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The Journal of Immunology, 2006, 177: 7139-7145.
Copyright © 2006 by The American Association of Immunologists, Inc.

Prenatal Malaria Immune Experience Affects Acquisition of Plasmodium falciparum Merozoite Surface Protein-1 Invasion Inhibitory Antibodies during Infancy1

Arlene Dent*, Indu Malhotra*, Peter Mungai*,{dagger}, Eric Muchiri{dagger}, Brendan S. Crabb{ddagger}, James W. Kazura* and Christopher L. King2,*,§

* Center for Global Health and Diseases, Case Western Reserve University, Cleveland, OH 44106; {dagger} Division of Vector Borne Diseases, Nairobi, Kenya; {ddagger} Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia; and § Veterans’ Affairs Medical Center, Cleveland, OH 44106


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
African infants are often born of mothers infected with malaria during pregnancy. This can result in fetal exposure to malaria-infected erythrocytes or their soluble products with subsequent fetal immune priming or tolerance in utero. We performed a cohort study of 30 newborns from a malaria holoendemic area of Kenya to determine whether T cell sensitization to Plasmodium falciparum merozoite surface protein-1 (MSP-1) at birth correlates with infant development of anti-MSP-1 Abs acquired as a consequence of natural malaria infection. Abs to the 42- and 19-kDa C-terminal processed fragments of MSP-1 were determined by serology and by a functional assay that quantifies invasion inhibition Abs against the MSP-119 merozoite ligand (MSP-119 IIA). Infants had detectable IgG and IgM Abs to MSP-142 and MSP-119 at 6 mo of age with no significant change by age 24–30 mo. In contrast, MSP-119 IIA levels increased from 6 to 24–30 mo of age (16–29%, p < 0.01). Infants with evidence of prenatal exposure to malaria (defined by P. falciparum detection in maternal, placental, and/or cord blood compartments) and T cell sensitization at birth (defined by cord blood lymphocyte cytokine responses to MSP-1) showed the greatest age-related increase in MSP-119 IIA compared with infants with prenatal exposure to malaria but who lacked detectable T cell MSP-1 sensitization. These data suggest that fetal sensitization or tolerance to MSP-1, associated with maternal malaria infection during pregnancy, affects the development of functional Ab responses to MSP-1 during infancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Falciparum malaria is one of the most important pediatric infectious diseases in sub-Saharan Africa, where it is estimated to kill at least 1 million children per year. In areas where malaria transmission is stable, infants and children typically experience clinical morbidity and high blood stage parasite densities. Infants <6 mo old appear to be relatively resistant to clinical malaria and high density parasitemia while those between 6 and 24 mo of age appear to have increased susceptibility to these clinical and parasitological outcomes (1, 2). Although evidence supporting a direct role for passively acquired maternal IgG Abs in mediating protection against infant malaria is inconsistent (3), epidemiologic studies show that the age-related increase in malaria susceptibility occurs after maternal Abs have waned, estimated to be 6–8 mo after birth, before immunity to blood stage Plasmodium falciparum develops as a result of repeated infections (4). Knowledge of the immune mechanisms underlying changes in infant malaria susceptibility may have important implications not only for fundamental understanding of human malaria immunobiology but also for how future malaria vaccines will be evaluated and delivered in this age group.

Understanding antimalaria immunity in human infants is complicated by the physiologic and immunologic effects of maternal malaria infection on the unborn fetus and its impact on infant acquisition of immune responses to blood stage infection. Unlike the other three malaria species that infect humans, P. falciparum-infected erythrocytes have the propensity to sequester in the intervillous blood of the placenta, a condition referred to as placental malaria. Placental malaria is most common in women with their first or second pregnancy, seemingly due to the lack of acquired immunity to P. falciparum clones that preferentially bind to the placental vasculature (5, 6, 7, 8). In this context, the unborn fetus may potentially be exposed to soluble malaria Ags or infected erythrocytes that gain access to the fetal circulation after crossing the placenta. Although it is not feasible to document this process in vivo, indirect evidence supporting its occurrence includes the demonstration of T and B cell responses to crude schizont extracts and blood stage Ags in cord blood lymphocytes (CBL),3 a cell population that represents circulating fetal lymphocytes present at the time of birth (9, 10, 11). The consequences of this in utero immune experience on infant malaria immunity are not known, particularly whether it accelerates the development of protective Abs by virtue of sensitization or impairs their acquisition by immune tolerance mechanisms. The potential health significance of this fetal malaria experience is underscored by epidemiologic studies suggesting that offspring of women with placental malaria are more susceptible to malaria during childhood compared with offspring of mothers without placental malaria (12, 13, 14, 15).

