Parity and placental infection affect antibody responses against

Parity and placental infection affect antibody responses against Plasmodium 1

falciparum during pregnancy 2

3

Running title: Malaria immunity during pregnancy 4

5

Alfredo Mayor1,2,3*

, Eduard Rovira-Vallbona1,3

, Sonia Machevo2, Quique Bassat

1,2,3, 6

Ruth Aguilar1,2

, Llorenç Quintó1,3

, Alfons Jiménez1, Betuel Sigauque

1,2, Carlota 7

Dobaño1,2,3

, Sanjeev Kumar4, Bijender Singh

4, Puneet Gupta

4, Virander S. Chauhan

4, 8

Chetan E. Chitnis4, Pedro L. Alonso

1,2,3, Clara Menéndez

1,2,3 9

1. Barcelona Centre for International Health Research (CRESIB), Hospital Clínic-10

Universitat de Barcelona, Barcelona, Spain. 11

2. Centro de Investigação em Saúde da Manhiça (CISM), Maputo, Mozambique. 12

3. CIBER Epidemiología y Salud Pública (CIBERESP), Spain 13

4. International Centre for Genetic Engineering and Biotechnology, New Delhi, India. 14

15

Key words: Plasmodium falciparum; pregnancy; placental infection; parity; antibodies. 16

17

Corresponding author: 18

Alfredo Mayor 19

Barcelona Centre for International Health Research (CRESIB), Hospital Clínic-20

Universitat de Barcelona, Rosselló 132, E-08036 Barcelona, Spain. Tel: 21

+34.932275706; Fax: +34.932279853. E-mail: [email protected] 22

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.01000-10 IAI Accepts, published online ahead of print on 7 February 2011

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ABSTRACT 23

24

Women are at higher risk of Plasmodium falciparum infection when pregnant. The 25

decreasing risk of malaria with subsequent pregnancies is attributed to parity-dependent 26

acquisition of antibodies against placental parasites expressing variant surface antigens, 27

VAR2CSA, that mediate placental sequestration through adhesion to chondroitin 28

sulphate A (CSA). However, modulation of immunity during pregnancy may also 29

contribute to increase the risk to malaria. We compared antibody responses between 30 30

Mozambican primigravidae and 60 multigravidae at delivery, 40 men and 40 children. 31

IgG levels were measured against the infected-erythrocyte surface of P. falciparum 32

isolates from 12 pregnant women (4 placental and 8 peripheral) and 26 non-pregnant 33

hosts. We also measured IgGs against merozoite recombinant antigens and total IgGs. 34

Placental P. falciparum infection was associated with increased levels of total IgGs as 35

well as IgG levels against merozoite antigens and parasite isolates from pregnant and 36

non-pregnant hosts. We therefore stratified comparisons of antibody levels by placental 37

infection. Compared to multigravidae, uninfected primigravidae had lower total IgGs as 38

well as lower IgGs against peripheral isolates from both pregnant and non-pregnant 39

hosts. These differences were not explained by use of bed nets, season at delivery, 40

neighbourhood of residence or age. Compared to men, infected primigravidae had 41

higher levels of IgGs against isolates from pregnant women and CSA-binding lines, but 42

not against other isolates, supporting the concept of a pregnancy-specific development 43

of immunity to these parasite variants. Results of this study show that parity and 44

placental infection can modulate immune responses during pregnancy against malaria 45

parasites. 46


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INTRODUCTION 47

48

Women are at higher risk of infection and disease when pregnant (10). This increased 49

susceptibility to infection is described for a broad spectrum of pathogens, including 50

bacteria (Listeria (29)), fungi (Coccidioides (5)), viruses (rubella, respiratory viruses 51

(28), H1N1 influenza virus (24)), and parasites (Toxoplasma (3), Leishmania (26) and 52

Plasmodium (9)). In particular, it has been suggested that the massive accumulation of 53

