Prenatal Immune Experience Affects Acquisition of Plasmodium falciparum Merozoite Surface Protein-1 Invasion Inhibitory Antibodies during Infancy This information is current as of September 26, 2021. Arlene Dent, Indu Malhotra, Peter Mungai, Eric Muchiri, Brendan S. Crabb, James W. Kazura and Christopher L. King J Immunol 2006; 177:7139-7145; ; doi: 10.4049/jimmunol.177.10.7139 Downloaded from http://www.jimmunol.org/content/177/10/7139

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

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

Arlene Dent,* Indu Malhotra,* Peter Mungai,*† Eric Muchiri,† Brendan S. Crabb,‡ James W. Kazura,* and Christopher L. King2*§

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 . Abs to the 42- and 19-kDa C-terminal processed fragments of MSP-1 were determined Downloaded from 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 http://www.jimmunol.org/ that fetal sensitization or tolerance to MSP-1, associated with maternal malaria infection during pregnancy, affects the develop- ment of functional Ab responses to MSP-1 during infancy. The Journal of Immunology, 2006, 177: 7139–7145.

alciparum malaria is one of the most important pediatric future malaria vaccines will be evaluated and delivered in this age infectious diseases in sub-Saharan Africa, where it is es- group. F timated to kill at least 1 million children per year. In areas Understanding antimalaria immunity in human infants is com- where malaria is stable, infants and children typically plicated by the physiologic and immunologic effects of maternal experience clinical morbidity and high blood stage parasite densi- malaria infection on the unborn fetus and its impact on infant ac- ties. Infants Ͻ6 mo old appear to be relatively resistant to clinical quisition of immune responses to blood stage infection. Unlike the by guest on September 26, 2021 malaria and high density parasitemia while those between 6 and 24 other three malaria species that infect humans, P. falciparum-in- mo of age appear to have increased susceptibility to these clinical fected erythrocytes have the propensity to sequester in the inter- and parasitological outcomes (1, 2). Although evidence supporting villous blood of the placenta, a condition referred to as placental a direct role for passively acquired maternal IgG Abs in mediating malaria. Placental malaria is most common in women with their protection against infant malaria is inconsistent (3), epidemiologic first or second pregnancy, seemingly due to the lack of acquired studies show that the age-related increase in malaria susceptibility immunity to P. falciparum clones that preferentially bind to the occurs after maternal Abs have waned, estimated to be 6–8 mo placental vasculature (5–8). In this context, the unborn fetus may after birth, before immunity to blood stage Plasmodium falciparum potentially be exposed to soluble malaria Ags or infected erythro- develops as a result of repeated (4). Knowledge of the cytes that gain access to the fetal circulation after crossing the immune mechanisms underlying changes in infant malaria suscep- placenta. Although it is not feasible to document this process in tibility may have important implications not only for fundamental vivo, indirect evidence supporting its occurrence includes the dem- understanding of human malaria immunobiology but also for how onstration of T and B cell responses to crude schizont extracts and blood stage Ags in cord blood lymphocytes (CBL),3 a cell popu- lation that represents circulating fetal lymphocytes present at the

*Center for Global Health and Diseases, Case Western Reserve University, Cleve- time of birth (9–11). The consequences of this in utero immune land, OH 44106; †Division of Borne Diseases, Nairobi, Kenya; ‡Walter and experience on infant malaria immunity are not known, particularly Eliza Hall Institute of Medical Research, Melbourne, Australia; and §Veterans’ Af- whether it accelerates the development of protective Abs by virtue fairs Medical Center, Cleveland, OH 44106 of sensitization or impairs their acquisition by immune tolerance Received for publication May 12, 2006. Accepted for publication August 21, 2006. mechanisms. The potential health significance of this fetal malaria The costs of publication of this article were defrayed in part by the payment of page experience is underscored by epidemiologic studies suggesting charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. that offspring of women with placental malaria are more suscep- 1 This work was supported by the U.S. Department of Health and Human Services tible to malaria during childhood compared with offspring of moth- (National Institutes of Health (NIH) Grants AI064687 and AI45473) and the Kenyan ers without placental malaria (12–15). 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 Fel- lowships (AI5206702 and AI0702427). 2 Address correspondence and reprint requests to Dr. Christopher L. King, Veterans’ Affairs Medical Center, Cleveland, OH 44106. E-mail address: christopher.king@ 3 Abbreviations used in this paper: CBL, cord blood lymphocyte; MSP, merozoite case.edu surface protein; RTQPCR, real-time quantitative PCR; AU, arbitrary unit.