Studies of infant immunity to merozoite surface protein-1 (MSP-1), an abundant merozoite surface ligand required for invasion of erythrocytes and a human malaria vaccine candidate, have been inconclusive regarding whether anti-MSP-1 Abs mediate or are at least a biomarker of protection against childhood blood-stage malaria infection and clinical morbidity (16, 17, 18, 19, 20). The reasons for these apparent discrepancies are ill-defined but taken together suggest that anti-MSP-1 Abs detected by serology alone are a poor surrogate of acquired immunity. Functional Ab assays may be a better measure of naturally acquired immunity, especially in relation to MSP-1 (21, 22, 23). We describe here the results of a prospective cohort study of infants born in a malaria holoendemic area of Kenya designed to test the hypothesis that prenatal malaria experience, reflected by newborn and maternal malaria infection at parturition and neonatal cord blood T cell sensitization to MSP-1, influences the development Abs to MSP-1 during infancy. We measured Abs to the 42- and 19-kDa C-terminal fragments of MSP-1, both by traditional serologic methods and by a functional assay that quantifies inhibition of invasion of erythrocytes by the membrane-anchored MSP-119 ligand required for entry of the parasite into RBC (23).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Study participants and study design

Approval for the study was obtained from the Kenya Medical Research Institute National Ethical Review Committee and the Institutional Review Board for Human Studies at Case Western Reserve University. Mothers provided witnessed oral informed consent for participation and assent for their infants. All women included in study were screened for HIV as part of a volunteer counseling and testing program supported by the International Center for Reproductive Health and the Kenyan Ministry of Health. Counseling and perinatal treatment with nevirapine was provided through this program. All HIV results were kept strictly confidential. Only HIV-negative women were included in this study to eliminate the impact HIV infection may have on maternal and fetal immune responses.

Pregnant women attending the antenatal clinic at Msambweni District Hospital (Kwale District, Coast Province, Kenya) were recruited. Malaria transmission in this area is stable with seasonal variation related to rainfall (24). Women enrolled in the study were given malaria prophylaxis consisting of single dose of sulfadoxine-pyrimethamine at the beginning of the second and third trimesters of pregnancy in accordance with the recommendations of the Kenya Ministry of Health. Paired maternal venous blood, placental intervillous blood, and umbilical cord blood from the newborns were collected at the time of birth. Infant venous blood samples anticoagulated with heparin were collected beginning at 6 mo of age and every 6 mo thereafter until age 24–30 mo. Plasma was stored at –70°C until Ab assays were performed. A total of 30 HIV-negative maternal-infant pairs were examined for malaria infection and cord blood T cell studies at birth (see below).

Malaria infection status

Maternal venous blood, intervillous placental blood, and cord blood were examined for malaria infection status by two methods. First, thin blood smears were prepared, fixed in methanol, stained with Giemsa, and examined by microscopy for P. falciparum-infected erythrocytes. Second, DNA was extracted from 200 µl of whole intervillous blood and the red cell pellet of anticoagulated maternal and cord blood (Qiagen). A total of 2.5 µl of DNA was used as a template for amplification of the multicopy P. falciparum 18S small subunit ribosomal RNA gene by real-time quantitative PCR (RTQPCR) (25, 26). The increased sensitivity of the RTQPCR assay in comparison to blood smear analysis for P. falciparum malaria has been described in detail (25). A newborn was considered "exposed" to malaria in utero if one or more of the blood smear preparations or RTQPCR results from the various compartments were positive. A newborn was considered to be "not exposed" when both diagnostic tests were negative for all three blood compartments.

