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The potential consequences of parasitic infections on a person’s immune responsiveness to unrelated antigens are often conjectured upon in relationship to allergic responses and autoimmune diseases. These considerations sometimes extend to whether parasitic infection of pregnant women can influence the outcomes of responses by their offspring to the immunizations administered during national Expanded Programs of Immunization. To provide additional data to these discussions, we have enrolled 99 close-to-term pregnant women in western Kenya and determined their Schistosoma mansoni and Plasmodium falciparum infection status. At 2 years of age, when the initial immunization schedule was complete, we determined their children’s IgG antibody levels to tetanus toxoid, diphtheria toxoid, and measles nucleoprotein (N-protein) antigens using a multiplex assay. We also monitored antibody responses during the children’s first 2 years of life to P. falciparum MSP119 (PfMSP119), S. mansoni Soluble Egg Antigen (SEA), Ascaris suum hemoglobin (AsHb), and Strongyloides stercoralis (SsNIE). Mothers’ infections with either P. falciparum or S. mansoni had no impact on the level of antibody responses of their offspring or the proportion of offspring that developed protective levels of antibodies to either tetanus or diphtheria antigens at 2 years of age. However, children born of S. mansoni-positive mothers and immunized for measles at 9 months of age had significantly lower levels of anti-measles N-protein antibodies when they were 2 years old (p = 0.007) and a lower proportion of these children (62.5 vs. 90.2%, OR = 0.18, 95% CI = 0.04-0.68, p = 0.011) were considered positive for measles N-protein antibodies. Decreased levels of measles antibodies may render these children more susceptible to measles infection than children whose mothers did not have schistosomiasis. None of the children demonstrated responses to AsHb or SsNIE during the study period. Anti-SEA and anti-PfMSP119 responses suggested that 6 and 70% of the children acquired schistosomes and falciparum malaria, respectively, during the first 2 years of life.
This longitudinal serologic study was conducted in western Kenya over a 4-year period, between 2013 and 2017. We enrolled late-term pregnant women and followed their children for 2 years for antibody responses to parasite antigens and standard immunizations. Pregnant women were diagnosed for malaria, soil-transmitted helminths (STH; A. lumbricoides; Trichuris trichiura, and hookworm), and schistosomiasis. Blood samples were collected from mothers, and from babies at or near birth, and subsequently until 2 years of age (see below). Pregnant women were recruited from the antenatal clinics at several county hospitals which were selected based on the average number of pregnant women visiting the facility, nearness of the facility to the Kenya Medical Research Institute (KEMRI) in Kisian, Kenya, and to represent multiple categorical levels of the hospitals. The six study hospitals used were: Jaramogi Oginga Odinga teaching and referral hospital (the largest facility in Kisumu offering comprehensive services in the region—level 5), Kisumu sub-county hospital (level 4), Port Florence and Ober Kamoth health centers (level 3), Usoma and Rota dispensaries (level 2). All the facilities used are government funded except Port Florence, which is private. Pregnant women who were in their third trimester (34–37 weeks gestation) were screened for eligibility and asked to participate in the study. Those who consented filled out a questionnaire and donated a single blood sample and three stool specimens collected on consecutive days. The name, detailed address, and phone number of each consented participant was recorded at the time of enrollment to facilitate home based follow-up of their child until 24 months postpartum. Each woman was assigned a unique study number that was then consistent with that of her child throughout the study. Pregnant women who were smear-positive for malaria were treated according to Kenya Ministry of Health guidelines. Mothers infected with schistosomes or intestinal helminths were treated soon after delivery. The objective of the study was explained in detail to all enrolled pregnant mothers in the local language (Luo) in the presence of community midwives who were not part of the study. Witnessed written informed consent was obtained from each subject for themselves and for their unborn child to participate over the entire 2-year follow-up period. A copy of the signed consent form was given to each enrollee and another copy was kept in a locked cabinet with restricted access in the offices of the KEMRI, Centre for Global Research at Kisian, in Kisumu County. Only infants born at term, defined as a gestational age of 37–42 weeks, were included in this study. When twins were born, both were enrolled in the study. Before birth, each mother