminor respects from that of the maternal antibody as a resultof prenatal immune sensitization in which the fetus wouldproduce malaria-specific IgG antibody. This was reported byVernes et al. (24), who advanced the argument for prenatalimmune stimulation in human malaria.While the occurrence of IgM antibodies in the sera of

malaria patients is well documented, there have been fewseroepidemiological studies on this class of Ig and fewerstudies still as it relates to malaria and pregnancy. There isan indication that IgM antibodies may commonly be presentin patients exposed to endemic malaria (6, 26). Similar to ourfindings, those earlier studies reported that all IgM-positivesamples had accompanying IgG antibody. However, thedifficulty in interpreting serological results for IgM assaybecause of methodological uncertainties is exemplified bythe study of Shehata et al. (19). Using the conventional IgMELISA method, these workers found an 84% positivity ratefor P. falciparum-specific IgM antibody in a serum samplefrom a Senegalese population. When they applied the re-verse ELISA to these sera (affinity-isolated anti-human IgMbound to the plate was incubated with the serum sample andthen was incubated with enzyme-conjugated P. falciparumantigen), none proved to be positive. We found that approx-imately 30% of the parturient women of our study had IgMantibody, as determined by the conventional ELISAmethod, the seropositivity rates not differing signficantlybetween the parity groups or between the placental para-sitemia-positive and -negative groups. The presence of ma-laria-specific IgM was confirmed by immunoblot, althoughthe serum samples showed a considerable heterogeneity inantigen recognition by this antibody.There had been no indication of congenital malaria in the

subjects of our study, all cord blood samples being parasi-tologically negative (10). The serological correlate of thisfinding was the absence of IgM ELISA positivity in all cordserum samples. This observation differs from that made byChizzolini et al. (6) from Gabon, Africa, who found P.falciparum-specific IgM antibody in 7 of 59 of their pairedcord blood samples. Those authors suggested that the pre-natal immune sensitization leading to IgM production by thefetus was probably facilitated by a placental parasitemiasevere enough to cause the histopathological alterations tothe maternal compartment found in six of the seven of thosecases. In contrast, all infected placentas in our study were ofa low level of parasitemia and there were no prominenthistological alterations such as an increase in syncytialknotting (1). Immunoblot analyses with the monoclonalanti-human IgM second antibody reagent support the ELISAfindings. However, although detectable congenital malariaappears to be rare in Papua New Guinea, prenatal immunesensitization, as evidenced by Plasmodium-specific cordblood lymphocyte blastogenesis, has been shown to be arelatively common phenomenon (8).Our observation of P. falciparum-specific IgE antibodies

in the maternal sera supports the earlier observation (9) ofthe occurrence of this class of Ig antibody in Papua NewGuinea populations exposed to endemic malaria. The immu-noblots for IgE tended to confirm the ELISA serologies andrevealed a heterogeneity of IgE-specific antigen recognition,although bands at the 38- and 48-kDa regions predominated.While there is still no direct experimental evidence for an

immunological role of anti-malaria IgE antibody, the signif-icant decrease in the placental parasitemia rate in womenwith >3 parity may reflect a progressively enhanced immu-nity with parity, and it is, therefore, of interest to note thatof the three Ig classes assayed for ELISA seropositivity,only IgE showed a progressively increasing rate with in-creasing parity. The recent finding (16) that deposition of IgEcomplexes in the placental fetal blood vessels occurred onlyin placentas with a low level of P. falciparum parasitemia inthe intervillous spaces and not in placentas with a high level(>20%) of parasitemia may have an important bearing onthis issue. Indeed, the authors of that study concluded that"the amount of deposition of IgE in the placenta fromwomen infected with P. falciparum correlates inversely withthe degree of parasitemia in the placenta."Perhaps the most unusual finding of this study was the

presence of P. falciparum-specific IgE antibody in 8.5% ofthe cord sera tested by ELISA. Because IgE does not crossthe placenta from the maternal circulation (la), there is theimplication that the cord antibody is from fetal production inresponse to immune stimulation-allergic sensitization. Thephenomenon of fetal production of antigen-specific IgE isnot without precedence, albeit for antigens other than thoseof the malaria parasite. It is known that the human fetus iscapable of producing IgE early in conception, by the 10th or11th week (15). However, while total cord blood IgE may beabnormally high with relative frequency and be predictive ofrisk to atopy to environmental or food allergens duringinfancy, the presence of specific IgE antibody to thoseantigens appears to be a rare occurrence. Of the 101 cordblood samples examined by Buscino et al. (3) only one hadspecific IgE antibody to milk protein. In contrast to theenvironmental allergens, parasitic antigens may have fargreater potential to induce specific prenatal allergic hyper-sensitization. Filaria-specific IgE antibody was detected in82% of cord serum samples of infants born in a setting inIndia where filariasis is endemic (27). Furthermore, similar

INFECT. IMMUN.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/i

ai o

n 15

Jan

uary

202

2 by

155

.133

.51.