Cord blood malaria Ag-driven T cell responses

CBL isolated from heparin anticoagulated blood obtained from the umbilical vein were used to evaluate cytokine production in response to known T cell epitopes within the C-terminal 83-kDa fragment of MSP-1 (GYRKPLDNIKDNVGKMEDYIKK, codons 250–271; KLNSLNNPHNVLQNFSVFFNK, codons 101–121) (27, 29). Peptides were synthesized and purified to >95% (Invitrogen Life Technologies). These peptides were subjected to analysis by MHC class II-binding peptide prediction algorithms (Propred and Tepitope) and found to have broad binding specificity to many MHC class II alleles including DRB1*0401, DRB1*0101, and DRB1*1501 which are common in Kenya (28). A newborn was considered to be "sensitized" to MSP-1 in utero when one of the following three conditions were met: 1) by IFN-{gamma} ELISPOT, there were more than four cytokine-secreting cells/106 CBL in response to MSP-1 peptides and no secreting cells were detected in negative control wells (containing medium alone); 2) by IFN-{gamma} ELISPOT, in cases where cytokine-secreting cells were observed in negative control wells, the number of spots generated by MSP-1-driven CBL was 2-fold greater than control wells; 3) by ELISA for IFN-{gamma}, IL-2, IL-5, or IL-13, net cytokine production by CBL in response to MSP-1 peptides was at least 2-fold greater than that of negative control wells (29). Using these criteria, no malaria-driven IFN-{gamma}-secreting cells were detected in CBL from 16 healthy North American newborns or PBMC from 10 malaria-naive adults.

IL-10 detection

ELISA quantification of IL-10 production was performed on culture supernatants collected after 72 h of MSP-1 peptide stimulation of CBL as previously described (29). Ab pairs for capture and detection (all biotinylated) for IL-10 used 18551D and 18652D (BD Pharmingen). Results were expressed in picograms per milliliter by interpolation from standard curves based on recombinant lymphokines.

Genotyping of P. falciparum MSP-1 alleles

DNA was extracted from venous blood of 12 women with P. falciparum infection detected by blood smear and used as a template for PCR amplification of the C-terminal region of the MSP-1 gene to determine the alleles present in the population at the time the study was conducted. Procedures identical with those described previously were followed (30).

IgG and IgM Abs to MSP-142 and MSP-119 measured by serology

IgG and IgM Abs to rMAD20 and Wellcome/K1 alleles of P. falciparum MSP-142 (provided by C. Long and S. Singh, Malaria Vaccine Development Unit, National Institute of Allergy and Infectious Diseases, Rockville, MD) and MSP-119 (MAD20 allele) were quantified by ELISA as described previously (22). rMSP-119 fused GST was expressed in Escherichia coli. To account for Abs to GST, Ab responses to Plasmodium chabaudi MSP-119 GST fusion protein were subtracted from P. falciparum MSP-119- fusion protein responses. IgG and IgM ELISAs were performed using standard methods (22). Briefly, Immunolon 4 plates (IgG ELISA) or Immunolon 1 plates (IgM ELISA) were coated with 0.1 µg/ml MSP-142 (MAD20 or Wellcome/K1 proteins) or 0.5 µg/ml MSP-119. Plasma from nine North American adults never exposed to malaria were used as the negative controls. Plasma from four known malaria-immune Kenyan adults was pooled to create a positive standard. A standard curve was performed for each plate tested. The value obtained with a 1/50 dilution of the positive pool was designated as 100 arbitrary units (AU), 1/100 dilution as 50 AU, 1/200 dilution as 25 AU, 1/500 dilution as 10 AU, 1/1000 dilution 5 AU, and 1/2000 dilution as 1 AU. A four-parameter standard fit curve was constructed from the positive control plasma pool and applied to sample values. Positive values were greater than the mean + 3 SD of the value of the individual negative control plasma samples.