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 7140 PRENATAL EXPOSURE AFFECTS INFANT-ACQUIRED MALARIA IMMUNITY

Studies of infant immunity to merozoite surface protein-1 PLDNIKDNVGKMEDYIKK, codons 250–271; KLNSLNNPHNVLQN (MSP-1), an abundant merozoite surface ligand required for inva- FSVFFNK, codons 101–121) (27, 29). Peptides were synthesized and pu- Ͼ sion of erythrocytes and a human malaria vaccine candidate, have rified to 95% (Invitrogen Life Technologies). These peptides were subjected to analysis by MHC class II-binding peptide prediction algo- been inconclusive regarding whether anti-MSP-1 Abs mediate or rithms (Propred and Tepitope) and found to have broad binding specificity are at least a biomarker of protection against childhood blood- to many MHC class II alleles including DRB1*0401, DRB1*0101, and stage malaria infection and clinical morbidity (16–20). The rea- DRB1*1501 which are common in Kenya (28). A newborn was considered sons for these apparent discrepancies are ill-defined but taken to- to be “sensitized” to MSP-1 in utero when one of the following three conditions were met: 1) by IFN-␥ ELISPOT, there were more than four gether suggest that anti-MSP-1 Abs detected by serology alone are cytokine-secreting cells/106 CBL in response to MSP-1 peptides and no a poor surrogate of acquired immunity. Functional Ab assays may secreting cells were detected in negative control wells (containing medium be a better measure of naturally acquired immunity, especially in alone); 2) by IFN-␥ ELISPOT, in cases where cytokine-secreting cells relation to MSP-1 (21–23). We describe here the results of a pro- were observed in negative control wells, the number of spots generated by spective cohort study of infants born in a malaria holoendemic area MSP-1-driven CBL was 2-fold greater than control wells; 3) by ELISA for IFN-␥, IL-2, IL-5, or IL-13, net cytokine production by CBL in response of Kenya designed to test the hypothesis that prenatal malaria ex- to MSP-1 peptides was at least 2-fold greater than that of negative control perience, reflected by newborn and maternal malaria infection at wells (29). Using these criteria, no malaria-driven IFN-␥-secreting cells parturition and neonatal cord blood T cell sensitization to MSP-1, were detected in CBL from 16 healthy North American newborns or influences the development Abs to MSP-1 during infancy. We PBMC from 10 malaria-naive adults. measured Abs to the 42- and 19-kDa C-terminal fragments of IL-10 detection MSP-1, both by traditional serologic methods and by a functional ELISA quantification of IL-10 production was performed on culture su- Downloaded from assay that quantifies inhibition of invasion of erythrocytes by the pernatants collected after 72 h of MSP-1 peptide stimulation of CBL as membrane-anchored MSP-119 ligand required for entry of the par- previously described (29). Ab pairs for capture and detection (all biotin- asite into RBC (23). ylated) for IL-10 used 18551D and 18652D (BD Pharmingen). Results were expressed in picograms per milliliter by interpolation from standard Materials and Methods curves based on recombinant lymphokines. Study participants and study design Genotyping of P. falciparum MSP-1 alleles Approval for the study was obtained from the Kenya Medical Research DNA was extracted from venous blood of 12 women with P. falciparum http://www.jimmunol.org/ Institute National Ethical Review Committee and the Institutional Review infection detected by blood smear and used as a template for PCR ampli- Board for Human Studies at Case Western Reserve University. Mothers fication of the C-terminal region of the MSP-1 gene to determine the alleles provided witnessed oral informed consent for participation and assent for present in the population at the time the study was conducted. Procedures their infants. All women included in study were screened for HIV as part identical with those described previously were followed (30). of a volunteer counseling and testing program supported by the Interna- tional Center for Reproductive Health and the Kenyan Ministry of Health. IgG and IgM Abs to MSP-142 and MSP-119 measured by Counseling and perinatal treatment with nevirapine was provided through serology this program. All HIV results were kept strictly confidential. Only HIV- negative women were included in this study to eliminate the impact HIV IgG and IgM Abs to rMAD20 and Wellcome/K1 alleles of P. falciparum MSP-1 (provided by C. Long and S. Singh, Malaria Vaccine Develop- infection may have on maternal and fetal immune responses. 