Plasmodium falciparum infected erythrocytes (IEs) in the intervillous spaces of the 54

placenta (11) triggers the deleterious effects of malaria in pregnant women and their 55

offspring (9). In P. falciparum endemic areas, parity has been consistently found to 56

reduce susceptibility to malaria during pregnancy (9). 57

58

There is growing evidence that malaria susceptibility in primigravidae could be largely 59

explained by the lack of antibodies that can block adhesion of IEs to placental 60

chondroitin sulfate A (CSA) (22). The CSA-adhesion phenotype is specific to placental 61

parasites (21) and has been linked to expression of a unique var gene (var2csa) (47). 62

Immunity to CSA-binding parasites is gender-specific (i.e., men exposed to malaria lack 63

these antibodies (44, 50)), parity-dependent (i.e., antibodies increase during successive 64

pregnancies (22, 44, 50)) and has been associated with lower risk of placental 65

parasitaemia (22), maternal anaemia (51) and low birth-weight (18, 51). In the light of 66

these experimental findings, it has been suggested that VAR2CSA may constitute an 67

attractive target for vaccination against malaria in pregnancy. However, antibodies 68

against P. falciparum antigens not specifically associated with pregnancy have also 69

been shown to increase with parity (12, 19, 34, 38). Moreover, a significant number of 70

women at delivery have antibodies against placental parasites but their placentas remain 71


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infected (22, 44) and several studies have failed to show an association between levels 72

of IgGs against CSA-binding IEs and reduced frequency of adverse consequences of 73

malaria during pregnancy (14, 20, 48). In some cases, poor pregnancy outcomes have 74

been associated with peripheral infection in the absence of placental malaria (36). 75

Finally, the high incidence of malaria episodes observed few weeks after delivery (16) 76

suggests that other mechanisms may also be involved in the susceptibility of pregnant 77

women to malaria. In particular, it has been proposed that modulation of immunity 78

induced by pregnancy might predispose women to malaria infection (32, 43, 45). 79

80

Although antibody responses against placental and CSA-binding P. falciparum parasites 81

have been extensively analysed (6, 7, 14, 18, 22, 44, 50, 51), immunity in pregnant 82

women against field isolates obtained from general population has not been examined in 83

such detail (22, 44, 51). Also, contradictory results have been reported for the 84

association between placental infection and antibody responses (8, 22, 31, 38, 39, 41, 85

51). The aim of this study was to describe pregnancy-specific and general anti-malarial 86

immunity in Mozambican pregnant women, men and children, taking into consideration 87

the effect of placental infection, gender and parity. To address this, antibodies were 88

measured not only against P. falciparum parasites isolated from placentas and 89

peripheral blood of pregnant women, but also against parasites infecting non-pregnant 90

individuals and merozoite recombinant antigens. Importantly, P. falciparum isolates 91

were used without in vitro expansion or selection to avoid changes of their var 92

expression profiles (42). 93

94

95

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MATERIALS AND METHODS 97

98

Study area 99

The study was carried out at the Centro de Investigação em Saúde de Manhiça (CISM) 100

in the Manhiça District, Mozambique. Adjacent to the CISM is the Manhiça District 101

Hospital (MDH). The characteristics of the area have been described in detail elsewhere 102

(1). Perennial malaria transmission with some seasonality is mostly attributed to P. 103

falciparum, and the estimated entomological inoculation rate for 2002 was 38 infective 104

bites per person per year (2). 105

106

Study participants and plasma samples 107

Between June 2006 and June 2007, 40 children 1-5 years of age (mean age: 3.2 years, 108

