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Comprehensive analysis of antibody responses to Plasmodium falciparum erythrocyte membrane protein 1 domains
Vaccine xxx (2018) xxx–xxx
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Comprehensive analysis of antibody responses to Plasmodium falciparum erythrocyte membrane protein 1 domains Bernard N. Kanoi a, Hikaru Nagaoka a, Masayuki Morita a, Michael T. White b, Nirianne M.Q. Palacpac c, Edward H. Ntege a, Betty Balikagala a, Adoke Yeka d,e, Thomas G. Egwang e, Toshihiro Horii c, Takafumi Tsuboi a,⇑, Eizo Takashima a,⇑ a
Division of Malaria Research, Proteo-Science Center, Ehime University, Matsuyama, Ehime 790-8577, Japan Unit of Malaria: Parasites and Hosts, Department of Parasites and Insect Vectors, Pasteur Institute, France Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, Suita 565-0871, Japan d Makerere University School of Public Health, Kampala, Uganda e Med Biotech Laboratories, Plot 4-6 Bell Close, Port Bell Road Luzira, Kampala, Uganda b c
a r t i c l e
i n f o
Article history: Received 2 May 2018 Received in revised form 3 August 2018 Accepted 22 August 2018 Available online xxxx Keywords: PfEMP1 Plasmodium falciparum Malaria Naturally acquired immunity Vaccine Uganda
a b s t r a c t Acquired antibodies directed towards antigens expressed on the surface of merozoites and infected erythrocytes play an important role in protective immunity to Plasmodium falciparum malaria. P. falciparum erythrocyte membrane protein 1 (PfEMP1), the major parasite component of the infected erythrocyte surface, has been implicated in malaria pathology, parasite sequestration and host immune evasion. However, the extent to which unique PfEMP1 domains interact with host immune response remains largely unknown. In this study, we sought to comprehensively understand the naturally acquired antibody responses targeting different Duffy binding-like (DBL), and Cysteine-rich interdomain region (CIDR) domains in a Ugandan cohort. Consequently, we created a protein library consisting of full-length DBL (n = 163) and CIDR (n = 108) domains derived from 62-var genes based on 3D7 genome. The proteins were expressed by a wheat germ cell-free system; a system that yields plasmodial proteins that are comparatively soluble, intact, biologically active and immunoreactive to human sera. Our findings suggest that all PfEMP1 DBL and CIDR domains, regardless of PfEMP1 group, are targets of naturally acquired immunity. The breadth of the immune response expands with children’s age. We concurrently identified 10 DBL and 8 CIDR domains whose antibody responses were associated with reduced risk to symptomatic malaria in the Ugandan children cohort. This study highlights that only a restricted set of specific domains are essential for eliciting naturally acquired protective immunity in malaria. In light of current data, tandem domains in PfEMP1s PF3D7_0700100 and PF3D7_0425800 (DC4) are recommended for extensive evaluation in larger population cohorts to further assess their potential as alternative targets for malaria vaccine development. Ó 2018 Elsevier Ltd. All rights reserved.
1. Introduction Naturally acquired immunity to Plasmodium falciparum malaria is characterized by a decrease in disease severity over several years after repeated infections. Antibodies directed against the variant surface antigens (VSAs) expressed on the surface of infected ery-
Abbreviations: WGCFS, wheat germ cell-free system; PPE, potential protective efficacy; DBL, duffy binding-like domain; CIDR, cysteine-rich interdomain regions; VSAs, variable surface antigens. ⇑ Corresponding authors. E-mail addresses: [email protected] (T. Tsuboi), takashima. [email protected] (E. Takashima).
throcytes have been suggested to be key targets of malaria immunity in people living in malaria-endemic regions [1–4] hence plausible targets of protective antibodies in malaria [4]. P. falciparum erythrocyte membrane protein 1 (PfEMP1) forms the major parasite component of the infected erythrocyte VSAs. PfEMP1s are encoded by 62 var genes per haploid genome and can be classified into groups A–E based on their chromosomal location [5]. The genes are mutually expressed at the individual parasite level [6]. Structurally, PfEMP1 has a large ectodomain consisting of multiple Duffy binding–like (DBL) and cysteine-rich interdomain regions (CIDR) that constitute the adhesive molecules. The DBL and CIDR extracellular domains are classified into several
https://doi.org/10.1016/j.vaccine.2018.08.058 0264-410X/Ó 2018 Elsevier Ltd. All rights reserved.