245.

ANTIBODIES TO P. FALCIPARUM IN MOTHER AND NEWBORN 993

to our finding that the paired maternal serum samples werenegative for four of the six IgE ELISA-positive cord serumsamples, these authors also noted that cord serum IgEantibody bore little or no relationship to that of the mother.The mother of the infant described by Buscino et al. (3) whohad cord serum IgE antibody to milk protein had no similarantibody. The maternal serum of Weil et al.'s (27) studywere all positive, as expected, for IgE antibody to filaria, butthe IgE cord blood titers were at least fourfold greater thanthose of the maternal sera. While the immunologic role offetal P. falciparum-specific IgE antibody requires elucida-tion, it is of interest to note that the immunoblots showedthese cord antibodies to be predominantly specific for 109-,48-, and 43-kDa antigens of the P. falciparum lysate. Anti-gens of these molecular masses have been shown to beimmunogenic and are considered to be vaccine candidates(11, 13, 18), but it is not known whether the antigensrevealed in our immunoblots and those described as vaccinecandidates are identical.

ACKNOWLEDGMENTS

We thank Carol Ryglewicz and the nursing staff of the obstetricalward of the Wewak (Boram) Hospital for their invaluable assistancein collecting the samples for this study. The work described hereinwas supported by the U.S. Agency for International Developmentand the American Institute of Biological Sciences under contractnumber DPE-053-C005066-00.

REFERENCES1. Aikawa, M., and R. S. Desowitz. Unpublished observations.la.Bazaral, M., H. A. Orgel, and R. N. Hamburger. 1971. IgE

levels in normal infants and mothers and an inheritance hypoth-esis. J. Immunol. 107:794-801.

2. Brabin, B. J. 1991. The risks and severity of malaria in pregnantwomen. UNDP/World Bank/WHO Special Programme for Re-search and Training in Tropical Diseases.

3. Buscino, L., F. Marchetti, G. Peliegrini, and R. Perlini. 1983.Predictive value of cord blood IgE levels in "at-risk" newbornbabies and influence of type of feeding. Clin. Allergy 13:503-508.

4. Campbell, C. C., J. M. Martinez, and W. E. Collins. 1980.Seroepidemiological studies of malaria in pregnant women andnewborns from costal El Salvador. Am. J. Trop. Med. Hyg.39:151-157.

5. Chang, S. P., G. S. Hui, A. Kato, and W. A. Siddiqui. 1989.Generalized immunological recognition of the major merozoitesurface antigen (gp195) of Plasmodium falciparum. Proc. Natl.Acad. Sci. USA 86:6343-6347.

6. Chizzolini, C., F. Trottein, F. X. Bernard, and M.-H. Kaufnann.1991. Isotypic analysis, antigen specificity, and inhibitory func-tion of maternally transmitted Plasmodium falciparum-specificantibodies in Gabonese newborns. Am. J. Trop. Med. Hyg.45:57-64.

7. Desowitz, R. S. 1987. The pathophysiology of malaria afterMaegraith. Ann. Trop. Med. Parasitol. 81:599-606.

8. Desowitz, R. S. 1988. Prenatal immune priming in malaria:antigen-specific blastogenesis of cord blood lymphocytes fromneonates born in a setting of holoendemic malaria. Ann. Trop.Med. Parasitol. 82:121-125.

9. Desowitz, R. S. 1989. Plasmodium-specific immunoglobulin E insera from an area of holoendemic malaria. Trans. R. Soc. Trop.Med. Hyg. 83:478-479.

10. Desowitz, R. S., and M. P. Alpers. 1992. Placental parasitaemiain East Sepik (Papua New Guinea) women of different parity:the apparent absence of acute effects on mother and foetus.Ann. Trop. Med. Parasitol. 86:95-102.

11. Franzen, L., B. WAhlin, M. Wahlgren, L. Aslund, P. Perlmann,H. Wigzell, and U. Petterson. 1989. Enhancement or inhibitionof Plasmodium falciparum erythrocyte reinvasion in vitro by

antibodies to asparagine rich protein. Mol. Biochem. Parasitol.32:201-212.

