Longitudinal analysis of naturally acquired antibodies to PfEMP1 CIDR domain variants and their association with malaria protection

Malaria pathogenicity is determined, in part, by the adherence of Plasmodium falciparum infected erythrocytes to the microvasculature mediated via specific interactions between PfEMP1 variant domains to host endothelial receptors. Naturally acquired antibodies against specific PfEMP1 variants can play an important role in clinical protection against malaria. We evaluated IgG responses against a repertoire of PfEMP1 CIDR domain variants to determine the rate and order of variant-specific antibody acquisition and their association with protection against febrile malaria in a prospective cohort study conducted in an area of intense, seasonal malaria transmission. Using longitudinal data, we found that IgG to the pathogenic domain variants CIDRα1.7 and CIDRα1.8 were acquired the earliest. Furthermore, IgG to CIDRγ3 was associated with reduced prospective risk of febrile malaria and recurrent malaria episodes. Future studies will need to validate these findings in other transmission settings and determine the functional activity of these naturally acquired CIDR variant-specific antibodies.

malaria transmission. Using longitudinal data, we found that IgG to the pathogenic domain 48 variants CIDRα1.7 and CIDRα1.8 were acquired the earliest. Furthermore, IgG to CIDRγ3 was 49 associated with reduced prospective risk of febrile malaria and recurrent malaria episodes. 50 Future studies will need to validate these findings in other transmission settings and determine 51 the functional activity of these naturally acquired CIDR variant-specific antibodies. 52

INTRODUCTION 53
Malaria due to Plasmodium falciparum causes greater than 400,000 deaths per annum (1). 54 Severe clinical manifestations of P. falciparum malaria are precipitated by widespread 55 sequestration of infected erythrocytes (IEs) in host microvasculature including in the brain and 56 placenta which can lead to cerebral malaria and placental malaria, respectively (2). 57 Cytoadherence of IEs occurs via specific interactions between host endothelial receptors and P. 58 falciparum erythrocyte membrane protein (PfEMP1), a parasite-derived protein expressed on the 59 surface of IEs that is a major target of naturally acquired immunity to malaria (3)(4)(5). The PfEMP1 60 adhesins are encoded by ~60 var gene variants that differ within and between parasite genomes 61 and that are expressed in a mutually exclusive manner within each IE (6-8). Switching between 62 var genes aids in parasite immune evasion and functional diversification of the PfEMP1 family 63 have resulted in mutually exclusive receptor binding phenotypes correlated to differences in 64 clinical severity (9, 10). 65 Members of the PfEMP1 family vary in the size and number of extracellular  like (DBL) and cysteine-rich interdomain region (CIDR) domains(11). DBL and CIDR domains are 67 classified based on sequence similarity into six (α, β, γ, δ, ε, ξ) and four (α, β, γ, δ) main classes, 68 respectively, of which some can be further divided into sub-classes (e.g. CIDRα1.1) (12,13). 69 PfEMP1 generally have a semi-conserved head structure near the N-terminus consisting of a 70 tandem DBLα-CIDR domain. This can be followed by a second DBLδ-CIDR tandem domain or 71 additional other types of DBL domains in larger proteins. Notably, however, the VAR2CSA PfEMP1 72 variants do not contain typical CIDR domains and bind placental chondroitin sulfate A via 73 specialized DBL domains (14,15). PfEMP1 have diversified to either bind endothelial protein C 74 receptor (EPCR) (10), the scavenger receptor CD36 (16) or yet undermined receptors via their 75 head structure CIDR domains. These phenotypes are maintained by the chromosomal 76 organization of the var genes (17). Among the subtelomeric var genes, Group A genes transcribed 77 toward the telomere encode DBLα1-CIDRα1 head structures binding to EPCR or DBLα1-78 CIDRβ/γ/δ head structures with unknown endothelial receptor specificities. Subtelomeric Group 79 B var genes transcribed toward the centromere as well as centromeric Group C var genes encode 80 DBLα0-CIDRα2-6 head-structures binding to CD36. In addition to this, chimeric group B/A var 81 genes encode EPCR-binding DBLα0-CIDRα1 head structures. The EPCR-binding phenotype has 82 been implicated in severe malaria (18-21), whereas CD36 binding has been associated with 83 uncomplicated malaria (22,23). Severe malaria has been associated with rosetting, a 84 phenomenon which involves binding between an IE and several uninfected erythrocytes but with 85 unclear clinical significance. A set of group A PfEMP1 with DBLα1-CIDRβ/γ/δ domains have been 86 shown to mediate rosettes. 87 Immunity to severe malaria is generally acquired after only one to two severe episodes (24) 88 with naturally acquired antibodies specific for PfEMP1 variants likely playing an important role in 89 clinical protection (25). Antibodies to group A PfEMP1 variants tend to be acquired prior to 90 antibodies to group B and C variants (26) and are associated with protection from severe malaria 91 (27). Similarly, antibodies to EPCR-binding CIDRα1 domains are acquired more rapidly than 92 antibodies to other CIDR domains in areas of high malaria transmission intensity and are boosted 93 by severe malaria but not uncomplicated malaria (28,29). However, a recent study showed that 94 antibodies to both rosetting-associated DBLα variants and CD36-binding CIDR domains predicted 95 reduced risk of severe malaria to a similar extent as antibodies to EPCR-binding CIDR domains 96 (30). The same study also showed that antibodies to group 2 DBLα variants, which are associated 97 with rosetting (31), also predicted protection from uncomplicated malaria. 98 To gain further insight into the role of PfEMP1-variant specific antibodies, we assessed IgG 99 responses against a repertoire of PfEMP1 CIDR domains to determine the rate and order of 100 variant-specific antibody acquisition and their association with protection against uncomplicated 101 febrile malaria in a prospective cohort study conducted in a Malian village with intense and 102 seasonal malaria transmission. 103