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major types (DBL; ɑ, b, c, d, e, f, and x, and CIDR; a, b, c, and d) and up to 147 subtypes [5]. The domains, which sometimes exist in combination as short tandem domain cassettes (DCs) of particular subtypes, mediate interactions with a variety of human cell surface receptors [5]. For instance, DBLb domains of PfEMP1 in groups A, B, and C bind intercellular adhesion molecule 1 (ICAM-1) [7–9]; group A PfEMP1 CIDRa domains bind endothelial protein C receptor (EPCR) [10,11]; and some group B and C PfEMP1 contain CIDRa domains that bind CD36 [12,13]. PfEMP1s extracellular domains are highly immunogenic and have been implicated in all forms of symptomatic and complicated malaria and in immune evasion. ICAM-1-binding DBLb domains found in PfEMP1s group A, B and C have been associated with risk of cerebral malaria [6] while individuals with uncomplicated malaria tend to have a wider breadth of antibodies against group A and B PfEMP1s [14]. Although CIDR domains are more complex with huge sequence diversity [10], expression of the EPCR binding subset of PfEMP1 CIDRa1 has been associated with severe malaria [6,15–17]. DC8 (DBLa2-CIDRa1.1-DBLb12-DBLc4/6) and DC13 (DBLa1.7-CIDRa1.4) found in group A PfEMP1 proteins have been implicated in the pathogenesis of cerebral malaria [18]. All these indicate that PfEMP1s have a defined role in malaria immunity and pathogenesis, providing a strong rationale that targeting these proteins may contribute to attenuating the disease. However, with the exception of var2csa in pregnancy malaria [19], the extent to which different individual PfEMP1 domains or DCs interact with host immune response and their potential as vaccine targets remain largely unknown. Several immuno-epidemiological studies examining the association between levels of antibodies and the risk of clinical malaria have pointed to an important role of P. falciparum antigens [6,20,21]. However, challenges associated with expression of the PfEMP1 cysteine-rich extracellular domains as well as complex Duffy binding–like folds [22] have made such screening for PfEMP1 a daunting task [20,23]. It has recently been demonstrated that wheat germ cell-free system (WGCFS), a robust eukaryotic alternative for expressing plasmodial proteins, overcomes limitations related to expression of functional recombinant plasmodial proteins in heterologous expression systems [reviewed in 24–27]. In this study, we leveraged this WGCFS platform to express a PfEMP1-domains-targeted genome-wide protein library that was assayed in an aqueous AlphaScreen system, a platform ideal for detection of protein-tertiary-structure-dependent antibody immunoreactions [28], to comprehensively characterize immunoreactivity of samples obtained from residents of a malaria hyperendemic region. We observed a high degree of antibody immunoreactivity to the recombinant proteins. Antibodies levels to several PfEMP1 domains were associated with reduced risk to symptomatic malaria in both children and young adults. The findings suggest that immune response to PfEMP1s is important in naturally acquired immunity and could be targeted as part of a strategy to prevent falciparum malaria infections.
2. Materials and methods 2.1. Study design and ethical statement Serum samples were collected in a prospective study of 66 nonvaccinated participants aged 6–20 years residing in Lira Municipality, Uganda; a region with perennial holoendemic malaria. The study, conducted in compliance with the International Conference on Harmonisation Good Clinical Practices, the Declaration of Helsinki, has been extensively described elsewhere [29–33]. For this study, a symptomatic malaria episode was defined as having fever >37.5 °C and asexual parasitemia of 2500/ml of blood [34] but
with no sign of complicated disease. All the individuals enrolled in this study were blood-smear negative at sampling [29]. Use of residual blood samples for this study was reviewed and approved by Lacor Hospital Institutional Research and Ethics Committee (LHIREC 023/09/13) and Uganda National Council for Science and Technology (HS1403) in Uganda. Use of 10 Thai malaria naïve sera from healthy adults in malaria-free Bangkok who had no history of travelling outside the region was approved by the Ethics Committee of the Thai Ministry of Public Health and the Institutional Review Board of Faculty of Medicine, Ramathibodi Hospital, Mahidol University (ID: 09-46-10) [35]. Written informed consent was obtained from all study participants and/or their parents or guardians. Approval to conduct the study in Japan was obtained from RIMD, Osaka University and Ehime University. 