42 by guest on September 26, 2021 Pregnant women attending the antenatal clinic at Msambweni District ment Unit, National Institute of Allergy and Infectious Diseases, Rockville, Hospital (Kwale District, Coast Province, Kenya) were recruited. Malaria MD) and MSP-119 (MAD20 allele) were quantified by ELISA as described transmission in this area is stable with seasonal variation related to rainfall previously (22). rMSP-119 fused GST was expressed in Escherichia coli. (24). Women enrolled in the study were given malaria prophylaxis con- To account for Abs to GST, Ab responses to Plasmodium chabaudi MSP- sisting of single dose of sulfadoxine-pyrimethamine at the beginning of the 119 GST fusion protein were subtracted from P. falciparum MSP-119- second and third trimesters of pregnancy in accordance with the recom- fusion protein responses. IgG and IgM ELISAs were performed using mendations of the Kenya Ministry of Health. Paired maternal venous standard methods (22). Briefly, Immunolon 4 plates (IgG ELISA) or Im- ␮ blood, placental intervillous blood, and umbilical cord blood from the new- munolon 1 plates (IgM ELISA) were coated with 0.1 g/ml MSP-142 ␮ borns were collected at the time of birth. Infant venous blood samples (MAD20 or Wellcome/K1 proteins) or 0.5 g/ml MSP-119. Plasma from anticoagulated with heparin were collected beginning at 6 mo of age and nine North American adults never exposed to malaria were used as the every 6 mo thereafter until age 24–30 mo. Plasma was stored at Ϫ70°C negative controls. Plasma from four known malaria-immune Kenyan adults until Ab assays were performed. A total of 30 HIV-negative maternal- was pooled to create a positive standard. A standard curve was performed infant pairs were examined for malaria infection and cord blood T cell for each plate tested. The value obtained with a 1/50 dilution of the positive studies at birth (see below). 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, Malaria infection status 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 Maternal venous blood, intervillous placental blood, and cord blood were values. Positive values were greater than the mean ϩ 3 SD of the value of examined for malaria infection status by two methods. First, thin blood the individual negative control plasma samples. smears were prepared, fixed in methanol, stained with Giemsa, and exam- ined by microscopy for P. falciparum-infected erythrocytes. Second, DNA MSP-119 invasion inhibitory Abs (MSP-119 IIA) levels was extracted from 200 ␮l of whole intervillous blood and the red cell Methods to quantify MSP-1 IIA were as described previously (22, 23). pellet of anticoagulated maternal and cord blood (Qiagen). A total of 2.5 ␮l 19 D10-PfM3Ј which encodes the MSP-1 MAD20 allele and an isogenic of DNA was used as a template for amplification of the multicopy P. 19 D10-PcMEGF parasite line in which the antigenically unrelated murine P. falciparum 18S small subunit ribosomal RNA gene by real-time quantita- chabaudi ortholog replaces the P. falciparum MSP-1 region were tested tive PCR (RTQPCR) (25, 26). The increased sensitivity of the RTQPCR 19 in parallel (31). Ring-stage parasites were synchronized twice by sorbitol assay in comparison to blood smear analysis for P. falciparum malaria has lysis and allowed to mature to late trophozoite/schizont stages. Purified been described in detail (25). A newborn was considered “exposed” to parasites were adjusted to 4% hematocrit with 0.5% infected red cells, and malaria in utero if one or more of the blood smear preparations or 50-␮ RTQPCR results from the various compartments were positive. A newborn l aliquots were placed in 96-well flat-bottom microtiter plates with an equal volume of 1/10 prediluted plasma in culture medium (final plasma was considered to be “not exposed” when both diagnostic tests were neg- ␮ ative for all three blood compartments. dilution 1/20, final volume 100 l). The same batch of prediluted plasma was added to the two parasite lines in the same assay. The cultures were Cord blood malaria Ag-driven T cell responses incubated for 26 h to allow for schizont rupture and merozoite invasion. Thin smears were made, fixed with methanol, and stained with Giemsa. CBL isolated from heparin anticoagulated blood obtained from the umbil- The number of ring-stage parasites per 1000 red cells was counted. Each ical vein were used to evaluate cytokine production in response to known slide was counted independently by two microscopists blinded to the study.