RESULTS 104
IgG specific for CIDRα1, CIDRδ, and CIDRγ domain variants are acquired rapidly. 105 Naturally acquired IgG antibody responses to 35 PfEMP1 CIDR domain variants representing 106 subtypes α, γ and δ CIDR, as well as three well-studied P. falciparum antigens (circumsporozoite 107 protein [PfCSP], apical membrane protein 1 [PfAMA1], and merozoite surface protein 1 108 [PfMSP1]), tetanus toxoid (non-malaria positive control), and bovine serum albumin (non-specific 109 background control; Table S1) were determined by multiplex bead-based immunoassay in 680 110 children and adults from the Kalifabougou, Mali cohort at their healthy baseline in May 2011 (Fig. 111 S1). Hierarchical clustering of baseline PfEMP1-specific IgG reactivity revealed distinct clustering 112 of samples by age, and by the presence of PCR-documented, asymptomatic P. falciparum 113 infection; as well as clustering of antigen targets by group (A, B, or B/A), binding phenotype 114 (EPCR, CD36, or unknown), and CIDR domain class (Fig. 1a); suggesting differential rates of 115 acquisition of IgG between PfEMP1 variants with cumulative P. falciparum exposure and the 116 acquisition of clinical immunity to malaria. PfEMP1-specific IgG reactivity increased rapidly up to 117 8 years of age, and within each age stratum, P. falciparum PCR-positive individuals exhibited 118 greater variant-specific IgG reactivity than uninfected individuals (Fig. 1b). 119 Categorization of PfEMP-1 variants by CIDR domain class suggested that IgG specific for 120 variants in the CIDRγ, CIDRα1, and CIDRδ classes was acquired rapidly whereas IgG specific for 121 group B variants of the CIDRα2-6 class was acquired slowly irrespective of P. falciparum infection 122 status (Fig. 1c). Indeed, when compared to variants of other domain classes within the linear 123 range of the fit curves (<8 years of age), IgG specific for variants within each of the CIDRγ, CIDRα1, 124 and CIDRδ classes increased significantly more rapidly with age, whereas IgG specific for variants 125 of the CIDRα2-6 classes increased significantly more slowly with age, independent of P. 126 falciparum infection status ( Fig. 1d and Table S2). Of note, IgG specific for AMA1, CSP, and MSP1 127 increased predictably with age in early childhood and plateaued in adolescence or young 128 adulthood, which is similar to what we previously observed in this cohort (32, 33) ( Fig. 1b-c). As 129 we observed previously by ELISA in a separate cohort in Mali (34), increases in tetanus toxoid-130 specific IgG in early childhood and adolescence corresponded with the primary childhood vaccine 131 series (diphtheria, tetanus, pertussis) and a subsequent booster of a tetanus toxoid-containing 132 vaccine in females of child-bearing age ( Fig. 1b-c). 133 With the exclusion of children <6 months of age whose IgG is most likely maternally derived, 134 ranking of antigens by decreasing seropositivity within each age group revealed 135 immunodominance of CIDRα1, CIDRδ, and CIDRγ domain classes, which are all either of the A or 136 B/A var group, in early childhood (<7 years) that is maintained to a large degree in adolescence 137 and early adulthood (Fig. S2). Notably, the most prevalent PfEMP1-specific IgG reactivity among 138 individuals greater than 1 year of age was against CIDRα1.7(c) with seroprevalence rapidly rising 139 from 25% in 2 to 3-year-old children to 60% in 4 to 6-year-old children and surpassing 95% in 140 older children and adults (Fig. S2). However, the majority of individuals within the oldest age 141 group (15-25 years) were also seropositive for several variants within the CIDRα2-6 domain 142 classes, suggesting that IgG antibodies against these variants are eventually acquired with 143 additional years of malaria exposure. 144 To assess the longitudinal acquisition of variant-specific IgG, we determined variant-specific 145 IgG reactivity across five annual cross-sectional surveys conducted just prior to each malaria 146 transmission season for an age-stratified random sample of 60 children from the entire cohort 147 (Fig. S1). Children in this subset experienced a median of 6 febrile malaria episodes (interquartile 148 range, 4-9 episodes) with a broad range of parasite densities and distributed widely but with 149 clear seasonal peaks in the number of episodes during the five-year surveillance period ( Fig. 2a-150 b). In the youngest children (6 months to 2 years), IgG specific for variants of the CIDRα1 and 151 CIDRδ domain classes began low and then increased rapidly over four malaria seasons, whereas 152 IgG specific for CIDRγ initially decreased during the first two years before rising during the third 153 year of surveillance ( Fig. 2c). In contrast, older children (3 to 8 years) appeared to maintain stable 154 levels of IgG specific for all PfEMP1 variants over four malaria seasons (Fig. 2c). 155