2.2. Production of P. falciparum parasite protein library We generated a protein library consisting of 271 PfEMP1 domains derived from 62 var genes based on 3D7 strain [5]. Specifically the library consisted of 163 DBL domains (DBLa (n = 60), DBLb (n = 14), DBLb2 (n = 3), DBLd (n = 50), DBLe (n = 14), DBLc (n = 14), DBLpam (n = 1), DBLpam2 (n = 1), DBLpam3 (n = 1), DBL2 (n = 4), DBLc2 (n = 1)) and 108 CIDR domains (CIDRa (n = 54), CIDRb (n = 35), CIDRd (n = 2), CIDRc (n = 16), CIDRpam (n = 1)) [5]. The transcription template was prepared in vitro as previously described [21]. In brief, the 271 domains were amplified from genomic DNA of P. falciparum 3D7 strain by polymerase chain reaction (PCR) using Primestar DNA polymerase (Takara Bio, Kusatsu, Japan) and domain specific primers (Table S1), and cloning into pEU vector (CellFree Sciences, Matsuyama, Japan) with In-Fusion HD Cloning Kit (Takara Bio, Kusatsu, Japan). The Nterminus mono-biotinylated recombinant proteins were expressed by the WGCFS as previously described [36]. Briefly, 1 ll (50 ng) WGCFS-expressed BirA was added to the translation lower layer and 500 nM D-biotin (Nacalai Tesque, Kyoto, Japan) added to both the translation and substrate upper layers. In vitro transcription and cell-free protein synthesis were carried out using GenDecoder1000 robotic protein synthesizer (CellFree Sciences). Successful expression of all domains as mono-biotinylated proteins was confirmed by Western blot analysis by probing with HRP conjugated streptavidin [36]. 2.3. Quantitation of PfEMP1-specific antibodies by AlphaScreen Human serum antibody levels to WGCFS-expressed PfEMP1 domains were quantified by AlphaScreen [36]. Briefly, the protocol, conducted in 384-well OptiPlate microtiter plates (PerkinElmer Life and Analytical Science, Boston, MA), was automated using JANUS Automated Workstation (PerkinElmer). The translation mixture (0.1 ml) containing the biotinylated recombinant P. falciparum proteins was diluted 50-fold (5 ml), mixed with 10 ll of 4000-fold diluted Ugandan sera in reaction buffer (100 mM TrisHCl [pH 8.0], 0.01% [v/v] Tween-20 and 0.1% [w/v] bovine serum albumin) and incubated for 30 min at 26 °C to form a proteinantibody (IgG) complex. Suspension of streptavidin-coated donor-beads (PerkinElmer) and acceptor-beads (PerkinElmer) conjugated with protein G (Thermo Scientific, Waltham, MA) in 10 ll reaction buffer was added to a final concentration of 12 lg/ml for both beads. The mixture was incubated in the dark for 1 h to allow optimal binding of the donor and acceptor-beads to biotinylated protein and human antibody, respectively. Upon excitation, the formed protein-antibody complex emitted a luminescence signal at 620 nm, detected by EnVision plate reader (PerkinElmer) and captured as raw AlphaScreen Counts (AlphaScreen-Countsraw). WGCFS translation mixture without mRNA template was included in each plate as a negative control. Each assay plate contained a
Please cite this article in press as: Kanoi BN et al. Comprehensive analysis of antibody responses to Plasmodium falciparum erythrocyte membrane protein 1 domains. Vaccine (2018), https://doi.org/10.1016/j.vaccine.2018.08.058
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standard curve of biotinylated rabbit IgG. This enabled standardisation between plates using a 5-parameter logistic standard curve. This standardised AlphaScreen-Countsraw was presented as AlphaScreen Counts. AlphaScreen assays were conducted in a randomised manner to avoid experimental bias. A seropositivity cut-off was set as half the lowest non-negative value from of the assayed samples [37]. Proteins were defined as reactive if more than 10% of the volunteers had levels above the seropositivity cut-off [37]. Immunoreactivity (AlphaScreen Counts) was compared between the 66 serum samples only for the same protein fragment, but not across the 271 protein fragments. 