SD 0.9) and 40 men more than 15 years of age (mean age: 26.5 years, SD 8.9) were 109

recruited into the study from patients attending the MDH with P. falciparum clinical 110

malaria. Before treatment, peripheral blood was collected by venipuncture in lithium 111

heparin tubes. Following centrifugation, plasma was stored at -20ºC. Ninety plasmas 112

collected in 2004 and 2005 from pregnant women at delivery (30 from PG [first 113

pregnancy; mean age: 19.1 years, SD 1.9] and 60 from MG [one or more previous 114

pregnancies; mean age: 22.9 years, SD 4.0]) were randomly selected from women who 115

received placebo in the context of an intermittent preventive treatment trial during 116

pregnancy conducted in the same study area (35). The subgroup of 90 women selected 117

for analysis here was comparable to the main group of pregnant women participating in 118

the trial (35) both in terms of prevalence of infection (p=0.599 and p=0.548 for 119

peripheral and placental infection, respectively) and parity (p=0.100). A panel of 14 120

negative-control plasmas from Spanish men and non-pregnant women without history 121


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of travel to malaria endemic areas and a pool of positive-control plasmas obtained from 122

10 pregnant-women with more than 3 previous pregnancies were tested in parallel. 123

124

Parasite isolates 125

A panel of 38 P. falciparum isolates collected from blood group O donors was used for 126

the study. Twenty-six of them were obtained from non-pregnant hosts (14 from children 127

1-5 years of age, 6 from men and 6 from non-pregnant women older than 15 years of 128

age) attending MDH with a primary clinical diagnosis of P. falciparum malaria and 129

asexual stage parasitaemia of 1-5% on thick blood film examination. Before treatment, 130

peripheral blood was collected by venipuncture in lithium heparin tubes and two drops 131

were spotted onto filter paper. Following centrifugation, 300 µl of the red blood cell 132

pellet was resuspended in 6 ml of Trizol (Invitrogen) and stored at -20ºC for RNA 133

isolation. Remaining red blood cell pellet was cryopreserved in liquid nitrogen. 134

135

Placental (n=4) and peripheral isolates (n=8) were collected from pregnant women 136

attending the Maternity Clinic of the MDH with microscopically detected P. falciparum 137

parasitaemia in their peripheral or placental blood. Placental blood was extracted from 138

freshly delivered placentas by making one-centimetre deep incisions in the endometrial 139

side of the placenta and by withdrawing blood into lithium heparin tubes. Peripheral 140

isolates and placental isolates previously cultured to ring stage were cryopreserved as 141

described above. The laboratory lines CS2CSA (MRA-96 from MR4, Manassas, VA, 142

USA), FCR3CSA, 193TCSA, R29Rosetting+, ITGICAM1 and E8BCD36/ICAM1 were also included 143

in the study. 144

145


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Parasitaemic individuals were treated following national guidelines at the time of study. 146

Participants were included in the study only if they or parents/guardians in the case of 147

children gave informed consent. The study was approved by the National Mozambican 148

Ethics Committee and the Hospital Clinic of Barcelona Ethics Review Committee. 149

150

Quantification of IgGs against the surface of infected erythrocytes 151

P. falciparum isolates from individuals with group O erythrocytes (to avoid blood group 152

incompatibility) and 1-5% parasitaemias were used to quantify the levels of IgGs in 153

plasmas against parasite antigens on the surface of IEs by flow cytometry. Cryo-154

preserved IEs were thawed and cultured to trophozoite stage. Ninety-five microliters of 155

the parasite suspension at 1% haematocrit in PBS-1% bovine serum albumin (BSA) 156

were sequentially incubated for 30 min with 5 µl of test plasma, 100 µl of rabbit anti-157

human IgG (DakoCytomation) diluted at 1/200 and 100 µl of AlexaFluor®

-conjugated 158

donkey anti-rabbit IgG diluted at 1/1000 (Invitrogen) plus 10 µg/ml ethidium bromide 159

(EtBr). Data from 1000 ethidium bromide positive events was acquired with a Becton-160

Dickinson FACSCalibur flow cytometer. Plasmas were tested in a single assay against 161

each particular parasite. The adjusted MFI was calculated by subtracting the MFI in 162

channel FL1 of the EtBr-negative cell population from that of the EtBr-positive cell 163

population. 164

165

Quantification of total IgGs and IgGs against merozoite antigens 166

Levels of IgGs in plasmas were measured by ELISA against the recombinant 19 kD 167

fragment of merozoite surface protein 1 (MSP119) from 3D7, F2 region of erythrocyte 168

binding antigen 175 (EBA175) from CAMP and full ecto-domain of the apical 169

membrane antigen 1 (AMA1 from 3D7), produced at ICGEB, New Delhi, India. 170


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Briefly, high-binding 96-well microplates (Nunc Maxisorp) were coated overnight at 171

