RTS,S Vaccination Is Associated With Serologic Evidence of Decreased Exposure to Plasmodium falciparum Liver- and Blood-Stage Parasites*
2014; Elsevier BV; Volume: 14; Issue: 3 Linguagem: Inglês
10.1074/mcp.m114.044677
ISSN1535-9484
AutoresJoe J. Campo, John J. Aponte, Jeff Skinner, Rie Nakajima, Douglas M. Molina, Li Liang, Jahit Sacarlal, Pedro L. Alonso, Peter D. Crompton, Philip L. Felgner, Carlota Dobaño,
Tópico(s)Malaria Research and Control
ResumoThe leading malaria vaccine candidate, RTS,S, targets the sporozoite and liver stages of the Plasmodium falciparum life cycle, yet it provides partial protection against disease associated with the subsequent blood stage of infection. Antibodies against the vaccine target, the circumsporozoite protein, have not shown sufficient correlation with risk of clinical malaria to serve as a surrogate for protection. The mechanism by which a vaccine that targets the asymptomatic sporozoite and liver stages protects against disease caused by blood-stage parasites remains unclear. We hypothesized that vaccination with RTS,S protects from blood-stage disease by reducing the number of parasites emerging from the liver, leading to prolonged exposure to subclinical levels of blood-stage parasites that go undetected and untreated, which in turn boosts pre-existing antibody-mediated blood-stage immunity. To test this hypothesis, we compared antibody responses to 824 P. falciparum antigens by protein array in Mozambican children 6 months after receiving a full course of RTS,S (n = 291) versus comparator vaccine (n = 297) in a Phase IIb trial. Moreover, we used a nested case-control design to compare antibody responses of children who did or did not experience febrile malaria. Unexpectedly, we found that the breadth and magnitude of the antibody response to both liver and asexual blood-stage antigens was significantly lower in RTS,S vaccinees, with the exception of only four antigens, including the RTS,S circumsporozoite antigen. Contrary to our initial hypothesis, these findings suggest that RTS,S confers protection against clinical malaria by blocking sporozoite invasion of hepatocytes, thereby reducing exposure to the blood-stage parasites that cause disease. We also found that antibody profiles 6 months after vaccination did not distinguish protected and susceptible children during the subsequent 12-month follow-up period but were strongly associated with exposure. Together, these data provide insight into the mechanism by which RTS,S protects from malaria. The leading malaria vaccine candidate, RTS,S, targets the sporozoite and liver stages of the Plasmodium falciparum life cycle, yet it provides partial protection against disease associated with the subsequent blood stage of infection. Antibodies against the vaccine target, the circumsporozoite protein, have not shown sufficient correlation with risk of clinical malaria to serve as a surrogate for protection. The mechanism by which a vaccine that targets the asymptomatic sporozoite and liver stages protects against disease caused by blood-stage parasites remains unclear. We hypothesized that vaccination with RTS,S protects from blood-stage disease by reducing the number of parasites emerging from the liver, leading to prolonged exposure to subclinical levels of blood-stage parasites that go undetected and untreated, which in turn boosts pre-existing antibody-mediated blood-stage immunity. To test this hypothesis, we compared antibody responses to 824 P. falciparum antigens by protein array in Mozambican children 6 months after receiving a full course of RTS,S (n = 291) versus comparator vaccine (n = 297) in a Phase IIb trial. Moreover, we used a nested case-control design to compare antibody responses of children who did or did not experience febrile malaria. Unexpectedly, we found that the breadth and magnitude of the antibody response to both liver and asexual blood-stage antigens was significantly lower in RTS,S vaccinees, with the exception of only four antigens, including the RTS,S circumsporozoite antigen. Contrary to our initial hypothesis, these findings suggest that RTS,S confers protection against clinical malaria by blocking sporozoite invasion of hepatocytes, thereby reducing exposure to the blood-stage parasites that cause disease. We also found that antibody profiles 6 months after vaccination did not distinguish protected and susceptible children during the subsequent 12-month follow-up period but were strongly associated with exposure. Together, these data provide insight into the mechanism by which RTS,S protects from malaria. The RTS,S malaria vaccine candidate provides partial protection against clinical malaria in African children, which has been repeatedly demonstrated in Phase IIb and Phase III clinical trials (1Alonso P.L. Sacarlal J. Aponte J.J. Leach A. Macete E. Milman J. Mandomando I. Spiessens B. Guinovart C. Espasa M. Bassat Q. Aide P. Ofori-Anyinam O. Navia M.M. Corachan S. Ceuppens M. Dubois M.C. Demoitié M.A. Dubovsky F. Menéndez C. Tornieporth N. Ballou W.R. 