12. Harvey, B., J. S. Remington, and A. J. Sulzer. 1969. IgM malariaantibodies in a case of congenital malaria in the United States.Lancet ii:333-335.

13. Inselburg, J., D. J. Bzik, W.-B. Li, K. M. Green, J. Kansopon,B. K. Hahm, I. C. Bathurst, P. J. Barr, and R. N. Rossan. 1991.Protective immunity induced inAotus monkeys by recombinantSERA proteins of Plasmodium fakciparum. Infect. Immun.59:1247-1250.

14. Lee, S. I., D. C. Heiner, and D. Wara. 1986. Development ofserum IgG subclass levels in children, p. 108-121. In F. Shakib(ed.), Basic and clinical aspects of IgG subclasses. Karger,Basel.

15. Miller, D. L., T. Hirvonett, and D. Gitlin. 1973. Synthesis of IgEby the human conceptus. J. Allergy Clin. Immunol. 52:182-188.

16. Nagatake, T., T. Tegoshi, R. S. Desowitz, R. W. Steketee, and M.Aikawa. 1992. Program Abstr. XIIIth Int. Congr. Trop. Med.Malaria, in press.

17. Quakyi, I. 1980. The development and validation of an enzymelinked immunosorbent assay for malaria. Tropenmed. Parasit.31:1-8.

18. Ramasamy, R., G. Jones, and R. Lord. 1990. Characterisation ofan inhibitory monoclonal antibody-defined epitope on a malariavaccine candidate antigen. Immunol. Lett. 23:305-310.

19. Shehata, M. G., W. E. Collins, A. J. Sulzer, E. L. F. Franco,J. C. Sldnner, and J. M. Roberts. 1988. Reversed enzyme-linkedimmunosorbent assay for detection of specific anti-Plasmodiumfalciparum immunoglobulin M antibodies. World Health Orga-nization document WHO/MAL/88.1050. World Health Organi-zation, Albany, N.Y.

20. Sholapurkar, S. L., A. N. Gupta, and R. C. Mahajan. 1988.Clinical course of malaria in pregnancy-a prospective con-trolled study from India. Trans. R. Soc. Trop. Med. Hyg.82:376-379.

21. Thelu, J., I. Sheick-Zakiuddin, C. Boudin, F. Peyron, S. Picot,and P. Ambroise-Thomas. 1991. Development of natural immu-nity in Plasmodium falciparnum malaria: study of antibodyresponse by Western blotting. J. Clin. Microbiol. 29:510-518.

22. Thomas, V., and C. W. Chit. 1980. A case of congenital malariain Malaysia with IgM malaria antibodies. Trans. R. Soc. Med.Hyg. 74:73-76.

23. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.USA 76:4350-4354.

24. Vernes, A., J. M. Pinon, J. F. Garin, P. Walter, and P. Ivanoff.1983. Materno-foetal relationships in Falciparum malaria: kinet-ics of inhibitory antibodies, p. 13. In Proceedings of the SecondInternational Conference on Malaria and Babesiosis.

25. Wahlgren, M., K. Berzins, H. Perimann, B. Wahlin, and M.Person. 1983. Characterization of the humoral immune responsein Plasmodium falciparum malaria. II. IgG-subclass levels ofanti-P. falciparum antibodies. Clin. Exp. Immunol. 53:135-142.

26. Wahlgren, M., H. Perlmann, K. Berzins, A. Bjorkkman, A.Larsson, I. [junngstrom, M. E. Patarroy, and P. Perlmann.1986. Characterization of the humoral immune response inPlasmodium falciparum malaria. III. Factors influencing thecoexpression of antibody isotypes (IgM and IgG-1 to 4). Clin.Exp. Immunol. 63:343-353.

27. Weil, G. J., R. Hussain, V. Kumarswami, S. P. Tripathy, K. S.Phillips, and E. A. Ottesen. 1983. Prenatal allergic sensitizationto helminth antigens in offspring of parasite-infected mothers. J.Clin. Invest. 71:1124-1129.

28. Williams, A. I. O., and H. McFarlane. 1969. Distribution ofmalarial antibody in maternal and cord sera. Arch. Dis. Child.44:511-514.

29. Williams, A. I. O., and H. McFarlane. 1970. Immunoglobulinlevels, malarial antibody titres and placental parasitemia inNigerian mothers and neonates. Br. J. Med. Sci. 1:369-376.

VOL. 61, 1993

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/i

ai o

n 15

Jan

uary

202

2 by

155

.133

.51.

245.