MSP-119 invasion inhibitory Abs (MSP-119 IIA) levels

Methods to quantify MSP-119 IIA were as described previously (22, 23). D10-PfM3' which encodes the MSP-119 MAD20 allele and an isogenic D10-PcMEGF parasite line in which the antigenically unrelated murine P. chabaudi ortholog replaces the P. falciparum MSP-119 region were tested in parallel (31). Ring-stage parasites were synchronized twice by sorbitol lysis and allowed to mature to late trophozoite/schizont stages. Purified parasites were adjusted to 4% hematocrit with 0.5% infected red cells, and 50-µl aliquots were placed in 96-well flat-bottom microtiter plates with an equal volume of 1/10 prediluted plasma in culture medium (final plasma dilution 1/20, final volume 100 µl). The same batch of prediluted plasma was added to the two parasite lines in the same assay. The cultures were incubated for 26 h to allow for schizont rupture and merozoite invasion. Thin smears were made, fixed with methanol, and stained with Giemsa. The number of ring-stage parasites per 1000 red cells was counted. Each slide was counted independently by two microscopists blinded to the study. Slides made from cord blood plasma MSP-119 IIA experiment were counted by one microscopist blinded to the exposure groups. The average count was used for each slide. The mean number ring-stage parasitemia for duplicate wells was calculated and results were expressed as a percentage of the ring-stage parasitemia of nonimmune control plasma (derived from nonmalaria-exposed adults) in parallel cultures. The percentage point change of invasion inhibition Abs specifically attributable to anti-MSP-119 Abs (MSP-119 IIA) was calculated by subtracting the percentage of invasion of D10-PfM3' relative to nonimmune controls from the percent invasion of D10-PcMEGF relative to nonimmune controls.

Statistics

Results are expressed as the mean ± SEM. The significance of differences between experimental groups was evaluated by the Student t test. Differences between proportions were examined by {chi}2 analysis. The relationship of age with MSP-119 IIA levels was evaluated using linear regression analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Malaria exposure and T cell responses at birth

Table I shows the malaria infection status of maternal-newborn pairs and MSP-1 driven cytokine production by CBL. Based on the presence or absence of malaria parasites and CBL responses to MSP-1, the newborns were divided into three groups. Nine newborns were "exposed" to malaria in utero (defined by positive smear or positive RTQPCR for malaria DNA in maternal, placental, or cord blood) with evidence of T cell "sensitization" (defined by positive CBL cytokine responses to MSP-1 T cell epitopes). Evidence for in utero T cell sensitization was primarily in the form of IFN-{gamma} production measured by ELISPOT (seven of nine subjects) and ELISA (six of nine). CBL from two newborns in this group failed to make IFN-{gamma} but were categorized as sensitized based on MSP-1-driven IL-2, IL-5, and/or IL-13 production. Another 10 newborns were also "exposed" but lacked MSP-1-driven T cell IFN-{gamma}, IL-2, IL-5, or IL-13 responses and were categorized as "not sensitized." Finally, 11 newborns were "not exposed" to malaria in utero (negative blood smears and negative RTQPCR for malaria DNA in all maternal, placental, and cord blood compartments) and were "not sensitized," (lacked CBL cytokine responses to MSP-1).


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  		Table I. Maternal-newborn malaria infection status at parturition and MSP-1-driven cytokine production by CBL

 
To examine whether active suppression may contribute to the lack of detectable MSP-1-driven IFN-{gamma}, IL-2, IL-5, and/or IL-13 response in the "exposed but not sensitized" group, MSP-1-driven IL-10 production by CBL was also examined in culture supernatants (Fig. 1). IL-10 production in the "exposed but not sensitized" group was higher than the other groups.


Figure 1
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  		FIGURE 1. Net MSP-1-driven IL-10 production by CBL. Samples were grouped according to perinatal malaria exposure and CBL sensitization to MSP-1 (measured by IFN-{gamma}, IL-2, IL-5, and/or IL-13 production) as described in Materials and Methods and Table I. Horizontal bars, Mean IL-10 detected for each exposure group. Based on the t test of log-transformed values, statistical differences between experimental groups are shown.

 
Table II presents the demographic characteristics of the three groups. Mothers of newborns who were "exposed" to malaria at birth and were either "sensitized" or "not sensitized" were younger than mothers of newborns who were "not exposed and not sensitized" (respective average ages 21.3, 20.7, and 24.9 years). The latter group included the lowest proportion of primigravida women. There were no differences among the three groups with respect to the proportion of female and male infants, infant malaria infection status at sampled time points, or mean number of samples per infant in follow-up.


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  		Table II. Demographic characteristics of maternal-fetal pairs according to newborn malaria exposure status and cord blood T cell responses to MSP-1

 
MSP-1 alleles and infant MSP-1 IgG and IgM Abs determined by serology

We sequenced venous blood samples from 12 P. falciparum-infected women to determine the distribution MSP-1 C-terminal alleles circulating in the population. Twelve amplicons corresponding to the MAD20 allele and two corresponding to the Wellcome/K1 alleles were observed (from mixed P. falciparum infections). Because the MAD20 allele was predominant, we report here results of studies performed with recombinant proteins corresponding to the MSP-1 proteins for this allele. IgG and IgM ELISAs described below were also performed with the Wellcome/K1 allele. No differences between reactivity relative to the Wellcome/K1 and MAD20 alleles were observed (data not shown).