Acquisition of IgG antibodies to CIDR domain classes is highly ordered with IgG against EPCR-156
binding domain variants CIDRα1.7 and CIDRα1.8 acquired first. 157 We next asked whether IgG antibodies to individual PfEMP1 variants were acquired in a particular 158 order. Here we used an approach called minimum violations ranking (MVR), where an algorithm 159 searches over different possible orders of acquisition of antibodies to PfEMP1 variants such that, 160 if a particular an order is assumed for each child, the number of order violations observed in the 161 data overall is minimized (refer to methods). We observed significantly less violations if we 162 assumed an ordered acquisition of antibodies compared to a model with randomized 163 seroconversion orders for each child, which highly suggests a hierarchical exposure to different 164 parasite CIDR domains in this population ( Fig. 3a-d). At the variant level, IgG specific to 165 CIDRα1.7(c) was acquired first followed by IgG to CIDRα1.8b(a), CIDRα1.8b(c), CIDRα1.7(a), 166 CIDRα6, and CIDRγ3 (Fig. 3a). Grouped by CIDR domain class, IgG was acquired against CIDRγ first 167 followed by CIDRα1, CIDRδ, and CIDRα2-6 ( Fig. 3b). Grouped on the basis of upstream sequence, 168 IgG was acquired against B/A first followed by A and B (Fig. 3c). Lastly, when variants were 169 grouped by binding phenotype, IgG against EPCR-binding domains were acquired first followed 170 by domains with unknown binding phenotypes and CD36-binding domains (Fig. 3d). Whether 171 this reflects differential prevalence of variants in the parasite population or age-specific 172 expression patterns remains an open question. 173

