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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is well recognized that infants in areas of stable malaria transmission have a reduced risk of clinical consequences of P. falciparum infection during the first few months of life. The presence of fetal hemoglobin, passively acquired malaria-specific antibodies, dietary absence of p-aminobenzoic acid and breast milk constituents such as lactoferrin may account for this phenomenon.10–12 Among these, transplacentally acquired antibodies are thought to play an important role.13
Although malaria in pregnancy is amenable to intervention through existing strategies such as intermittent preventive treatment,1 this may be undermined by resistance of P. falciparum to antimalarials, poor compliance, and other logistical problems.14 In our study area in western Kenya, we demonstrated that in an area with intense malaria transmission, insecticide (permethrin)-treated bed nets (ITNs) reduce the densities of malaria-transmitting mosquitoes by 90%.15 The ITNs were also found to be a promising tool for malaria control in pregnancy, since they markedly reduced malaria and its adverse effects during the first four pregnancies.16
One concern with the use of ITNs, especially in high-transmission areas, is whether their use might interfere with the development and maintenance of antibody responses to malarial antigens in pregnant women, thereby potentially affecting passive immune protection in early infancy. Previous studies in children and non-pregnant adults have found that use of ITNs was associated with a reduction in antibody responses to malarial antigens.17–19 In a companion study conducted in young children, we found that ITNs were associated with a reduction in IgG responses to pre-erythrocytic circumsporozoite surface protein (CSP) and liver stage antigen-1 (LSA-1), but not to the blood stage antigen merozoite surface protein-1 (MSP-1).20 The current investigation focused on the antibody responses in mothers at delivery and cord blood samples to determine the effect of ITNs on antibody responses to P. falciparum pre-erythrocytic (CSP and LSA-1) and erythrocytic (MSP-1) antigens. A subsidiary goal of the investigation was to evaluate the effect of ITNs on the transplacental transfer of malaria-specific IgG.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sample collection. At delivery, approximately 500 µl of maternal capillary blood and 5 mL of cord blood were collected in EDTA-containing tubes and heparinized tubes, respectively. Blood samples were transported to the laboratory at 4°C for determination of hemoglobin levels and immunologic studies. Two thick blood smears on the same slide and a thin blood smear on a separate slide were prepared from maternal, cord, and placental blood for detection of malaria parasites.
Antigens. The same antigens used in our study in young children20 were used for the assessment of antibody reactivity in maternal and cord plasma samples. These are Escherichia coli–expressed full-length recombinant CSP, synthetic peptides corresponding to the repeat region of LSA-1 and Saccharomyces cerivisae–expressed MSP-119kD protein. Clostridium tetani tetanus toxoid (TT) (Accurate Chemical and Scientific Corp., Westbury, NY) was used as a non-malarial control antigen to test for any underlying difference in the general antibody responses in maternal and cord plasma from ITN and control villages.
Laboratory procedures. Sample processing, hematologic, and parasitologic examinations. A complete blood cell count was done on maternal and cord blood using an AcT 10 Coulter-Counter® (Coulter Corporation, Miami, FL). Maternal and cord plasma were obtained by centrifuging blood at 700 x g for five minutes in a Micro 7® microcentrifuge (Fisher Scientific, Pittsburgh, PA), aliquoted into sterile vials, and stored at -70°C until testing for antibodies. Maternal, cord, and placental blood smears were stained with Giemsa and examined for the presence of malaria parasites and/or pigment with a 100x oil-immersion objective.