4ºC with 200 ng per well of recombinant antigen diluted in 100 µL of 0.05 mol/L 172

carbonate-bicarbonate buffer. After blocking with 2% BSA at 4ºC for 8 h, 100 µL of 173

plasma diluted at 1:500 were tested in duplicate. After incubation with peroxidase-174

conjugated goat anti-human IgG antibodies (SIGMA) at 1/30,000, H2O2 and o-175

phenylendiamine chromagen were added and OD measured at 492 nm. Total IgGs in 176

plasmas were measured by coating 96-well microplates with plasmas diluted at 177

1/160,000 in PBS-0.1% BSA. After blocking for 4 hours with PBS-2% BSA and 178

washing with PBS, peroxidase conjugated goat anti-human IgG was added at 1/50,000. 179

Reaction was developed as described above. 180

181

Parasite genotyping and quantification of var2csa transcription 182

Parasite DNA was extracted from filter papers (QIAamp DNA Blood kit, Qiagen) and 183

used to estimate the multiplicity of infection (MOI) by PCR-typing based on 184

polymorphic regions of msp1 and msp2 genes (49). RNA was extracted from Trizol 185

samples (PureLink Micro-to-Midi RNA Purification Kit, Invitrogen). After DNAse-I 186

(Invitrogen) treatment for 1 hour at 37ºC, cDNAs were prepared using Superscript III 187

First Strand Synthesis System (Invitrogen). Quantitative PCR was performed on an ABI 188

PRISM 7500 Real-Time System (Applied Biosystems) using 5 µl of cDNA in a final 189

volume of 20 µl, including 10 µl of Power SYBR Green Master Mix (Applied 190

Biosystems) and 200 nmol/L of primers for var2csa gene fragment encoding DBL3X 191

(17) and seryl-tRNA-synthetase gene as endogenous control (47). Level of var2csa 192

transcription was expressed as the difference between the cycle threshold (Ct) value of 193

var2csa and the Ct value of the endogenous gene (dCt). 194

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Definitions and statistical methods 196

Placental malaria infection was defined by the presence of parasites and/or pigment on 197

histological examination of placental tissue (23). Age was categorized as ≤20, 21-25 198

and >25 years. ODs for merozoite recombinant antigens and MFIs for P. falciparum 199

isolates collected from pregnant and non-pregnant hosts, as well as laboratory lines, 200

were pooled after subtraction of the mean values of negative controls (background) to 201

allow for comparisons between plasma samples (13, 15, 52). The associations of age, 202

parity, placental infection and gender of plasma donor with pooled MFIs and ODs were 203

log-transformed and analyzed among responders by linear regression with a robust 204

variance estimator to account for within-subject correlation. Analysis was also done for 205

each isolate and merozoite recombinant antigen and is presented in Supplementary 206

Tables, both in terms of IgG levels (linear regression analysis of log-transformed data) 207

and high/low responders (defined as being above or below the median value of all 208

samples measured for each antigen or parasite; logistic regression). Both crude and 209

multivariate models were used. Differences between var2csa transcription levels (dCt) 210

and MOI among groups of parasite isolates were evaluated by Kruskall-Wallis test and 211

Poisson regression, respectively. Data was analyzed with Stata version 9.0 (Stata 212

Corporation). A p-value < 0.05 was considered statistically significant. 213

214

RESULTS 215

216

Characteristics of P. falciparum isolates 217

Thirty-eight parasite field isolates, 3 CSA-binding lines (CS2, 193T and FCR3CSA), 3 218