T cell epitopes within the C-terminal 83-kDa fragment of MSP-1 (GYRK Slides made from cord blood plasma MSP-119 IIA experiment were The Journal of Immunology 7141 counted by one microscopist blinded to the exposure groups. The average Evidence for in utero T cell sensitization was primarily in the form of count was used for each slide. The mean number ring-stage parasitemia for IFN-␥ production measured by ELISPOT (seven of nine subjects) and duplicate wells was calculated and results were expressed as a percentage ELISA (six of nine). CBL from two newborns in this group failed to of the ring-stage parasitemia of nonimmune control plasma (derived from nonmalaria-exposed adults) in parallel cultures. The percentage point make IFN-␥ but were categorized as sensitized based on MSP-1- change of invasion inhibition Abs specifically attributable to anti-MSP-119 driven IL-2, IL-5, and/or IL-13 production. Another 10 newborns Abs (MSP-119 IIA) was calculated by subtracting the percentage of inva- were also “exposed” but lacked MSP-1-driven T cell IFN-␥, IL-2, sion of D10-PfM3Ј relative to nonimmune controls from the percent inva- sion of D10-PcMEGF relative to nonimmune controls. IL-5, or IL-13 responses and were categorized as “not sensitized.” Finally, 11 newborns were “not exposed” to malaria in utero (negative Statistics blood smears and negative RTQPCR for malaria DNA in all maternal, Results are expressed as the mean Ϯ SEM. The significance of differences placental, and cord blood compartments) and were “not sensitized,” between experimental groups was evaluated by the Student t test. Differ- (lacked CBL cytokine responses to MSP-1). ␹2 ences between proportions were examined by analysis. The relationship To examine whether active suppression may contribute to the of age with MSP-1 IIA levels was evaluated using linear regression 19 ␥ analysis. lack of detectable MSP-1-driven IFN- , IL-2, IL-5, and/or IL-13 response in the “exposed but not sensitized” group, MSP-1-driven Results IL-10 production by CBL was also examined in culture superna- Malaria exposure and T cell responses at birth tants (Fig. 1). IL-10 production in the “exposed but not sensitized” Table I shows the malaria infection status of maternal-newborn group was higher than the other groups. Table II presents the demographic characteristics of the three pairs and MSP-1 driven cytokine production by CBL. Based on the Downloaded from presence or absence of malaria parasites and CBL responses to groups. Mothers of newborns who were “exposed” to malaria at MSP-1, the newborns were divided into three groups. Nine new- birth and were either “sensitized” or “not sensitized” were younger borns were “exposed” to malaria in utero (defined by positive than mothers of newborns who were “not exposed and not sensi- smear or positive RTQPCR for malaria DNA in maternal, placen- tized” (respective average ages 21.3, 20.7, and 24.9 years). The tal, or cord blood) with evidence of T cell “sensitization” (defined latter group included the lowest proportion of primigravida by positive CBL cytokine responses to MSP-1 T cell epitopes). women. There were no differences among the three groups with http://www.jimmunol.org/

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

Blood Compartment with P. falciparuma Cytokine Production by ELISA (pg/ml)

Category Maternal venous Intervillous placenta CordIFN-␥ ELISPOTb IFN-␥ (IL-2)c IL-5 (IL-13)c