was confirmed to have been provided with a vaccination clinic booklet that was to be used to record and track vaccination history of the infant and the health status of both the mother and child. In accordance with KEPI, infants were vaccinated with BCG vaccine at birth; 3 doses of [diphtheria, pertussis, tetanus (DPT)] vaccine, at 6, 10, and 14 weeks; oral polio vaccine (OPV) at or within 2 weeks of birth and 6, 10, 14 weeks; and measles vaccine at 9 months. BCG, DPT, OPV, and measles vaccines, all of which were recommended and pre-qualified by WHO, were manufactured by Serum Institute of India PVT, Hadapsar, India. Vaccine manufacturers provided the study product but had no role in the conduct of the study, analysis of the data, or preparation of this report. These vaccines were provided free-of-charge by the Ministry of Health officials through the Division of Vaccines and Immunization under KEPI. All immunizations were recorded on an infant birth vaccination clinic booklet brought by the mother and all dates of vaccinations were verified for all children by our field study team. The research protocol was approved by the Scientific Steering Committee of the Kenya Medical Research Institute (SSC-KEMRI), KEMRI/Scientific Ethical Review Unit (Protocol No. 2303) and the Institutional Review Board at the University of Georgia (Protocol No. 00004091). The Centers for Disease Control and Prevention (CDC) also reviewed the protocol; CDC personnel were considered not to be engaged because they had no contact with study participants or access to personal identifiers. All subjects provided written informed consent in accordance with the Declaration of Helsinki. Maternal venous blood was collected at the time of the neonate’s first bleed, at or a few days after birth. Subsequent blood collections from children were at 6 and 20 weeks and 9, 12, 18, and 24 months of age. At least 400 µL of heel prick or finger blood was collected by capillary blood collection using lithium heparin anticoagulant (Kabe Labortechnik GMBH, Germany). Blood was centrifuged to isolate the plasma fraction from the cell pellet and plasma specimens were stored at −20°C. Antibody assays were performed at the same time after all plasmas were collected. Detection and enumeration of S. mansoni eggs were determined by Kato Katz fecal examination (12) based on two slides from each of the three stool samples collected from women upon enrollment. Intensity of infection was obtained for S. mansoni as eggs per gram of feces (epg) and the presence or absence of eggs of the three STH was recorded. Blood from all pregnant women was examined for malaria parasites. Thick and thin blood smears were prepared, stained with 10% Giemsa for 15 min, and examined by light microscopy for Plasmodium-infected erythrocytes. At least 200 microscope fields were scanned before a smear was considered as negative. Children were not assayed for malaria parasites in their blood, nor were they evaluated by stool examination for schistosome infection. Fluorescent bead–based multiplex bead immunoassays were performed using Luminex technology (Luminex Corp., Austin, TX, USA) to analyze plasma antibody levels to multiple antigens from mothers and infants. Expression and purification of S. stercoralis NIE (SsNIE) (13) and P. falciparum 19-kDa subunit of Merozoite Surface Protein 1 (MSP119) (14) antigens as fusion proteins with Schistosoma japonicum glutathione-S-transferase (GST) have been described (15, 16). Recombinant GST with no fusion partner was also expressed and purified as previously described (17) for use as a negative control in the multiplex assays. The conditions for coupling these antigens to the beads have been described (16) as has coupling of S. mansoni soluble egg antigen (SEA) (18). Native hemoglobin Ascaris suum hemoglobin (AsHb) purified from A. suum worms was a kind gift of Peter Geldhof (Ghent University, Belgium) (19, 20). SEA and AsHb were coupled to SeroMap microsphere beads (Luminex Corp., Austin, TX, USA) in phosphate-buffered saline (PBS) at pH 7.2 using 120 µg protein for 12.5 × 106 beads as previously described (18). The following antigens were purchased from commercial sources: tetanus toxoid (Massachusetts Biological Laboratories, Boston, MA, USA), diphtheria toxoid from Corynebacterium diphtheriae (List Biological Laboratories, Campbell, CA, USA), and recombinant measles nucleoprotein (MV-N, Meridian Life Sciences, Memphis, TN, USA) (21). Tetanus toxoid was coupled to SeroMap beads as previously described (16). Diphtheria toxoid was coupled in buffer containing 50 mM 2-N-morpholinoethanesulfonic acid (MES) at pH 5.0 with 0.85% NaCl using 60 µg of protein per 12.5 × 106 beads in 1 mL final volume. Measles nucleoprotein (N-protein) was partially purified by MonoQ column chromatography (GE Healthcare, Piscataway, NJ, USA) and coupled to beads in buffer containing 50 mM MES/NaCl buffer at pH 5.0 using 6 µg of protein per 12.5 × 106 beads in 1 