CSA-nonbinding lines (R29, E8B and ItG) and 3 P. falciparum merozoite antigens 219

produced as recombinant proteins (MSP119, F2 region of EBA175 and AMA1) were 220


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included in the study of antibody responses among pregnant women (n=90), men (n=40) 221

and children (n=40). MOI did not differ significantly between isolates from pregnant 222

and non-pregnant hosts (p=0.687), nor between peripheral and placental isolates from 223

pregnant women (p=0.558, Table 1). Transcription levels of var2csa (dCt in Table 1) 224

were similar for placental and peripheral isolates from pregnant women (p=0.396). 225

However, var2csa transcription was lower in parasites isolated from non-pregnant hosts 226

(median dCt= 5.80, range [2.33, 7.24]) compared to isolates from pregnant women 227

(median dCt= -2.36, range [-3.45, -1.13]; p<0.001). 228

229

IgG reactivity with IEs and merozoite antigens among children and men 230

Forty plasmas from children and 40 from men were used to measure levels of total IgG 231

as well as IgGs against the panel of parasites and recombinant antigens described above. 232

Compared to children, men had higher levels of IgGs against isolates from non-pregnant 233

hosts (26 out of 26 [100%] isolates) and peripheral isolates from pregnant women (5 out 234

of 8 [62%] isolates); FIG. 1 and Supplementary Table 1). A similar trend, although not 235

statistically significant, was found for total IgGs (p=0.058) and merozoite antigens (2 236

out of 3 [67%] merozoite antigens in Supplementary Table 1). In contrast, levels of 237

IgGs against CSA-binding lines and placental isolates were similar in men and children 238

(FIG. 1). 239

240

Placental infection and IgG reactivity with IEs and merozoite antigens 241

Ninety plasmas from pregnant women collected at delivery (30 PG and 60 MG) were 242

used to measure levels of IgGs against the panel of parasites and recombinant antigens. 243

Among these 90 pregnant women, 40 (44%) were infected in their placentas (20 out of 244

30 PG [67%] and 20 out of 60 MG [33%], p=0.004). Eight (9%) women were also 245


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infected in their peripheral blood (3 of the 30 PG [10%] and 5 of the 60 MG [8%], 246

p=1.000). There was no difference between PG and MG in their use of bed nets (20 out 247

of 30 PG [67%]; 35 out of 60 MG [58%]; p=0.645), nor in the proportion of deliveries 248

during rainy season (21 out of 30 PG [70%]; 29 out of 60 MG [48%]; p=0.117), nor in 249

their neighbourhood of residence (p=0.598). Placental infection was associated with an 250

increase in total IgG and levels of IgGs against merozoite recombinant antigens (2 out 251

of 3 [67%] antigens), CSA-binding lines (3 out of 3 [100%] antigens) and CSA-non-252

binding lines (3 out of 3 [100%] lines), isolates from pregnant women (10 out of 12 253

[83%] isolates) and from non-pregnant hosts (22 out of 26 [85%] isolates) (FIG. 2 and 254

Supplementary Table 2). The analysis stratified by parity showed that placental 255

infection was associated with an increase in antibody levels both in PG and MG women 256

(data not shown). 257

258

Parity and IgG reactivity with IEs and merozoite antigens 259

Given the effect placental infection can have on IgG levels, we separated pregnant 260

women with and without placental infection for the analysis of immune responses by 261

parity. To account for the effect of age on IgG levels, all analyses were adjusted by age. 262

Among women without placental infection, levels of IgGs were lower in PG than MG 263

for CSA-binding lines (2 out of 3 [33%] lines), for both placental and peripheral isolates 264

from pregnant women (10 out of 12 [83%] isolates), and for peripheral isolates from 265

non-pregnant hosts (10 out of 26 [38%] isolates), as well as for total IgGs (FIG. 3A and 266

Supplementary Table 3). Among women with placental infection, levels of IgGs were 267

significantly higher in MG than PG only for placental isolates (FIG. 3B and 268

Supplementary Table 3). There was no statistical evidence of an increase in the IgG 269