Exposed and sensitized

14 ϩϪϩϩ222 0 by guest on September 26, 2021 21 ϩ (32) ϩϩϩ 147 (218) 81 ϩϩϩϩ535 0 87 ϩϩ(40) ϩϩ 241 0 102 Ϫϩϩϩ192 0 129 ϩ (134) ϩϩϩ (74) 128 134 ϩϩϪϪ0 447 145 ϪϪϩϩ1963 880 230 ϪϩϪϪ(208) (145) Exposed and not sensitized 1 ϩϩndd Ϫ 00 27 ϩϩϪϪ00 75 ϩ (240) ϩϩϪ 00 95 ϪϪϩϪ00 107 ϩϩ(Ͼ60) ϪϪ 00 142 ϪϩϩϪ00 157 ϩϩϪϪ00 168 ϩϪϩϪ00 177 ϩϩϪϪ00 186 nd ϩϩϪ 00 Not exposed and not sensitized 10 ϪϪnd Ϫ 00 28 ϪϪϪϪ00 34 ϪϪnd Ϫ 00 37 ϪϪnd Ϫ 00 72 ϪϪϪϪ00 79 ϪϪϪϪ00 105 ϪϪϪϪ00 146 ϪϪϪϪ00 152 ϪϪϪϪ00 166 ϪϪϪϪ00 181 ϪϪϪϪ00

a Assessed by RTQPCR. Values in parentheses indicate the number of asexual P. falciparum per microliter of blood detected by thin blood smear. b More than four IFN-␥-producing cells per 4 ϫ 105 CBL following stimulation with either or both MSP-1 peptides (GYRKPLDNIKDNVGKMEDYIKK, codons 250–271; KLNSLNNPHNVLQNFSVFFNK; codons 101–121) when negative control wells lacked detectable cytokine-secreting cells, or Ͼ2-fold number of IFN-␥-secreting cells compared to negative control wells when the latter had cytokine-secreting cells. c Value in column represents net cytokine production: outside parentheses, IFN-␥ and IL-5; inside parentheses, IL-2 and IL-13. d nd, Not done. 7142 PRENATAL EXPOSURE AFFECTS INFANT-ACQUIRED MALARIA IMMUNITY

sitized (n ϭ 29) to MSP-1 at birth. In general, there were no statis-

tically 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). Stratifica- tion 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).

Infant MSP-119-specific IIA Fig. 3 describes age-related percent-invasion inhibition due to MSP- FIGURE 1. Net MSP-1-driven IL-10 production by CBL. Samples were 119 IIA for all infants participating in the study. MSP-119 IIA levels grouped according to perinatal malaria exposure and CBL sensitization to gradually increased significantly between 6 and 24 mo. The average ␥ MSP-1 (measured by IFN- , IL-2, IL-5, and/or IL-13 production) as described percent inhibition due to MSP-1 IIA was 16% at 6 mo and 29% at in Materials and Methods and Table I. Horizontal bars, Mean IL-10 detected 19 24–30 mo. For all time points combined, infants with malaria infec- for each exposure group. Based on the t test of log-transformed values, sta- tistical differences between experimental groups are shown. tion at the time points plasma was obtained had similar percent inhi- Ϯ bition due to MSP-119 IIA (19 3.3%) compared with children who were not infected when plasma was obtained (21.7 Ϯ 2.6%). The

results were similar for values at birth (cord blood), 6, 12, 18, and Downloaded from respect to the proportion of female and male infants, infant malaria 24–30 mo of age (data not shown). There was no correlation observed infection status at sampled time points, or mean number of samples between percent invasion inhibition due to MSP-1 IIA and MSP-1 per infant in follow-up. 19 42 and MSP-119 IgG or IgM Ab levels measured by serology as ob- MSP-1 alleles and infant MSP-1 IgG and IgM Abs determined served in earlier studies in humans (21–23). by serology To examine whether prenatal sensitization to MSP-1 affected