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Issifou S. Kremsner P.G. Sacarlal J. Aide P. Lanaspa M. Aponte J.J. Machevo S. Acacio S. Bulo H. Sigauque B. Macete E. Alonso P. Abdulla S. Salim N. Minja R. Mpina M. Ahmed S. Ali A.M. Mtoro A.T. Hamad A.S. Mutani P. Tanner M. Tinto H. D'Alessandro U. Sorgho H. Valea I. Bihoun B. Guiraud I. Kaboré B. Sombié O. Guiguemdé R.T. Ouédraogo J.B. Hamel M.J. Kariuki S. Oneko M. Odero C. Otieno K. Awino N. McMorrow M. Muturi-Kioi V. Laserson K.F. Slutsker L. Otieno W. Otieno L. Otsyula N. Gondi S. Otieno A. Owira V. Oguk E. Odongo G. Woods J.B. Ogutu B. Njuguna P. Chilengi R. Akoo P. Kerubo C. Maingi C. Lang T. Olotu A. Bejon P. Marsh K. Mwambingu G. Owusu-Agyei S. Asante K.P. Osei-Kwakye K. Boahen O. Dosoo D. Asante I. Adjei G. Kwara E. Chandramohan D. Greenwood B. Lusingu J. Gesase S. Malabeja A. Abdul O. Mahende C. Liheluka E. Malle L. Lemnge M. Theander T.G. Drakeley C. Ansong D. Agbenyega T. Adjei S. Boateng H.O. Rettig T. Bawa J. Sylverken J. Sambian D. Sarfo A. Agyekum A. Martinson F. Hoffman I. Mvalo T. Kamthunzi P. Nkomo R. Tembo T. Tegha G. Tsidya M. Kilembe J. Chawinga C. Ballou W.R. Cohen J. Guerra Y. Jongert E. Lapierre D. Leach A. Lievens M. Ofori-Anyinam O. Olivier A. Vekemans J. Carter T. Kaslow D. Leboulleux D. Loucq C. Radford A. Savarese B. Schellenberg D. Sillman M. Vansadia P. A phase 3 trial of RTS,S/AS01 malaria vaccine in African infants.N. Engl. J. Med. 2012; 367: 2284-2295Crossref PubMed Scopus (542) Google Scholar). The RTS,S target is the Plasmodium falciparum circumsporozoite protein (CSP), and it has been shown to generate high antibody titers that remain above levels acquired naturally for years (6Regules J.A. Cummings J.F. Ockenhouse C.F. The RTS,S vaccine candidate for malaria.Expert Rev. Vaccines. 2011; 10: 589-599Crossref PubMed Scopus (78) Google Scholar). However, it remains unclear how the vaccine, which targets sporozoites, provides protection against disease caused by blood-stage parasites. 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We and others hypothesized that partial blockage of pre-erythrocytic development would result in low-level blood-stage infections that go untreated in RTS,S vaccinees and that this would boost the blood-stage immune response, contributing to protection from malaria disease (8Guinovart C. Aponte J.J. Sacarlal J. Aide P. Leach A. Bassat Q. Macete E. Dobaño C. Lievens M. Loucq C. Ballou W.R. Cohen J. Alonso P.L. Insights into long-lasting protection induced by RTS,S/AS02A malaria vaccine: further results from a phase IIb trial in Mozambican children.PLoS One. 2009; 4: e5165Crossref PubMed Scopus (73) Google Scholar, 9Campo J.J. Dobaño C. Sacarlal J. Guinovart C. Mayor A. Angov E. Dutta S. Chitnis C. Macete E. Aponte J.J. Alonso P.L. Impact of the RTS,S malaria vaccine candidate on naturally acquired antibody responses to multiple asexual blood stage antigens.PLoS One. 2011; 6: e25779Crossref PubMed Scopus (27) Google Scholar, 10Sutherland C.J. Drakeley C.J. Schellenberg D. How is childhood development of immunity to Plasmodium falciparum enhanced by certain antimalarial interventions?.Malar. J. 2007; 6: 161Crossref PubMed Scopus (38) Google Scholar). We set out to address the question of how the vaccine works by investigating the response to malaria parasites in the context of RTS,S vaccination. However, until recently, the means of assessing the response to malaria parasites has been limited to a sparse selection of recombinant proteins or parasite lysates. The P. falciparum (Pf) proteome contains more than 5,300 proteins, and, until recently, less than 0.5% of them have been closely investigated (11Doolan D.L. Plasmodium immunomics.Int. J. Parasitol. 