CIDRγ-specific IgG associates with protection from uncomplicated, febrile malaria. 174
We focused on the risk of uncomplicated malaria given that severe malaria was rarely observed 175 in the Kalifabougou cohort due to early diagnosis and treatment. We specifically evaluated 176 whether baseline seropositivity for each variant could predict protection from febrile malaria 177 after subsequent PCR-confirmed P. falciparum parasitemia in individuals who began the study 178 PCR-negative using a Cox regression model that included age, presence of the malaria-protective 179 HbS allele, gender, IgG reactivity to AMA1 (as a surrogate for prior malaria exposure), and 180 seropositivity to each of the 35 PfEMP-1 variants as covariates. Notably, seropositivity to CIDRγ3 181 (IT4var08), which has an unknown binding phenotype was significantly associated with reduced 182 risk of febrile malaria (Table 1). CIDRγ domains have been associated with rosetting of 183 erythrocytes (11), a phenomenon associated with severe forms of malaria (35) except in 184 individuals with blood group O erythrocytes which appear to exhibit reduced rosetting (36). We 185 therefore hypothesized that the reduced risk afforded by CIDRγ-specific IgG might occur via the 186 inhibition of rosette formation and may therefore be negatively affected by blood group O. When 187 included as a covariate in a reduced Cox regression model, group O blood type affected neither 188 malaria risk itself nor the association between CIDRγ-specific IgG and risk of febrile malaria ( Table  189 S3). Notably, baseline CIDRγ3-specific IgG reactivity did not significantly correlate with decreased 190 parasite density at the first malaria episode after controlling for age and the presence of the HbS 191 allele (data not shown), suggesting that CIDRγ-specific IgG may not have anti-parasite activity. 192 Given the association between CIDRγ-specific IgG and delay in malaria fever during the first year 193 of the study, we specifically examined if CIDRγ3 serostatus at the beginning of each malaria 194 season affected the risk of recurrent malaria episodes in the 60 children who were longitudinal 195 evaluated for PfEMP1 IgG responses over five malaria seasons. Presence of CIDRγ3-specific IgG 196 prior to each season predicted a reduction in febrile malaria episodes even after controlling for 197 AMA1-specific IgG serostatus and the HbS allele (Table 2). 198

DISCUSSION 199
PfEMP1 variants containing domains of the CIDRα1 class generally bind to EPCR on 200 endothelial cells and are associated with severe malaria (10), whereas variants containing 201 domains of the CIDRα2-6 classes bind to CD36 present on several host cell types, including 202 microvascular endothelial cells, mononuclear phagocytes, and platelets (16,37). Antibodies 203 targeting these PfEMP1 domains can potentially disrupt adhesion of IEs to host receptors but can 204 also facilitate IE clearance via opsonization and phagocytosis or antibody-mediated cytotoxicity 205 (10, 38, 39). Consistent with a prior study conducted in a Tanzanian cohort (28), we observed 206 early acquisition of IgG antibodies against EPCR-binding PfEMP1 variants of the CIDRα1 domain 207 class relative to CD36-binding variants in both age-stratified cross-sectional and longitudinal 208 analyses. This is also consistent with studies that investigated acquisition of antibodies to PfEMP1 209 classified by upstream sequence group and found that antibodies to DBL and CIDR domains 210 belonging to group A and B/A are acquired earlier in life than group B and C variants among 211 individuals living in malaria-endemic settings (26,40). Importantly, the antigen panel used in the 212 current study contained unique CIDR domains not covered by these prior studies. 213 Among the 35 distinct CIDR domains evaluated here, CIDRα1.7(c) elicited the most robust 214 and prevalent IgG responses in early childhood, eventually approaching 100% seroprevalence in 215 adolescents and adults in this cohort. Longitudinal analysis to assess hierarchical acquisition 216 confirmed that IgG antibodies specific for CIDRα1.7(c) were acquired first, with IgG against the 217 related CIDRα1.7(a) variant acquired fourth. Transcripts encoding CIDRα1.7 domains have been 218 found to predominate among the most severe cases of pediatric cerebral malaria-those that 219 lead to brain swelling and death (19). The immunodominance of CIDRα1.7(c) may be a 220 consequence of epitopes targeted by cross-reactive CIDRα1 antibodies (41, 42). Moreover, 221 PfEMP1 with CIDRα1.4 and CIDRα1.7 domains frequently contain ICAM1-binding DBLβ domains 222 (43). The dual EPCR-and ICAM1-binding phenotype is thought to be particularly pathogenic, and 223 antibodies to these DBLβ domains have been associated with reduced risk of clinical malaria with 224 parasite densities of ≥10,000 parasites/μl (44). We also observed early acquisition of IgG specific 225 for CIDRα1.8 domains. Expression of these domains, as well as EPCR-binding CIDRα1 domains in 226 general, is associated with severe malaria including cerebral malaria in African children (18)(19)(20)(21)227