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levels against merozoite antigens or isolates from non-pregnant hosts with age of 270

pregnant women (Supplementary Table 4). 271

272

Gender and reactivity with IEs and merozoite antigens 273

Levels of IgGs against the panel of parasites and recombinant antigens were compared 274

between the 40 men and the 40 pregnant women with placental infection. Plasma from 275

PG showed better reactivity with merozoite antigens, CSA-binding parasite lines and 276

isolates from pregnant women compared to infected men. However, no differences were 277

found in the reactivity of plasmas from infected PG and men with isolates from non-278

pregnant hosts (FIG. 4A). Plasma from infected MG showed better reactivity compared 279

to plasma from men with merozoite antigens, CSA and non-CSA binding parasite lines 280

as well as parasite isolates from pregnant women and non-pregnant hosts (FIG. 4B). 281

282

283

DISCUSSION 284

285

Natural immunity against P. falciparum malaria appears to depend on the gradual 286

acquisition of a broad repertoire of IgGs against the surface of erythrocytes infected by 287

mature forms of the parasite (30). This immunity is acquired as a result of antigenic 288

stimulation through repeated parasite infections from early childhood onwards (33). In 289

agreement with this, results of the present study show that IgGs against IEs isolated 290

from children, men and non-pregnant women, as well as against non-CSA binding lines 291

and EBA175, are higher in men than in children from Manhiça. 292

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The currently accepted model of pregnancy-specific immunity to P. falciparum malaria 294

predicts that exposure to placental parasites leads to acquisition of antibody responses 295

against the VAR2CSA family of variant surface antigens (8, 51). This study confirms 296

that var2csa is uniquely transcribed by placental and peripheral isolates from pregnant 297

women as well as CSA-binding laboratory lines. The observation that men and children 298

have equally poor IgG levels against parasite isolates from pregnant women and CSA-299

binding laboratory lines is consistent with the concept that immunity against VAR2CSA 300

is acquired specifically during pregnancy. 301

302

Previous studies have differed in the association between placental infection and 303

antibodies (8, 22, 31, 38, 39, 41, 51). In this study, analysis of plasma from pregnant 304

women with and without placental infection revealed that placental infection boosts 305

antibody responses against isolates from both pregnant as well as from non-pregnant 306

hosts, and CSA-binding as well as CSA non-binding laboratory lines. Placental 307

infection also boosted total IgGs and IgGs against merozoite antigens that are expressed 308

by all isolates. This observation suggests that placental parasites may stimulate the 309

production of antibodies that cross-react with parasites infecting non-pregnant hosts. 310

Alternatively, placental infection might consist of parasites expressing var genes other 311

than var2csa (7) that can stimulate the production of antibodies with different 312

specificities. Non-specific stimulation of B lymphocytes (4) by placental infection 313

might also be responsible for the increase in pregnant women of IgGs against diverse P. 314

falciparum isolates, and even against other pathogens, as previously reported (27, 40). 315

Finally, the profound effect of placental infection on antibody responses suggests that 316

IgG levels in plasmas collected from pregnant women at delivery may reflect exposure 317

to P. falciparum during pregnancy. The analysis of immune responses in pregnant 318


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women should thus take into consideration the presence of placental P. falciparum 319

infection. For this reason, further analysis of the effect of gender and parity on IgG 320

levels was stratified by the infection status of the placenta. 321

322

Plasma from PG with placental infection exhibited higher levels of IgGs against isolates 323

from pregnant women and CSA binding parasite lines, compared to plasma from 324

infected men. However, no differences were found in the reactivity of plasmas from 325

infected PG and men with isolates from non-pregnant hosts. Analyses were adjusted for 326

age to correct for the effect of different durations of exposure. These observations are 327

consistent with current models for development of immunity against malaria in 328

pregnancy in which antibodies against placental isolates and CSA-binding parasite lines 329

develop following exposure to such isolates during pregnancy. 330

331

Results of this study also show that, compared to MG, PG without placental infection 332

had lower IgG levels against isolates from pregnant as well as from non-pregnant hosts. 333