2011; 41: 3-20Crossref PubMed Scopus (81) Google Scholar). Similar to the approach taken with gene expression microarrays, protein arrays offer the opportunity to screen antibody responses to partial or complete proteomes (12Templin M.F. Stoll D. Schwenk J.M. Pötz O. Kramer S. Joos T.O. Protein microarrays: promising tools for proteomic research.Proteomics. 2003; 3: 2155-2166Crossref PubMed Scopus (205) Google Scholar). This approach was taken in this study to identify the breadth and magnitude of naturally acquired immune responses in Mozambican children vaccinated with RTS,S/AS02 1The abbreviations used are:AS02adjuvant system associated with RTS,S malaria vaccine candidateCSPcircumsporozoite protein, the immunodominant surface protein of Plasmodium sporozoitesHAhemagglutinin epitopeHISpolyhistidine epitopeORFopen reading framePfPlasmodium falciparumRTSrapid translation systemRTSSmalaria vaccine candidate. 1The abbreviations used are:AS02adjuvant system associated with RTS,S malaria vaccine candidateCSPcircumsporozoite protein, the immunodominant surface protein of Plasmodium sporozoitesHAhemagglutinin epitopeHISpolyhistidine epitopeORFopen reading framePfPlasmodium falciparumRTSrapid translation systemRTSSmalaria vaccine candidate., the predecessor to the RTS,S/AS01 formulation used in the current Phase III trial, or comparator vaccine. adjuvant system associated with RTS,S malaria vaccine candidate circumsporozoite protein, the immunodominant surface protein of Plasmodium sporozoites hemagglutinin epitope polyhistidine epitope open reading frame Plasmodium falciparum rapid translation system malaria vaccine candidate. adjuvant system associated with RTS,S malaria vaccine candidate circumsporozoite protein, the immunodominant surface protein of Plasmodium sporozoites hemagglutinin epitope polyhistidine epitope open reading frame Plasmodium falciparum rapid translation system malaria vaccine candidate. In addition to characterizing the RTS,S mode of action, we aimed to identify biomarker correlates of protection against clinical malaria. Malaria vaccinology is lacking in surrogate markers of protection, and such biomarkers would be a highly useful measure for assessment of vaccine efficacy, especially when control or placebo vaccine groups are no longer available (13Hudgens M.G. Gilbert P.B. Self S.G. Endpoints in vaccine trials.Stat. Methods Med. Res. 2004; 13: 89-114Crossref PubMed Scopus (27) Google Scholar). This could mitigate the current inefficient means of measuring efficacy in clinical trials. In the post-genomic era, with systems approaches employed for questions to complex problems in biology and medicine, perhaps alternative thinking is required to tackle the question of how to assess vaccines (14Pulendran B. Li S. Nakaya H.I. Systems vaccinology.Immunity. 2010; 33: 516-529Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 15Tran T.M. Samal B. Kirkness E. Crompton P.D. Systems immunology of human malaria.Trends Parasitol. 2012; 28: 248-257Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In this study, we took steps in that direction in order to identify antibody signatures of protection that contribute toward a surrogate marker for the RTS,S and other vaccines. The study was approved by the Mozambican National Health and Bioethics Committee (Ref. 237/CNBS), the Hospital Clínic of Barcelona Ethics Committee (Registro 2008/4444), and the PATH Research Ethics Committee (Study file number HS 482) and written informed consent was gathered from parents/guardians. Serum samples were obtained from a Phase IIb randomized, controlled trial of the RTS,S/AS02 vaccine administered to 1–4-year-old Mozambican children (ClinicalTrials.gov registry number NCT00197041). A nested case-control study was designed using the cross-sectional survey at study month 8.5 (M8.5), 6 months after the third dose, as the sampling time point (Fig. 1). Cases of clinical malaria, irrespective of treatment group, were identified during the 12-month follow-up period from M8.5 to study month 21 (M21), following the trial secondary case definition of P. falciparum asexual blood-stage parasitemia of >0 parasite/μl of blood and an axillary temperature ≥ 37.5 °C. For the cases that had available serum samples at the time of the study, controls were matched to cases 2 to 1 by random selection of non-cases. A total of 623 samples (207 cases and 416 controls), 588 (196 cases and 392 controls) of which passed