acquisition of MSP-1 IIA during infancy, the data were stratified http://www.jimmunol.org/ We sequenced venous blood samples from 12 P. falciparum-in- 19 according to whether the infant was or was not sensitized in utero fected women to determine the distribution MSP-1 C-terminal al- according to CBL responses to MSP-1 T cell epitopes (see Table leles circulating in the population. Twelve amplicons correspond- I). Sensitized infants displayed higher MSP-1 IIA levels at 18 ing to the MAD20 allele and two corresponding to the Wellcome/K1 19 and 24–30 mo compared with infants who were not sensitized at alleles were observed (from mixed P. falciparum infections). Because birth ( p Ͻ 0.01; the Student t test). Additionally, the sensitized the MAD20 allele was predominant, we report here results of infants demonstrated a significant increase in MSP-1 IIA levels studies performed with recombinant proteins corresponding to the 19 with increasing age (r2 ϭ 0.21, F ϭ 6.1, p ϭ 0.02; Fig. 4A). When MSP-1 proteins for this allele. IgG and IgM ELISAs described the not-sensitized group was further divided into infants who were below were also performed with the Wellcome/K1 allele. No by guest on September 26, 2021 or were not exposed to malaria detected at birth, additional differ- differences between reactivity relative to the Wellcome/K1 and ences in MSP-1 IIA were observed (Fig. 4B). Notably, at 18 and MAD20 alleles were observed (data not shown). 19 24–30 mo, the exposed but not sensitized infants had significantly Fig. 2 describes the mean levels of MSP-1 and MSP-1 IgG and 42 19 ( p Ͻ 0.01) lower MSP-1 IIA compared with the other two IgM Abs and the percentage of positive infants over time grouped 19 groups. Exposed and sensitized infants had significantly ( p Ͻ according to whether the infants were sensitized (n ϭ 9) or not sen- 0.05) higher MSP-119 IIA compared those exposed but not sensi- tized infants.

Table II. Demographic characteristics of maternal-fetal pairs Discussion according to newborn malaria exposure status and cord blood T cell responses to MSP-1 Results of our study lend further support to the idea that fetal malaria experience is important in the regulation of childhood ma- Not laria immunity in areas where placental malaria is com- Exposed Exposed Exposed mon. We show that infants born of mothers who live in a malaria and and Not and Not holoendemic area of Kenya develop Abs with functional invasion Newborn Category Sensitized Sensitized Sensitized inhibitory activity directed against MSP-119. The levels of these Maternal/newborn infection Yes Yes No functional Abs increase over the first 2 years after birth. This is with malaria most pronounced in infants with evidence of exposure to malaria Newborn T cell response to Yes No No at parturition and T cell sensitization to MSP-1 in utero. In con- malaria Ags Number of children 9 10 11 trast, infants who lack detectable MSP-1-driven CBL cytokine re- Males/females 4/5 5/5 7/4 sponses fail to develop MSP-119 IIA despite having equivalent Average age of mother at 21.3 20.7 24.9 evidence of perinatal exposure to P. falciparum. We speculate that delivery (years) infants in the exposed but not-sensitized group failed to develop Primigravida/multigravida 4/5 4/6 2/9 mothers high levels of MSP-119 IIA because they became immunologically Mean number samples per 2.9 3.2 2.8 tolerant by developing T regulatory cells specific for MSP-1. This infant in follow-up putatively tolerant group of infants demonstrated elevated produc- Fraction of infants with tion of MSP-1-driven IL-10 and no detectable Th1 or Th2-type positive malaria blood cytokine production by CBL supporting the presence of T regula- Smear at each follow-up 0.29 0.39 0.35 time pointa tory cells. Whether MSP-1-driven IL-10 originated from T cells in the current study was not addressed. However, we have previously a Venous blood was examined for P. falciparum DNA by RTQPCR as described in Materials and Methods. The calculation was adjusted for the number of follow-up shown MSP-1-driven IL-10 in CBL from other newborns in the ϩ samples examined for each infant. same population originates from CD4 T cells (29). Other studies The Journal of Immunology 7143

FIGURE 2. IgG and IgM Ab lev- els to the MAD20 allele of MSP-142 and MSP-119 determined by ELISA. Infants were grouped according to perinatal malaria exposure and neo- natal CBL sensitization to MSP-1 as described in Materials and Methods and Table I. Plasma from the four Downloaded from times of collection was not available for all infants, i.e., at 6 mo, n ϭ 24; 12 mo, n ϭ 21; 18 mo, n ϭ 22; 24–30 mo, n ϭ 19. Œ, Infants sensi- tized to MSP-1 in utero based on positive responses of CBL to MSP-1; http://www.jimmunol.org/ E, infants not sensitized to MSP-1 in utero. Values for anti-MSP-142/19 IgG/IgM Abs are presented as mean arbitrary units Ϯ SEM. A and B, and E and F, MSP-142 IgG and IgM levels and the percentage of posi- tive infants. C and D, and G and H,