Parity groups were comparable in terms of use of insecticide-treated nets, 334

neighbourhood of residence and season at delivery, suggesting that there was no 335

difference in exposure between PG and MG. Age was also discarded as a possible 336

confounding factor by adjusting the analysis for this variable and by showing no 337

difference in IgG levels between age groups in pregnant women. The lower level of 338

antibody responses among PG compared to MG against all type of parasite isolates (i.e., 339

those of placental origin but also parasites from non-pregnant hosts), may reflect 340

previous placental exposure to a broad range of PfEMP1 proteins (both VAR2CSA and 341

others) in MG, but also a non-specific modulation of immune responses during first 342

pregnancies. Pregnancy-associated immunomodulation may be needed to prevent 343


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immune responses against foetal antigens (37, 46, 53) and might explain poor 344

pregnancy outcomes in the absence of placental infection (36) and the increased 345

susceptibility to malaria during the early postpartum period (16). Of importance, 346

placental infection in PG was still associated with a boosting of IgGs, suggesting that 347

first-time mothers can produce antibodies in response to plasmodia infection, and that 348

other immune mechanisms, such as cell-mediated immunity (43) and 349

opsonization/phagocytosis (25), might be modulated during pregnancy (43). 350

351

In conclusion, this study highlights that placental infection boosts antibody responses 352

against a wide range of parasite antigens. Prospective studies using plasma samples 353

collected from pregnant women in early stages of pregnancy and analysis of the 354

functional properties of the antibodies (i.e., inhibition of CSA-adhesion (44)) are needed 355

to understand the role of antibody responses against VAR2CSA and other P. falciparum 356

antigens in protection against malaria in pregnancy. Our results confirm that immunity 357

to parasites transcribing var2csa is pregnancy-specific but, importantly, also show that 358

PG have lower immune responses against parasites not specifically associated to 359

pregnancy (i.e., those infecting children, men and non-pregnant women) as compared to 360

women of higher parities. This generalized low IgG response in primigravidae, together 361

with the lack of antibodies specific against placental parasites expressing VAR2CSA 362

may both predispose women to malaria in their first pregnancies. 363


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ACKNOWLEDGMENTS 364

365

This article is dedicated to the memory of Nivedita Bir, whose work on CSA binding 366

DBL domains contributed to studies of malaria in pregnancy. We are grateful to the 367

individuals who agreed to participate in the study; the staff of the Manhiça District 368

Hospital and the CISM; G. Cabrera, Mauricio H. Rodríguez, L. Mussacate, N. Ernesto 369

José, A. Nhabomba, L. Puyol and P. Cisteró for their laboratory work; and J. Ordi for 370

histological diagnosis of placentas. We thank MR4 for providing us with CS2 malaria 371

parasite contributed by S.J. Rogerson, and J Gysin for the 193T parasite line. 372

373

The study received financial support from the Instituto de Salud Carlos III (grant 374

PS09/01113, and salary support CP-04/00220 for AM and FI06/00019 for ERV), Banco 375

de Bilbao-Vizcaya-Argentaria Foundation (BBVA 02-0) and Ministerio de Ciencia e 376

Innovación (RYC-2008-02631 for CD). The Manhiça Health Research Center receives 377

core support from the Spanish Agency for International Cooperation. 378

379

Authors do not have any commercial or other association that might pose a conflict of 380

interest. The funders had no role in study design, data collection and analysis, decision to 381

publish, or preparation of the manuscript. 382

383


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385

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587


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Table 1. Characteristics of the P. falciparum isolates used in the study. 588

589

Isolates from n

Mean years

of age (SD)

Mean MOI

(range)

Median var2csa

dCt (range)

Children 14 3.2 (0.9) 3.9 (2-8) 6.0 (3.4-7.9)

Men 6 32.0 (9.8) 2.8 (2-5) 6.1 (4.7-8.2)

Non-pregnant women 6 28.0 (8.9) 3.0 (2-6) 4.9 (2.3-5.7)