filtering criteria, was probed at the Protein Microarray Laboratory at the University of California Irvine (UCI). The clinical trial enrolled two study cohorts from different areas of Manhiça District to measure different efficacy endpoints, cohort 1 in Manhiça and Maragra for efficacy against clinical malaria and cohort 2 from Ilha Josina for efficacy against time to first infection (1Alonso P.L. Sacarlal J. Aponte J.J. Leach A. Macete E. Milman J. Mandomando I. Spiessens B. Guinovart C. Espasa M. Bassat Q. Aide P. Ofori-Anyinam O. Navia M.M. Corachan S. Ceuppens M. Dubois M.C. Demoitié M.A. Dubovsky F. Menéndez C. Tornieporth N. Ballou W.R. Thompson R. Cohen J. Efficacy of the RTS, S/AS02A vaccine against Plasmodium falciparum infection and disease in young African children: randomised controlled trial.Lancet. 2004; 364: 1411-1420Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). Only cohort 1 of the trial was selected since efficacy had waned in cohort 2 (16Alonso P.L. Sacarlal J. Aponte J.J. Leach A. Macete E. Aide P. Sigauque B. Milman J. Mandomando I. Bassat Q. Guinovart C. Espasa M. Corachan S. Lievens M. Navia M.M. Dubois M.C. Menendez C. Dubovsky F. Cohen J. Thompson R. Ballou W.R. Duration of protection with RTS,S/AS02A malaria vaccine in prevention of Plasmodium falciparum disease in Mozambican children: single-blind extended follow-up of a randomised controlled trial.Lancet. 2005; 366: 2012-2018Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar), and the time point was selected to allow 6 months of post-vaccination natural exposure before sampling and a 1-year follow-up timeframe after sampling. This was chosen as opposed to a longer follow-up to increase the specificity of antibody responses measured at M8.5 and association with subsequent clinical cases. At the pre-vaccination baseline time point, serum samples were tested for antibody titers to infected red blood cells by immunofluorescence antibody test, as previously described (1Alonso P.L. Sacarlal J. Aponte J.J. Leach A. Macete E. Milman J. Mandomando I. Spiessens B. Guinovart C. Espasa M. Bassat Q. Aide P. Ofori-Anyinam O. Navia M.M. Corachan S. Ceuppens M. Dubois M.C. Demoitié M.A. Dubovsky F. Menéndez C. Tornieporth N. Ballou W.R. Thompson R. Cohen J. Efficacy of the RTS, S/AS02A vaccine against Plasmodium falciparum infection and disease in young African children: randomised controlled trial.Lancet. 2004; 364: 1411-1420Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). These data were used to demonstrate the higher transmission intensity in cohort 2, and these data are used herein to compare basal levels of blood-stage antibody responses to control for bias. Proteins were expressed using the Escherichia coli cell-free Rapid Translation System (RTS) kit (5 Prime, Gaithersburg, MD). A library of Pf partial or complete open reading frames (ORFs) cloned into a T7 expression vector pXT7 has been established at Antigen Discovery, Inc. (ADi, Irvine, CA). This library was created through an in vivo recombination cloning process with PCR-amplified Pf ORFs, and a complementary linearized expressed vector transformed into chemically competent E. coli was amplified by PCR and cloned into pXI vector using a high-throughput PCR recombination cloning method described elsewhere (17Davies D.H. Liang X. Hernandez J.E. Randall A. Hirst S. Mu Y. Romero K.M. Nguyen T.T. Kalantari-Dehaghi M. Crotty S. Baldi P. Villarreal L.P. Felgner P.L. Profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen discovery.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 547-552Crossref PubMed Scopus (335) Google Scholar). Each expressed protein includes a 5′ polyhistidine (HIS) epitope and 3′ hemagglutinin (HA) epitope. After expressing the proteins according to manufacturer instructions, translated proteins were printed onto nitrocellulose-coated glass AVID slides (Grace Bio-Labs, Inc., Bend, OR) using an Omni Grid Accent or Omni Grid 100 robotic microarray printer (Digilabs, Inc., Marlborough, MA). Microarray chip printing and protein expression were quality checked by probing random slides with anti-HIS and anti-HA monoclonal antibodies with fluorescent labeling, as shown in Fig. S1. A down-selected array was designed (PF11+ chip, ADi) to include eight "pads," or replicated grids of spotted proteins totaling 960 features per pad, each available to probe a single specimen. Each pad contains 