Enzyme-linked immunosorbent assay (ELISA). Total IgG reactivity to CSP, LSA-1, and MSP-1 antigens was determined by a standard ELISA as described elsewhere.20 The IgG subclass 1–4 reactivity to LSA-1 and MSP-1 was as described for total IgG with minor modifications. Briefly, mouse anti-human monoclonal antibodies diluted in phosphate-buffered saline containing 0.05% Tween-20 and 2.5% nonfat milk powder (PBS-T-M): IgG1 at 1:2,000, IgG2 at 1:6,000, IgG3 at 1:50,000, and IgG4 at 1:20,000 were used as the primary antibodies. After four washes with PBS containing 0.05% Tween-20 (PBS-T), peroxidase-conjugated goat anti-mouse antibodies (Biosource International, Camarillo, CL) diluted at 1:10,000 in PBS-T-M was added and allowed to react for one hour at room temperature. The rest of the procedure was as described for the total IgG ELISA.20 The procedure for evaluating IgG reactivity to TT antigen was similar to that described for total IgG to the malarial antigens CSP and LSA-1,20 except that the coating antigen was used at a concentration of 1 µg/ml and in 0.01 M PBS, pH 7.2. A positive control, consisting of pooled hyperimmune sera was included in each experiment. The cut-off values for each antigen were determined by assaying samples from 40 malarianaive individuals. Samples that showed an optical density (OD) greater than the mean plus two standard deviations of the negative control plasma were scored as positive.
Statistical analyses. To eliminate zeros and negative ELISA OD values, we added the sum of the lowest negative value for each antigen plus 0.001 to each of the corresponding antibody data. The resulting ELISA OD values were log transformed to normalize the data. Analyses were performed using SUDAAN version 8.0, SAS callable version (Research Triangle Institute, Research Triangle Park, NC) and SAS version 8.0 (SAS Institute, Cary, NC). Since in multivariate analysis there was no effect modification by gravida, the data were pooled for all gravidae. To assess differences in mean levels of antibodies (maternal and cord blood) between those from ITN and control villages, analysis of variance methods were implemented. The association between maternal and corresponding cord blood antibody levels and possible differences in transplacental transfer of antibodies between women from the ITN and control villages were assessed through fitting multiple regression models. Log-binomial regression models were used to estimate ratios of antibody prevalence for ITN versus control. Models were fit using GEE methods in the GENMOD procedure in SAS. Since the data were collected as part of a group randomized trial, reported 95% confidence intervals are based on empirical SE estimates obtained by adjusting for clustering at the village level. A two-sided P value < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Overall, the demographic characteristics in women from both intervention and control villages were comparable, with the exception of the number of years of education, which was significantly higher in women living in ITN villages. The prevalence of maternal anemia and parasitemia was lower in women from the ITN villages, although the differences were not statistically significant. The birth characteristics of infants from ITN and control villages were also comparable with the exception of cord blood hemoglobin levels, which were lower in infants from ITN villages (P = 0.06) (Table 2).
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and Table 3). For CSP, only total IgG reactivity was assessed. The frequency and levels of total IgG to CSP were significantly higher in women from ITN villages than in women from control villages. In contrast, there was no significant difference in the prevalence and levels of IgG antibodies to CSP in cord blood samples (Figure 1A and Table 3).
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and Table 3). The pattern of cord blood antibody reactivity to LSA-1 was similar to that seen in maternal samples, although the difference was only of marginal statistical significance for total IgG and IgG2 levels and prevalence of total IgG (Figure 1B and Table 3).
Similar to the LSA-1 antigen, the prevalence and levels of IgG3 to the blood stage antigen MSP-1 were significantly lower in maternal and cord plasma from ITN villages than control villages. There was no significant difference in the prevalence and levels of total IgG and IgG1 to MSP-1 antigen in both maternal and cord blood samples from ITN villages and those from control villages (Figure 1C and Table 3).
Association between maternal and cord blood antibody levels.