45) and Indian adults (46). 228
Given that all CIDRα1 variants have been linked to severe malaria in African children, the early 229 acquisition of IgG specific to CIDRα1.7 and CIDRα1.8 domains may just be a reflection of local 230 parasite population dynamics rather than enhanced pathogenicity conferred by these specific 231 CIDR variants. However, the potential lethality of parasites expressing CIDRα1 in general 232 underscores why a vigorous host antibody response against these variant domains in early 233 childhood may be advantageous. This study builds on older work (4,47,48) showing an age-234 specific acquisition of antibodies to particular parasite strains, and we are able to statistically 235 confirm this pattern for the first time, and identify key genetic underpinnings of those 236 observations. We still cannot address the slippery problem of whether this order reflects the 237 circulation of genotypes with different transmissibility; under this scenario, high fitness 238 genotypes lead to high prevalence and therefore low age of first infection, and coincidentally 239 cause more disease in relatively non-immune children compared to low fitness genotypes as a 240 result. In contrast, it is possible that the ordered expression of PfEMP1 variants across strains, 241 potentially in response to the immune status of the parasite's immediate host, leads to the 242 hierarchical acquisition of antibodies observed. 243 Due to the low incidence of severe disease in the cohort, we could not assess the impact of 244 CIDRα1.7-specific or CIDRα1.8-specific antibodies on the risk of severe malaria in the study. 245 However, when all 35 CIDRs were assessed for association with the prospective risk of 246 uncomplicated, febrile malaria, IgG specific to CIDRγ3 (IT4var08) was significantly associated with 247 reduced malaria risk. PfEMP1 variants encoding CIDRβ, CIDRγ, or CIDRδ domains have been 248 associated with rosetting (11, 46), which can enhance microvasculature obstruction thereby 249 increasing malaria severity. However, direct evidence that any of these CIDR domains have 250 intrinsic rosetting properties is lacking (49). Rather, their association with rosetting may be 251 related to their tandem expression with an adjacent DBLα1 at the N-terminal head (50). Rosetting 252 frequency has been correlated with severity of malaria with the highest levels in cerebral malaria 253 (35, 51, 52) but is still commonly observed in uncomplicated malaria. Thus, the role of rosetting 254 in severity of malarial disease remains unclear. Nevertheless, disruption of rosettes by targeting 255 DBL1α has been used as a vaccine strategy (53), and antibodies to rosetting-associated group 2 256 DBLα domains predicted protection from uncomplicated malaria, suggesting a protective role for 257 these antibodies in less severe disease (30, 31). Although speculative, it is possible that naturally 258 acquired CIDRγ-specific IgG confers protection from febrile malaria by blocking rosette 259 formation. However, this mechanism is not supported by the current study given that the 260 protection attributable to CIDRγ-specific IgG is unchanged after controlling for blood group O, 261 which has been shown to be protective against severe falciparum malaria through the reduction 262 of rosetting (36). It also must be noted that reduced malaria risk was not observed for IgG-specific 263 for variants of the CIDRδ class, which is also predicted to have rosetting activity. Furthermore, as 264 CIDRγ3 was the only CIDRγ domain variant tested in this study, it remains unknown whether the 265 protective effect observed here would be generalizable to IgG targeting other CIDRγ variants. 266 A limitation of the study is that we did not sequence var transcripts from individuals with P. 267 falciparum infections in the longitudinal analysis. This may have allowed us to prospectively 268 assess if seroconversion against specific CIDRs such as CIDRα1.7, CIDRα1.8, or CIDRγ reliably led 269 to the absence of parasites expressing the corresponding var transcript during clinical malaria 270 episodes. In addition to our limited assessment of CIDRγ domains, we also did not evaluate CIDRβ 271 domains, which also have been associated with the rosetting phenotype. 272 In summary, this longitudinal study provides evidence that acquisition of IgG antibodies to 273 PfEMP1 variants is ordered and demonstrates that antibodies to CIDRα1 domains, specifically the 274 pathogenic domain variants CIDRα1.7 and CIDRα1.8, are acquired the earliest in children residing 275 in an area of intense, seasonal malaria transmission. We also show that IgG antibodies to the 276 rosetting-associated CIDRγ3 domain is acquired early and is associated with protection from 277 febrile malaria. Future studies will need to validate these findings in other transmission settings 278 and determine the functional activity of these naturally acquired CIDR variant-specific antibodies. 279