94 control spots, composed of the following: (i) 16 purified human IgG or a mouse anti-human IgG positive control spots, printed in quadruplicate at two different concentrations; (ii) 38 tris-based buffering solution "blanks" with or without Tween-20 (12 and 16 spots, respectively) or nothing at all (four spots); (iii) 40 RTS reactions without Pf ORFs (NoDNA), as shown in Fig. S2. The NoDNA controls were randomly distributed across the pad subarrays. Printed features do not vary in position between pads or slides. Each pad contains 824 peptide fragments expressed from Pf ORFs representing 702 unique proteins and three concentrations of 14 full-length proteins expressed using clinical-grade manufacturing procedures. The protein targets on the array were selected from published studies done on a larger microarray containing 2,323 protein features after interrogating specimens from naturally exposed individuals (18Crompton P.D. Kayala M.A. Traore B. Kayentao K. Ongoiba A. Weiss G.E. Molina D.M. Burk C.R. Waisberg M. Jasinskas A. Tan X. Doumbo S. Doumtabe D. Kone Y. Narum D.L. Liang X. Doumbo O.K. Miller L.H. Doolan D.L. Baldi P. Felgner P.L. Pierce S.K. A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 6958-6963Crossref PubMed Scopus (327) Google Scholar), and specimens from sporozoite vaccine trials (19Trieu A. Kayala M.A. Burk C. Molina D.M. Freilich D.A. Richie T.L. Baldi P. Felgner P.L. Doolan D.L. Sterile protective immunity to malaria is associated with a panel of novel P. falciparum antigens.Mol. Cell Proteomics. 2011; 10M111.007948 Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The top 824 most immunoreactive antigens from these studies were printed on this down-selected array. Serum samples were diluted 1:100 in a 10% E. coli lysate solution in protein arraying buffer (Maine Manufacturing, Sanford, ME) and incubated at room temperature for 30 min. Chips were rehydrated in blocking buffer for 30 min. Blocking buffer was removed, and chips were probed with preincubated serum samples using sealed chambers fitted to eight-pad slides to ensure no cross-contamination of sample between pads. Chips were incubated overnight at 4 °C with agitation. Chips were washed five times with TBS-0.05% Tween 20, followed by incubation with biotin-conjugated goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA) diluted 1:200 in blocking buffer at room temperature. Chips were washed three times with TBS-0.05% Tween 20, followed by incubation with streptavidin-conjugated SureLight P-3 (Columbia Biosciences, Frederick, MD) at room temperature protected from light. Chips were washed three times with TBS-0.05% Tween 20, three times with TBS, and once with water. Chips were air dried by centrifugation at 1,000 × g for 4 min and scanned on a ScanArray Express HT spectral scanner (Perkin-Elmer, Waltham, MA), and spot and background intensities were measured using an annotated grid file (.GAL). Data were exported in Microsoft Excel. A standardized data processing pipeline for protein array technology has not been established, and most analysis techniques to date have employed methods applied to gene expression microarrays (20Sundaresh S. Doolan D.L. Hirst S. Mu Y. Unal B. Davies D.H. Felgner P.L. Baldi P. Identification of humoral immune responses in protein microarrays using DNA microarray data analysis techniques.Bioinformatics. 2006; 22: 1760-1766Crossref PubMed Scopus (83) Google Scholar). However, the assumptions of gene microarrays, particularly that the vast majority of features should be equally expressed between subjects, may not be valid for protein microarrays (21Sboner A. Karpikov A. Chen G. Smith M. Mattoon D. Dawn M. Freeman-Cook L. Schweitzer B. Gerstein M.B. Robust-linear-model normalization to reduce technical variability in functional protein microarrays.J. Proteome Res. 2009; 8: 5451-5464Crossref PubMed Scopus (51) Google Scholar). In fact, whereas gene expression experiments assume that all genes are expressed to some level, protein arrays assume that antibody reactivity can be positive at any level or zero and vary naturally between individuals. Therefore, an analysis pipeline that can be applied to protein array datasets was established (Skinner et al. in preparation). Briefly, raw spot and local background intensities, protein annotation, and sample phenotypes were imported and merged in the R statistical environment (www.r-project.org), where all subsequent procedures were performed unless specified otherwise (22R Development Core Team R: a language and environment for statistical computing.R Foundation for Statistical Computing. 