mL final volume. Test plasma samples were diluted 1:400 in PBS buffer (pH 7.2) containing 0.3% Tween 20, 0.02% sodium azide, 0.5% casein, 0.5% polyvinyl alcohol, 0.8% polyvinylpyrrolidone, and 3 µg/mL Escherichia coli extract (15, 18, 22). Multiplex bead assays for total IgG antibodies were performed as previously described (15, 23). Each assay plate included a buffer only blank, as well as positive and negative controls, and all samples were tested in duplicate. Data were collected using a BioPlex 200 instrument with BioPlex Manager version 6.1.1 software (BioRad, Hercules, CA, USA). Responses are reported as the average of the median fluorescent intensity minus background for the duplicate wells (MFI-BKG). Samples having a coefficient of variation of >15% between the duplicate wells for >3 positive antibody responses were repeated. All samples collected from one child were assayed on the same plate to minimize potential impacts caused by variability in assay performance. Cutoffs for the responses to the vaccine antigens were determined using reference standards TE-3 (tetanus) and 10/262 (diphtheria) purchased from National Institute for Biological Standards and Control in the Hertfordshire, UK. Twofold serial dilutions were performed, and a log–log plot used to fit a line to the data below the response plateau. Correlations were all consistently high. Median fluorescence intensity (MFI) cutoffs were as follows: diphtheria cutoff for complete protection = 0.1 IU/mL = 4,103 MFI-BKG (24, 25); tetanus cutoff for protection = 10 mIU/mL = 43 MFI-BKG (16, 26). The choice of measles N-protein assay cutoff was based on a receiver-operating characteristic curve analysis of 140 sera comparing multiplex bead assay to the “gold standard” plaque reduction neutralization assay, a live virus infection assay that measures all classes of virus-neutralizing immunoglobulin. The 149 MFI-BKG cutoff value calculated at the CDC in Atlanta, GA, USA, was translated to the data generated at KEMRI using a twofold serial dilution standard curve that was run at both locations. The resulting measles N-protein assay cutoff for the KEMRI BioPlex 200 instrument was 67 MFI-BKG units. The optimal cutoff points for positive antibody levels against PfMSP1 were determined by assigning all mothers’ responses in this holoendemic area as positive and all children’s responses at 20 weeks of age as negative and calculating an ROC curve and J-Index (GraphPad Prism version 6 for Windows, San Diego, CA, USA). The cutoff for anti-SEA antibodies was determined by using the mixture model reported in Cutoff Finder using R (27). The final dataset used for analysis was comprised of mother/neonate pairs for which the mother and her neonate were concordant for either presence or absence of anti-SEA IgG and for mother/neonate pairs where the mother’s plasma contained anti-SEA IgG and her stool specimens were positive for S. mansoni eggs. Egg-negative mothers were Kato Katz-negative but could be anti-SEA IgG antibody negative or positive with a consistent antibody response in the neonate. Mothers in this category were assumed either to be: (a) uninfected; (b) previously infected; or (c) infected with low eggs per gram feces below the Kato Katz limit of detection. Mother/neonate pairs for which the mother and her neonate were discordant for the presence of any anti-SEA IgG (i.e., one member of the pair had specific antibody and the other was completely negative) were deemed to be mistakenly matched during data collection or entry and were, therefore, excluded from final analyses. Data were cleaned and analyzed using IBM SPSS Statistics for Windows, Version 24.0 (IBM Corp., Armonk, NY, USA). GraphPad Prism version 6 for Windows (La Jolla California) was used for statistical analyses as well as for graph preparation. Characteristics of the participating mothers were summarized using descriptive statistics. Anemia was categorized using WHO-recommended cutoffs (28) adjusted by age and altitude with the following hemoglobin values: normal (>11.1 g/dL), mild (10.2–11.1 g/dL), moderate (7.2–10.1 g/dL), and severe (<7.2 g/dL). Correlations between a mother’s and her infant’s antibody levels at birth were examined using Spearman’s correlation test and simple linear regression. Antibody levels were log-transformed due to skewed distribution. The Mann–Whitney U test was used to compare vaccine antibody responses in S. mansoni egg positive and negative mothers. To determine whether an infant reached a protective antibody level, the following cutoffs were used: diphtheria: 4,103 MFI (mean fluorescence intensity) minus background, tetanus: 43 MFI minus background, and Measles: 67 MFI minus background. Chi square analysis was used to determine whether the proportion of vaccine-protected infants differed between S. mansoni egg positive and negative mothers. Tests were considered statistically significant at p < 0.05.
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