Ethics 281
The Ethics Committee of the Faculty of Medicine, Pharmacy and Dentistry at the University 282 of Sciences, Techniques, and Technology of Bamako, and the Institutional Review Board of the 283 National Institute of Allergy and Infectious Diseases, National Institutes of Health approved this 284 study (ClinicalTrials.gov identifier: NCT01322581). Written, informed consent was obtained from 285 the parents or guardians of participating children or from adult participants. 286

Study Site 287
The study was conducted in the village of Kalifabougou, Mali, which is located 40 km 288 northwest of Bamako, Mali within the savanna ecoclimatic zone. Within this community, 289 Bambara is the predominant ethnic group, and ~90% of residents engage in subsistence farming. 290 Malaria transmission is intense and seasonal, reliably occurring from June through December, 291 with the vast majority of malaria cases caused by P. falciparum (54). 292

Study Population and Study Design 293
Recruitment and enrollment procedures of participants for this study have been previously 294 described (55). Briefly, exclusion criteria at enrollment included a hemoglobin level <7 g/dL, 295 axillary temperature ≥37.5°C, acute systemic illness, underlying chronic disease, use of 296 antimalarial or immunosuppressive medications in the past 30 days, or pregnancy. The study 297 design and selection of subjects are summarized in Fig. S1. 298

Human samples 299
At the beginning and end of the malaria-transmission season, blood samples were drawn by 300 venipuncture into sodium-citrate-containing Vacutainer tubes (Becton Dickinson). Plasma was 301 separated by centrifugation and cryopreserved. Hemoglobin typing was performed using a D-10 302 instrument (Bio-Rad). Blood for ABO typing was collected in EDTA containing microtainers. ABO 303 typing was conducted with forward typing using Cypress Diagnostics Reagents. Anti-A, Anti-B, 304 and Anti-AB IgM reagents were mixed with the sample, and blood type was determined by 305 agglutination. During the first malaria season, blood was collected by finger-prick onto 903 filter 306 paper (Whatman) for PCR analysis at each scheduled clinic visit (occurring at 2-week intervals for 307 7 months) and sick visit for subsequent molecular diagnostics. 308

Diagnosis and Treatment of Infections 309
Clinical malaria episodes. Individuals were initially enrolled in May 2011 and have been 310 followed continuously since unless withdrawn or lost to follow-up. During the first malaria 311 season, clinical malaria episodes were detected prospectively by self-referral and weekly active 312 clinical surveillance visits which alternated between the study clinic and the participants' homes.