Fig. 2 describes the mean levels of MSP-142 and MSP-119 IgG and IgM Abs and the percentage of positive infants over time grouped according to whether the infants were sensitized (n = 9) or not sensitized (n = 29) to MSP-1 at birth. In general, there were no statistically significant differences in MSP-142 and MSP-119 IgG and IgM Ab levels detected in infants over time or in relation to exposure group. No statistically significant differences in cord blood Ab levels were observed between exposure groups (data not shown). Stratification of the not sensitized group into newborns who were exposed or not exposed to malaria in utero indicated there were no age-related differences in IgG and IgM Ab trends (data not shown).


Figure 2
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  		FIGURE 2. IgG and IgM Ab levels to the MAD20 allele of MSP-142 and MSP-119 determined by ELISA. Infants were grouped according to perinatal malaria exposure and neonatal CBL sensitization to MSP-1 as described in Materials and Methods and Table I. Plasma from the four times of collection was not available for all infants, i.e., at 6 mo, n = 24; 12 mo, n = 21; 18 mo, n = 22; 24–30 mo, n = 19. {blacktriangleup}, Infants sensitized to MSP-1 in utero based on positive responses of CBL to MSP-1; {circ}, infants not sensitized to MSP-1 in utero. Values for anti-MSP-142/19 IgG/IgM Abs are presented as mean arbitrary units ± SEM. A and B, and E and F, MSP-142 IgG and IgM levels and the percentage of positive infants. C and D, and G and H, Anti-MSP-119 IgG and IgM levels and the percentage of positive infants. There was no significant difference between the two groups at any age (Student’s t test).

 
Infant MSP-119-specific IIA

Fig. 3 describes age-related percent-invasion inhibition due to MSP-119 IIA for all infants participating in the study. MSP-119 IIA levels gradually increased significantly between 6 and 24 mo. The average percent inhibition due to MSP-119 IIA was 16% at 6 mo and 29% at 24–30 mo. For all time points combined, infants with malaria infection at the time points plasma was obtained had similar percent inhibition due to MSP-119 IIA (19 ± 3.3%) compared with children who were not infected when plasma was obtained (21.7 ± 2.6%). The results were similar for values at birth (cord blood), 6, 12, 18, and 24–30 mo of age (data not shown). There was no correlation observed between percent invasion inhibition due to MSP-119 IIA and MSP-142 and MSP-119 IgG or IgM Ab levels measured by serology as observed in earlier studies in humans (21, 22, 23).


Figure 3
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  		FIGURE 3. Mean percent inhibition due to MSP-119 IIA as described in Materials and Methods. Plasma samples were obtained from 30 infants at birth (cord blood; n = 30), 6 (n = 24), 12 (n = 21), 18 (n = 22), and 24–30 (n = 19) mo after birth. Bars indicate the mean percent inhibition due to MSP-119 IIA + SEM. There was a significant difference in mean MSP-119 IIA levels for infants 6 mo vs 24–30 mo of age (p < 0.01, Student’s t test).

 
To examine whether prenatal sensitization to MSP-1 affected acquisition of MSP-119 IIA during infancy, the data were stratified according to whether the infant was or was not sensitized in utero according to CBL responses to MSP-1 T cell epitopes (see Table I). Sensitized infants displayed higher MSP-119 IIA levels at 18 and 24–30 mo compared with infants who were not sensitized at birth (p < 0.01; the Student t test). Additionally, the sensitized infants demonstrated a significant increase in MSP-119 IIA levels with increasing age (r2 = 0.21, F = 6.1, p = 0.02; Fig. 4A). When the not-sensitized group was further divided into infants who were or were not exposed to malaria detected at birth, additional differences in MSP-119 IIA were observed (Fig. 4B). Notably, at 18 and 24–30 mo, the exposed but not sensitized infants had significantly (p < 0.01) lower MSP-119 IIA compared with the other two groups. Exposed and sensitized infants had significantly (p < 0.05) higher MSP-119 IIA compared those exposed but not sensitized infants.