Periphery of pregnant women 8 22.7 (3.9) 2.8 (2-4) -2.1 (-3.2-4.1)

Placenta of pregnant women 4 21.8 (4.0) 3.5 (3-4) -2.7 (-3.4-1.3)

590

MOI: Multiplicity of infection; SD: standard deviation; dCt: difference in the cycle 591

threshold for var2csa and seryl-tRNA synthetase. 592


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Figure 1. IgG levels (MFI or ODs) in plasmas from children and men residing in 593

Manhiça (Mozambique) against merozoite antigens, P. falciparum laboratory lines and 594

field isolates. Vertical bars represent geometric mean levels of pooled MFIs or ODs, 595

error bars the 95% confidence interval and p the statistical significance of the univariate 596

regression analysis with a robust variance estimator. 597

598

599

600

601

602

603

604

605

Note: Mean IgG recognition by negative controls was as follows: Merozoite antigens, 606

0.24; CSA-binding lines, 7.60; CSA-nonbinding lines, 6.83; Placental isolates, 7.40; 607

Peripheral isolates from pregnant women, 3.89; Peripheral isolates from non-pregnant 608

hosts, 2.15. 609

120

100

80

60

40

20

0

p 0.058 0.175 0.961 <0.001 0.107 0.001 <0.001

ChildrenMen

OD

(×× ××

100)

MF

I

Total-IgG Merozoite CSA NonCSA Placenta Periph. Periph. pregnant non-pregnant


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Figure 2. IgG levels (MFI or ODs) in plasmas from pregnant women with or without 610

placental infection against merozoite recombinant antigens, P. falciparum laboratory 611

lines and field isolates. Vertical bars represent geometric mean levels of pooled MFIs or 612

ODs, error bars the 95% confidence interval and p the statistical significance of the 613

regression analysis with a robust variance estimator (adjusted by age and parity). 614

615

616

617

618

619

620

621

622

120

100

80

60

40

20

0Total-IgG Merozoite CSA NonCSA Placenta Periph. Periph.

pregnant non-pregnant

OD

(×× ××

100)

MF

I

Pregnant women with placental infectionPregnant women without placental infection

P 0.016 0.006 <0.001 0.001 <0.001 <0.001 < 0.001


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Figure 3. IgG levels (MFI or ODs) in plasmas from pregnant women with and without placental P. falciparum infection by parity against 623

merozoite recombinant antigens, P. falciparum laboratory lines and isolates. Vertical bars represent geometric mean levels of pooled MFIs or 624

ODs, error bars the 95% confidence interval and p the statistical significance of the regression analysis with a robust variance estimator (adjusted 625

by age). 626

627 120

100

80

60

40

20

0

A. Pregnant women without placental infection B. Pregnant women with placental infection

Multigravidae

Primigravidae

OD

(×× ××1

00)

M

FI

P 0.003 0.152 0.026 0.089 0.021 <0.001 0.011 P 0.296 0.661 0.242 0.846 0.031 0.190 0.681




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Figure 4. IgG levels (MFI or ODs) in plasmas from men and pregnant women with placental infection against merozoite recombinant antigens, 628

P. falciparum laboratory lines and parasite isolates. Vertical bars represent geometric mean levels of pooled MFIs or ODs, error bars the 95% 629

confidence interval and p the statistical significance of the regression analysis with a robust variance estimator (adjusted by age). 630

631

632

120

100

80

60

40

20

0

Men

Pregnant women

B. Multigravidae with placental infection and men

OD

(×× ××100)

MF

I

P 0.358 0.008 <0.001 0.043 <0.001 <0.001 0.020

A. Primigravidae with placental infection and men

P 0.222 0.011 <0.001 0.332 <0.001 0.003 0.648

OD

(×× ××100)

MF

I

Total-IgG Merozoite CSA NonCSA Placenta Periph. Periph.


Total-IgG Merozoite CSA NonCSA Placenta Periph. Periph.



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Documents

Parity and placental infection affect antibody responses against