Protein Expression and Multiplex Immunoassays 328
The 35 recombinant His-tagged CIDR domains (Table S1) were expressed in baculovirus-329 transfected insect cells, and purified by nickel affinity chromatography as previously described 330 (28,42,56). AMA1, CSP, and MSP1 recombinant proteins were kindly provided by David Narum 331 (Laboratory of Malaria Immunology and Vaccinology, NIAID, NIH). AMA1 and CSP were expressed 332 from P. falciparum 3D7 in P. pastoris as previously described (57, 58). MSP1 was expressed from 333 P. falciparum 3D7 in Escherichia coli as previously described (59). Purified tetanus toxoid was 334 provided the staff at Biologic Laboratories, University of Massachusetts Medical School. Bovine 335 serum albumin (BSA) was obtained from Sigma. These proteins were coupled to MagPlex-C 336 micospheres (Luminex) and mixed to form a protein bead array in which IgG reactivity to each 337 antigen could be measured in multiplex, as previously described(60) with minor modifications. 338 Briefly, plasma samples were diluted 1:500 and 1:2000 (to better assess highly reactive antigens) 339 in Assay Buffer E (ABE: 0.1% BSA, 0.05% Tween-20 in PBS, pH7.4). For each plate, pooled malaria-340 hyperimmune plasma was serially diluted in ABE at 1:50, 1:158, 1:500, 1:1580, 1:5000, 1:1580, 341 1:50000, and 1:158000 to generate an 8-point dilutional standard curve. 50 µl of beads and 50 µl 342 of diluted plasma was added to 96-well microtiter plates (MSBVS 1210, Millipore, USA) pre-343 wetted with ABE. 50 µl of phycoerythrin-conjugated Goat Anti-Human IgG (Jackson 344 ImmunoResearch Laboratories), diluted 1:3000 was added, and mean fluorescent intensities 345 were measured using the Luminex 200 system. To account for plate-to-plate variation, 346 fluorescence intensities were normalized using the median reactivity for each antigen on each 347 plate. Normalized intensities were then scaled to the mean reactivity for each antigen to allow 348 comparison between antigens. Using the ncal function within the nCal package (61) Note that the more that individuals' seroconversions occur strictly in their rank order, the 362 smaller v will be. In this way, the number of violations v provides a convenient test statistic for a 363 standard one-tailed p-value test: our null hypothesis is that there is no meaningful order to 364 seroconversions, and thus, randomly permuting the order of seroconversions for each individual 365 and recomputing v should make no difference. In other words, the null hypothesis is that the Computations were performed according to the following details. Let matrix entry Aij be the 373 number of times, over each individual, that a seroconversion to i was observed prior to a 374 seroconversion to j. If the matrix's rows and columns are sorted according to some re-ordering r, 375 then the number of violations v can be computed as the sum of the lower triangle of A(r). Finding 376 the r that minimizes v can be done by beginning from a random r, and then sequentially proposing 377 swaps of pairs of indices in which any swap that increases v is rejected and otherwise swaps are 378 accepted. This MVR algorithm exits after a large number of proposed swaps have been rejected 379 without any decrease in v, and the output is both v and the order of seroconversion that 380 corresponds to that v. Permutation tests were then performed by shuffling the seroconversions 381 and years, independently for each individual, and then applying the computation above. 382

Statistical Analysis 383
The use of specific statistical tests and methods are indicated in the Results and/or figure  384 legends. Statistical significance was defined as a 2-tailed P value of <.05. Analyses were 385 performed in R version 3.6.1 (http://www.R-project.org). Plots were generated with the ggplot2 386 package. Cox regression was performed using the survival and glmnet packages. For the time to 387 febrile malaria analysis ( a Hierarchical clustering heatmap showing IgG reactivity to each of the 35 PfEMP-1 variants in 680 subjects at enrollment (May 2011 healthy baseline). Clustering was performed using the Ward.D method and the Pearson distance metric. AU refers to arbitrary concentration units, which was calculated by fitting data to a dilutional standard curve of pooled hyper-immune plasma from malaria-exposed Malian adults. b-c IgG reactivity obtained at May 2011 healthy baseline versus age for each PfEMP-1 variant (solid lines) or grouped by CIDR domain class (dashed lines) with loess fit curves and 95% confidence intervals. Control antigens shown as dotted colored lines. d Linear portion of plot in b (age range 3 months to 8 years) with linear fit curves and 95% confidence intervals (see Table S2). For comparison, regression line for all variants together is represented by the black dotted line.

421
IgG reactivity specific to PfEMP-1 variants was determined for 60 children ages 6 months to 8 years (see