2013; Google Scholar). Spot intensities were adjusted for local background using the "normexp" method, which produces a monotonic transformation of positive, local background-subtracted foreground intensities (23Ritchie M.E. Silver J. Oshlack A. Holmes M. Diyagama D. Holloway A. Smyth G.K. A comparison of background correction methods for two-colour microarrays.Bioinformatics. 2007; 23: 2700-2707Crossref PubMed Scopus (730) Google Scholar). This method is available in the "limma" package of R using the "backgroundCorrect" function, and an additional offset value of 50 was applied to all spots (24Smyth G.K. Gentleman R. Carey V. Huber W. Irizarry R. Dudoit S. Bioinformatics and computational biology solutions using R and Bioconductor. Springer, New York2005: 397-420Google Scholar). Next, all foreground values were transformed using the base 2 logarithm (Log2). The dataset was normalized to remove systematic effects using linear models, as previously demonstrated by Sboner et al. (21Sboner A. Karpikov A. Chen G. Smith M. Mattoon D. Dawn M. Freeman-Cook L. Schweitzer B. Gerstein M.B. Robust-linear-model normalization to reduce technical variability in functional protein microarrays.J. Proteome Res. 2009; 8: 5451-5464Crossref PubMed Scopus (51) Google Scholar). For this dataset, we used a linear mixed model, adjusting signal intensities by printing batch, array chip, and pad (which also adjusted for print tip), including random effects to account for array chip nested in printing batch. Importantly, the linear model was fit to only the 94 control spots of each array, which includes a dynamic range of responses. All samples were included in the model, which was then applied to the entire dataset for adjustment. Additional linear methods, such as median scaling, and nonlinear methods, such as variance stabilizing normalization (VSN) (25Huber W. von Heydebreck A. Sultmann H. Poustka A. Vingron M. Variance stabilization applied to microarray data calibration and to the quantification of differential expression.Bioinformatics. 2002; 18: S96-S104Crossref PubMed Scopus (1617) Google Scholar) and quantile normalization (26Bolstad B.M. Irizarry R.A. Astrand M. Speed T.P. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias.Bioinformatics. 2003; 19: 185-193Crossref PubMed Scopus (6393) Google Scholar), were tested with the dataset but not applied due to inferior or inappropriate scaling of systematic effects found in distribution plots or principal component analysis plots (data not shown). A seropositivity threshold was established as the mean plus two standard deviations of NoDNA signals across all arrays in the dataset, and antigen reactivity was calculated. Reactive antigens were defined as those that had seropositive responses in at least 1% of the study population. Antibody breadth was defined as the number of reactive antigens with seropositive antibody responses per individual, and an antibody "breadth score" was calculated as the sum of seropositive Pf target probes for each individual. Antibody magnitude was defined as signal intensity of reactive Pf antigens with respect to NoDNA probes. To calculate the magnitude of antibody responses, the median normalized signal intensity (Log2 scale) of the NoDNA probes per individual was subtracted from Pf target probe normalized signal intensities for each individual. For each Pf target probe, a life cycle stage was assigned based on maximal gene expression detected by Affymetrix gene microarray databases available on PlasmoDB (www.plasmodb.org) (27The Plasmodium Genome Database Collaborative PlasmoDB: An integrative database of the Plasmodium falciparum genome.Nucleic Acids Res. 2001; 29: 66-69Crossref PubMed Google Scholar) or by multidimensional protein identification technology from the database published by Florens and colleagues (28Florens L. Washburn M.P. Raine J.D. Anthony R.M. Grainger M. Haynes J.D. Moch J.K. Muster N. Sacci J.B. Tabb D.L. Witney A.A. Wolters D. Wu Y. Gardner M.J. Holder A.A. Sinden R.E. Yates J.R. Carucci D.J. A proteomic view of the Plasmodium falciparum life cycle.Nature. 2002; 419: 520-526Crossref PubMed Scopus (1089) Googl
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