Anti-MSP-119 IgG and IgM levels and the percentage of positive in- fants. There was no significant dif- by guest on September 26, 2021 ference between the two groups at any age (Student’s t test).

of T cells of newborns born to mothers with malaria and neonatal tive immunity might be affected by exposure to maternal malaria pups of mice infected with Plasmodium yoelii demonstrate that infection. Consistent with the concept that in utero exposure to immunoregulatory lymphocytes of newborns are capable of sup- malaria is linked with placental sequestration of infected RBC, pressing malaria Ag-driven T cell responses or inducing T cell newborns who were deemed exposed to malaria were more likely anergy (29, 32, 33). Taken together with the demonstration of im- to have primigravida mothers (44%, 9 of 19) relative to not- mune tolerance to filarial parasites by a mechanism of prenatal exposed infants (18%, 2 of 11). Recently, we observed that in exposure in the context of maternal infection during gestation (34, women with placental malaria, primi/secundigravid women 35), these findings suggest that intravascular that chron- compared with multigravid women were more likely to have ically infect women during their childbearing years may result in microtransfusions antenatally that would favor transplacental de facto immune tolerance when infants and children are chal- passage of infected RBC and/or malaria Ags (36). Multigravid lenged with natural infection later in life. women with prior placental malaria experience have protective Given that exposure of the unborn human fetus to malaria-in- Abs directed against P. falciparum clones that preferentially fected RBC or Ags cannot be evaluated directly for technological bind to placental endothelial cells of the placenta thereby re- and ethical reasons, we cannot know precisely when during ges- ducing the parasite burden in the intervillous space of the pla- tation or for how long before parturition the fetal innate and adap- centa (5–8). 7144 PRENATAL EXPOSURE AFFECTS INFANT-ACQUIRED MALARIA IMMUNITY

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 poly- clonal 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 intraerythro- cytic growth of parasites. In contrast to our earlier studies of older children and adults from a highland area of Kenya where trans- mission of malaria is low and unstable (22), we found that MSP- 1 IIA detected in plasma from infants residing in this malaria FIGURE 3. Mean percent inhibition due to MSP-1 IIA as described in 19 19 holoendemic of Coast Province was relatively low (average of all Materials and Methods. Plasma samples were obtained from 30 infants at infant groups combined was ϳ20 vs ϳ50–80% for residents of the birth (cord blood; n ϭ 30), 6 (n ϭ 24), 12 (n ϭ 21), 18 (n ϭ 22), and 24–30 (n ϭ 19) mo after birth. Bars indicate the mean percent inhibition due to highlands). The reasons for these differences aside from age (in- ϩ fants younger than 2 years were not included in the highlands 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 Ͻ 0.01, Student’s t test). study) are currently being investigated.

There are several implications of our findings with respect to how Downloaded from infant immunity to a malaria vaccine or natural blood stage infection Results reported here confirm and extend earlier work describ- might be affected by prenatal immune priming or tolerance. Fetal Ag ing the biological significance of MSP-119-specific IIA in human exposure and T cell priming are associated with acquisition of func- malaria immunity and its relationship to serologic measures of tional 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 http://www.jimmunol.org/ to modulate infant immune responses. This contrasts with other stud- ies 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 re- combinant protein. This inhibition was specific to Ab development and did not affect T cell responses. The differential effect of maternally by guest on September 26, 2021 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 transpla- centally transferred to the fetus during the last trimester of pregnancy whereas mouse Abs are transferred to pups primarily through co- lostrums (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. Im- mune 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 ex-