Figure 4
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  		FIGURE 4. Relationship of infant MSP-119 IIA to newborn malaria exposure and sensitization status. A, Groups divided into newborns sensitized to MSP-1 ({diamondsuit} and solid line) or not sensitized to MSP-1 ({square} and dashed line) and MSP-119 IIA levels. Temporal changes in MSP-119 IIA in the two groups were statistically different using regression analysis (p = 0.02). B, Groups further divided into newborns who were exposed and sensitized to malaria ({diamondsuit} and solid line), exposed and not sensitized ({triangleup} and dotted line) or not exposed and not sensitized ({circ} and dashed line) as described in Table I. Plasma samples were obtained from 30 infants at birth (cord blood), 6, 12, 18, and 24–30 mo after birth (samples were not collected from study participants at every time point). There was a significant difference in mean MSP-119 IIA between the groups at 18 and 24–30 mo of age. *, p < 0.05 and **, p < 0.01 by Student’s t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Results of our study lend further support to the idea that fetal malaria experience is important in the regulation of childhood malaria immunity in endemic areas where placental malaria is common. We show that infants born of mothers who live in a malaria holoendemic area of Kenya develop Abs with functional invasion inhibitory activity directed against MSP-119. The levels of these functional Abs increase over the first 2 years after birth. This is most pronounced in infants with evidence of exposure to malaria at parturition and T cell sensitization to MSP-1 in utero. In contrast, infants who lack detectable MSP-1-driven CBL cytokine responses fail to develop MSP-119 IIA despite having equivalent evidence of perinatal exposure to P. falciparum. We speculate that infants in the exposed but not-sensitized group failed to develop high levels of MSP-119 IIA because they became immunologically tolerant by developing T regulatory cells specific for MSP-1. This putatively tolerant group of infants demonstrated elevated production of MSP-1-driven IL-10 and no detectable Th1 or Th2-type cytokine production by CBL supporting the presence of T regulatory cells. Whether MSP-1-driven IL-10 originated from T cells in the current study was not addressed. However, we have previously shown MSP-1-driven IL-10 in CBL from other newborns in the same population originates from CD4+ T cells (29). Other studies of T cells of newborns born to mothers with malaria and neonatal pups of mice infected with Plasmodium yoelii demonstrate that immunoregulatory lymphocytes of newborns are capable of suppressing malaria Ag-driven T cell responses or inducing T cell anergy (29, 32, 33). Taken together with the demonstration of immune tolerance to filarial parasites by a mechanism of prenatal exposure in the context of maternal infection during gestation (34, 35), these findings suggest that intravascular pathogens that chronically infect women during their childbearing years may result in de facto immune tolerance when infants and children are challenged with natural infection later in life.

Given that exposure of the unborn human fetus to malaria-infected RBC or Ags cannot be evaluated directly for technological and ethical reasons, we cannot know precisely when during gestation or for how long before parturition the fetal innate and adaptive immunity might be affected by exposure to maternal malaria infection. Consistent with the concept that in utero exposure to malaria is linked with placental sequestration of infected RBC, newborns who were deemed exposed to malaria were more likely to have primigravida mothers (44%, 9 of 19) relative to not-exposed infants (18%, 2 of 11). Recently, we observed that in women with placental malaria, primi/secundigravid women compared with multigravid women were more likely to have microtransfusions antenatally that would favor transplacental passage of infected RBC and/or malaria Ags (36). Multigravid women with prior placental malaria experience have protective Abs directed against P. falciparum clones that preferentially bind to placental endothelial cells of the placenta thereby reducing the parasite burden in the intervillous space of the placenta (5, 6, 7, 8).

Results reported here confirm and extend earlier work describing the biological significance of MSP-119-specific IIA in human malaria immunity and its relationship to serologic measures of anti-MSP-142 and MSP-119 Abs (22, 23). As described previously for older children and adults, there was no association between MSP-119 IIA and IgG or IgM Abs to recombinant MSP-119 or MSP-142 proteins measured by serology (22). These data along with reports by other investigators (16, 20) demonstrate that polyclonal B cell responses to MSP-1 that develop over time and with repeated natural malaria infections may include those that produce Abs with specificities that may be functionally neutral or inhibitory in terms of their ability to impair RBC invasion and intraerythrocytic growth of parasites. In contrast to our earlier studies of older children and adults from a highland area of Kenya where transmission of malaria is low and unstable (22), we found that MSP-119 IIA detected in plasma from infants residing in this malaria holoendemic of Coast Province was relatively low (average of all infant groups combined was ~20 vs ~50–80% for residents of the highlands). The reasons for these differences aside from age (infants younger than 2 years were not included in the highlands study) are currently being investigated.