To further examine the effect of ITNs on the association between maternal and cord blood antibody levels, we assessed the slope estimates of ITN and control groups from a regression model fitting maternal antibodies to cord blood antibodies while controlling for gravida and rainfall 90 days prior to birth. There was a significant positive association between maternal and cord blood total IgG levels to TT, LSA-1, and MSP-1 in both ITN and control villages (Table 4). Furthermore, the slopes for total IgG to these three antigens between ITN and control villages were not significantly different (Table 4). In contrast, there was a significant negative association between maternal and cord blood total IgG levels to CSP in ITN villages and no significant association in control villages (Table 4). The slopes for total IgG to CSP were significantly different between ITN and control villages (Table 4), suggesting that ITNs modified the relationship between maternal and cord blood antibody levels.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We found lower antibody levels against the MSP-1 and against peptides complimentary to the pre-erythrocytic antigen LSA-1 and higher antibody response to the pre-erythrocytic antigen CSP in maternal samples from ITN villages compared with control villages. This effect was not dependent on pregnancy order and was observed in all gravida groups. The lower antibody response to the LSA-1 antigenic determinant is consistent with the antibody reactivity to LSA-1 observed in young children in our cross-sectional surveys,20 and presumably indicates reduced exposure to malaria parasites in women from ITN villages compared with women from control villages. Our observation is also in agreement with a previous study showing a relatively lower prevalence of antibodies to the repeat region of LSA-1 in individuals residing in two areas of low malaria transmission compared with those from an area of moderately higher transmission.21
The increased antibody responses to CSP and decreased responses to MSP-1 in this study differ from the immune response we observed in children.20 A possible explanation for these inconsistencies are differences in parasite densities and/or the predominant parasite stage-specific immune effector mechanism in children vs. adults. Several studies have shown that parasite densities decrease as a function of age.22,23 In endemic areas, young children are more likely to have high parasite densities compared with adults, who develop protective immunity and are capable of limiting malaria blood stage parasite densities to very low levels.24 This observation has led to the hypothesis that children first develop anti-toxic (disease) immunity, followed by the development of anti-parasite immunity with an increase in age.25 The predominant immune effector mechanisms in adults could mainly be directed at limiting parasite invasion/multiplication at the pre-erythrocytic stage, while in children the mechanisms could be directed at the blood stages, which are responsible for clinical disease. This hypothesis is supported by recent findings in Dielmo, Senegal, suggesting that adults are able to resist a higher sporozoite challenge than are children.26
The observation of increased levels of antibody to CSP in women in the ITN group was surprising, but may be explained by cross-regulation of immune responses by different parasite stages, which may be further complicated by immune suppression during pregnancy. Studies in both humans and animal models have shown that blood stage malaria parasites cause an immunosuppressive effect on the anti-sporozoite antibody response.27,28 In addition, a negative correlation has been shown between malaria-induced polyclonal B cell activation and anti-sporozoite antibody responses in vitro.29 However, whether and how the higher parasitemia rates resulting from the more intense malaria transmission in control villages lead to immunosuppressive effects on antibody responses to CSP in pregnant women remains unclear and requires further investigation.
Our previous studies conducted in children from the same area have demonstrated that MSP-119 kD antibody responses, particularly IgG1, are associated with clinical protection.30,31 There was no evidence from our epidemiologic investigations that the diminished antibody responses to MSP-119 kD had any clinical implications. Pregnant women in their first four pregnancies from ITN villages were significantly less likely to have maternal or placental malaria parasitemia resulting in a reduced risk of maternal anemia and increased birth weight in the newborn.16 It is of note that IgG1 responses were not affected by bed net use, and that only IgG3 responses to MSP-119 kD were diminished. It is possible that switching antibody subclasses may occur during pregnancy. Consequently, the antibody subclasses to MSP-1 could play different roles in malaria infection in pregnant women, in whom IgG3 may be associated with exposure.
Consistent with other studies, we found that antibody reactivity to the malarial antigens LSA-1 and MSP-1 in cord blood were positively and linearly correlated with the maternal responses32,33 and thus lower in cord blood from newborns in ITN villages than in control villages. The ITNs did not affect this relationship between maternal/cord blood antibodies to LSA-1 and MSP-1. Interestingly, there was no significant difference in the levels of IgG to CSP in cord blood responses despite the difference observed in maternal plasma. We further observed no association between maternal and cord blood total IgG antibodies to CSP in control villages, which is consistent with other studies.33 However, we found a negative linear correlation in ITN villages. This difference between ITN and control villages was statistically significant, suggesting that ITNs modified the maternal/cord blood anti-CSP antibody relationship.