FIGURE 4. Relationship of infant MSP-119 IIA to newborn malaria ex- perience and subsequent malaria susceptibility in endemic popula- posure and sensitization status. A, Groups divided into newborns sensitized tions will ideally include a greater emphasis on the possible influence to MSP-1 (ࡗ and solid line) or not sensitized to MSP-1 (Ⅺ and dashed of placental and external environmental factors. Studies are underway line) and MSP-119 IIA levels. Temporal changes in MSP-119 IIA in the two to assess whether the severity of placental malaria correlates with ϭ groups were statistically different using regression analysis (p 0.02). B, prenatal sensitization or tolerance and the relationship of these new- Groups further divided into newborns who were exposed and sensitized to born immune phenotypes with the time to first infection, malaria (ࡗ and solid line), exposed and not sensitized (‚ and dotted line) density of parasitemia during infancy, and the frequency of clinical or not exposed and not sensitized (E and dashed line) as described in Table I. Plasma samples were obtained from 30 infants at birth (cord blood), 6, malaria in the first 1 or 2 years after birth. It will also be important to 12, 18, and 24–30 mo after birth (samples were not collected from study include a greater number of participants than those described here to participants at every time point). There was a significant difference in mean account for variability in outcomes attributable to individual malaria Ͻ ء MSP-119 IIA between the groups at 18 and 24–30 mo of age. , p 0.05 exposure history, use of bed nets by pregnant women and their chil- .p Ͻ 0.01 by Student’s t test. dren, and frequency of use of antimalarial drugs ,ءء and The Journal of Immunology 7145

Acknowledgments (PfMSP1) of Plasmodium falciparum are associated with reduced malaria mor- bidity. Parasite Immunol. 14: 321–337. Publication of this study was approved by Dr. Davy Koech, Director of the 19. Branch, O. H., V. Udhayakumar, A. W. Hightower, A. J. Oloo, W. A. Hawley, Kenya Medical Research Institute. We acknowledge Adams Omollo, B. L. Nahlen, P. B. Bloland, D. C. Kaslow, and A. A. Lal. 1998. A longitudinal Kephar Otieno, and Elton K. Mzungu for technical help, and Grace Watutu investigation of IgG and IgM antibody responses to the merozoite surface pro- for data entry. We are especially grateful to the maternity nurses who tein-1 19-kilodalton domain of Plasmodium falciparum in pregnant women and infants: associations with febrile illness, parasitemia, and anemia. Am. J. Trop. facilitated the conduct of the study and the women and children who par- Med. Hyg. 58: 211–219. ticipated. We appreciate the help of Jackson Abuya and Livingstone 20. Okech, B. A., P. H. Corran, J. Todd, A. Joynson-Hicks, C. Uthaipibull, T. G. Egwang, A. A. Holder, and E. M. Riley. 2004. Fine specificity of serum 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 antibodies to Plasmodium falciparum merozoite surface protein, PfMSP-1(19), predicts protection from malaria infection and high-density parasitemia. Infect. Ag used for this study, along with helpful advice for this study. Immun. 72: 1557–1567. 21. de Koning-Ward, T. F., R. A. O’Donnell, D. R. Drew, R. Thomson, T. P. Speed, Disclosures and B. S. Crabb. 2003. A new rodent model to assess blood stage immunity to the Plasmodium falciparum antigen merozoite surface protein 119 reveals a protec- The authors have no financial conflict of interest. tive role for invasion inhibitory antibodies. J. Exp. Med. 198: 869–875. 22. John, C. C., R. A. O’Donnell, P. O. Sumba, A. M. Moormann, T. F. de Koning-Ward, References C. L. King, J. W. Kazura, and B. S. Crabb. 2004. Evidence that invasion-inhibitory 1. Macdonald, G. 1950. The analysis of malaria parasite rates in infants. Trop. Dis. antibodies specific for the 19-kDa fragment of merozoite surface protein-1 (MSP-1 Bull. 47: 915–938. 19) can play a protective role against blood-stage Plasmodium falciparum infection 2. McGuinness, D., K. Koram, S. Bennett, G. Wagner, F. Nkrumah, and E. Riley. 1998. in individuals in a malaria endemic area of Africa. J. Immunol. 173: 666–672. Clinical case definitions for malaria: clinical malaria associated with very low para- 23. O’Donnell, R. A., T. F. de Koning-Ward, R. A. Burt, M. Bockarie, J. C. Reeder, site densities in African infants. Trans. R. Soc. Trop. Med. Hyg. 92: 527–531. A. F. Cowman, and B. S. Crabb. 2001. Antibodies against merozoite surface protein (MSP)-1(19) are a major component of the invasion-inhibitory response

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