There are several implications of our findings with respect to how infant immunity to a malaria vaccine or natural blood stage infection might be affected by prenatal immune priming or tolerance. Fetal Ag exposure and T cell priming are associated with acquisition of functional MSP-1 Ab activity during infancy in this study. However, cord blood levels of MSP-119 IIA did not differ between the exposure groups indicating that maternally derived MSP-1 Abs were not likely to modulate infant immune responses. This contrasts with other studies suggesting that maternally derived Abs may modify subsequent immune responses. Persisting maternal antimalarial Abs have been found to affect the development of Abs in neonatal mice by generating suppressor T cells (37). In addition, Stanisic et al. (33) have shown that maternal P. yoelii MSP-119-specific Abs inhibit the development of Abs to MSP-119 in pups immunized with the corresponding recombinant protein. This inhibition was specific to Ab development and did not affect T cell responses. The differential effect of maternally derived Abs on the fetal response in mice and humans may have occurred because of differences in gestational age and mechanisms by which Abs are transferred to the fetus. In humans, Abs are transplacentally transferred to the fetus during the last trimester of pregnancy whereas mouse Abs are transferred to pups primarily through colostrums (38). A recent study of infants born in a holoendemic area of western Kenya suggests that placental malaria infection correlates with subsequent infant humoral immune responses to malaria (13). In that study, infants up to 1 year of age born to mothers with placental malaria had significantly reduced Ab levels to blood stage epitopes compared with offspring of women without placental malaria. Immune status of infants at the time of birth was not evaluated (13). Future studies may be able to shed light on the mechanistic basis for differential MSP-119 IIA development in some infants but not others by examining how prenatal Ag exposure and concomitant T and B cell responses to various MSP-1 epitopes influence MSP-119 IIA development.

Further efforts to understand the significance of fetal malaria experience and subsequent malaria susceptibility in endemic populations will ideally include a greater emphasis on the possible influence of placental and external environmental factors. Studies are underway to assess whether the severity of placental malaria correlates with prenatal sensitization or tolerance and the relationship of these newborn immune phenotypes with the time to first infection, incidence density of parasitemia during infancy, and the frequency of clinical malaria in the first 1 or 2 years after birth. It will also be important to include a greater number of participants than those described here to account for variability in outcomes attributable to individual malaria exposure history, use of bed nets by pregnant women and their children, and frequency of use of antimalarial drugs.


   Acknowledgments
 
Publication of this study was approved by Dr. Davy Koech, Director of the Kenya Medical Research Institute. We acknowledge Adams Omollo, Kephar Otieno, and Elton K. Mzungu for technical help, and Grace Watutu for data entry. We are especially grateful to the maternity nurses who facilitated the conduct of the study and the women and children who participated. We appreciate the help of Jackson Abuya and Livingstone Wanyama for reading the blood smear slides for the MSP-119 IIA assay. Dr. Sanjay Singh and Dr. Carole Long provided the MSP1-42 recombinant Ag used for this study, along with helpful advice for this study.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the U.S. Department of Health and Human Services (National Institutes of Health (NIH) Grants AI064687 and AI45473) and the Kenyan Ministry of Health. B.S.C. is an International Research Fellow of the Howard Hughes Medical Institute. A.D. was supported by National Institutes of Health Training Fellowships (AI5206702 and AI0702427). Back

2 Address correspondence and reprint requests to Dr. Christopher L. King, Veterans’ Affairs Medical Center, Cleveland, OH 44106. E-mail address: christopher.king{at}case.edu Back

3 Abbreviations used in this paper: CBL, cord blood lymphocyte; MSP, merozoite surface protein; RTQPCR, real-time quantitative PCR; AU, arbitrary unit. Back

Received for publication May 12, 2006. Accepted for publication August 21, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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