It is well accepted that malaria-specific transplacental antibodies play an important role in the passive immune protection in newborns, and concentrations of antibody to MSP-119 kD at birth have been associated with protection against parasitemia, clinical malaria, and anemia in young infants.30,34 Similar to the results in pregnant women,16 there was no indication from our larger birth cohort analysis that, with continued ITN use, newborns from ITN villages were at an increased risk of malaria morbidity in the first few months of life.35 Follow-up of these newborns showed they had 70% less clinical malaria episodes and anemia than young infants in control villages.35 However, the direct relationship between malaria clinical end points during early and late infancy and the consequences of the effect of ITNs on transplacental transfer of the different subclasses of antibodies requires further investigation.
In summary, we have observed that the use of ITNs leads to decrease in antibody levels to LSA-1 and MSP-1 antigenic determinants in pregnant women with associated reductions in levels of the same antibodies in cord blood. In contrast, ITN use was found to be associated with increased antibody responses to CSP in pregnant women, but had no effect on antibody levels to CSP in cord blood. The marked improvements in clinical outcomes observed in mother and infant15,35 do not suggest that the altered levels of antibodies resulting from ITN use adversely affect the risk of malaria in pregnant women and their newborns.
Acknowledgments: We thank the people of Rarieda Division and all the field workers who made this study possible. We are grateful to Philip Onyona, Franklin Komino, Patroba Anyona, Thomas Otieno and Malachi Ogalo for technical assistance. We express our gratitude to Dr. David Lanar (Walter Reed Army Institute of Research, Silver Springs, MD) and Dr. David Kaslow (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD) for the kind donation of recombinant CSP and MSP-1 antigens, respectively. We thank Dr. Victor Tsang (Centers for Disease Control and Prevention, Atlanta, GA) for providing us mouse anti-human IgG subclass monoclonal antibodies. We acknowledge Dr. Laurence Slutsker (Director, Centers for Disease Control and Prevention/Kenya Medical Research Institute Research Station, Kisumu, Kenya) and Dr. Richard Steketee (Centers for Disease Control and Prevention, Atlanta, GA) for reviewing this manuscript. We also thank the Director of the Kenya Medical Research Institute for his permission to publish this paper.
Financial support: The ITN project was funded by the United States Agency for International Development. The laboratory work was supported by the UNDP/World Bank/World Health Organization/Special Program for Research and Training in Tropical Diseases. Feiko O. ter Kuile and Dianne J. Terlouw were partially supported by a grant from the Netherlands Foundation for the Advancement of Tropical Research (WOTRO) (The Hague, The Netherlands). Kathleen Wannemuehler was supported in part by an appointment to the research participation program at the Centers for Disease Control and Prevention that was administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the Centers for Disease Control and Prevention.
Disclaimer: The opinions or assertions contained in this manuscript are the private ones of the authors and are not to be construed as official or reflecting the views of the U.S. Public Health Service or Department of Health and Human Services. Use of trade names is for identification only and does not imply endorsement by the U.S. Public Health Service or Department of Health and Human Services.
Authors’ addresses: Simon K. Kariuki, Centre for Vector Biology and Control Research, Kenya Medical Research Institute, PO Box 1578, Kisumu, Kenya. Feiko O. ter Kuile, Kathleen Wannemuehler, Dianne J. Terlouw, Margarette S. Kolczak, William A. Hawley, Penelope A. Phillips-Howard, Altaf A. Lal, and Ya Ping Shi, Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Mailstop F-22, 4770 Buford Highway, Atlanta, GA 30341. Alloys SS. Orago, Zoology Department, Kenyatta University, PO Box 43844, Nairobi, Kenya. Bernard L. Nahlen, Roll Back Malaria, World Health Organization, Avenue Appia 20, 1211 Geneva 27, Switzerland.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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