2.4. Statistical analysis Antibody associations with age and clinical outcome were examined by Mann–Whitney U test or Kruskal-Wallis with Bonferroni-Dunn as post hoc test. For analysis of effects of an individual antigen, individuals with AlphaScreen Counts above the population median for that antigen were categorized as ‘High Responders’ while the rest as ‘Low Responders’. Kaplan–Meier was used to generate curves for time-to-first clinical episode. Risk of acquiring clinical malaria was assessed using Cox proportional hazard model. Although some children had multiple episodes of parasitemia or symptomatic malaria, we considered only the time-to-first symptomatic episode. This was akin to previous approaches used in this type of studies [21,38–40]; and analysis based on multiple episodes was precluded because of low incidence. Potential protective efficacy (PPE) was computed as 1- hazard ratio (PPE% = (1 HR) 100%). Multivariate survival analysis while adjusting for confounders focused on bed-net use and age as a categorical variable (6–10 years, 11–15 years and 16– 20 years). For sensitivity analysis, the data was also analyzed with modified Poisson Regression Model for prospective study with binary outcome associating High Responders vs. Low Responders to asymptomatic or symptomatic malaria. Analysis while adjusting for different antibody responses as covariates was limited by their highly correlated nature. p-value < 0.05 was considered statistically significant without correction for multiple comparisons. Because of the large number of antigens tested (n = 271), the clinical outcome data was randomly permutated 1000 times amongst
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all participants to assess the number of associations that expected to be significant whilst still accounting for the correlated structure of the data. All analysis was by R software (Version 3.2.3; R Foundation for Statistical Computing) or Prism 6 (GraphPad Software Inc., La Jolla, CA). 3. Results 3.1. WGCFS expresses quality PfEMP1 proteins In this study, we established a PfEMP1 library consisting of 271 domains derived from 3D7-strain sequence, whose genome sequence is a broad representative of West African isolates [41]. All 163 DBL and 108 CIDR domains were successfully expressed by WGCFS [24]. The DBL and CIDR domains showed strong immunoreactivity with sera from malaria-exposed volunteers compared to the pooled sera from 10 malaria-naïve individuals (Fig. S1). Immunoreactivity to sera was observed in all PfEMP1 domains (Fig. 1A). Seroprevalence varied widely between DBL domains (12–100%) and CIDR domains (22–92%) (Mann-Whitney test, p = 0.022; Fig. 1A), with no difference among PfEMP1 groups (Fig. S2). This could be due to high level of exposure to these abundantly expressed proteins or cross-reactivity among the conserved regions of the domains [4]. 3.2. Anti-PfEMP1 antibody repertoire increased with age The probability of an individual responding to a specific domain increased with age for both CIDR and DBL with individuals aged 16–20-year-olds, but not 6–10 or 11–15-year-olds, responding significantly more to CIDR than DBL domains (Fig. 1B). Magnitude of antibody responses increased with age except for 16.6% (45/271) of the domains that did not associate with age and seems to have been acquired early in life and remaining stable with age (Table S1). The overall breadth of immune response associated with age, with serum samples from young adults (16 years) recognizing more PfEMP1 domains (Fig. 2A, p < 0.001) potentially due to increasing yearly exposure to P. falciparum antigens hence expanding the antibody repertoire. The breadth of immune
Fig. 1. Seroprevalence of antibodies to 271 PfEMP1 domains in Uganda. (A) Antibodies immunoreactivity to CIDR (n = 108) are represented in green while DBL (n = 163) are in blue. The dashed line indicates the protein immunoreactivity cut-off point, set at 10% of volunteers. The horizontal line in each group denotes the overall median per group (p = 0.022). (B) Proportion of individuals according to age groups (n = 22 per age group) whose antibodies recognized a given CIDR domain (green columns) or DBL domains (blue columns). A total of 163 DBL and 108 CIDR domains were tested. Box plots illustrate medians with 25th and 75th and whiskers for 10th and 90th percentiles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Kanoi BN et al. Comprehensive analysis of antibody responses to Plasmodium falciparum erythrocyte membrane protein 1 domains. Vaccine (2018), https://doi.org/10.1016/j.vaccine.2018.08.058
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Fig. 2. Breadth of the antibody response per person at the time of enrolment. (A) The number of seropositive antigens in each individual is shown, with box-plots indicating the median and ranges for each age cohort. Seropositivity was set at the limit of detection of the assay, equal to half the lowest non-negative AlphaScreen Counts value. Statistical difference was assessed using the Kruskal-Wallis test (p < 0.001); with Dunn’s test as post hoc test, p < 0.01 between age 6–10 and other age groups (11–15, and 16–20 years). (B) There was no statistical difference between individuals who developed symptomatic malaria and those who did not experience symptomatic malaria.
response did not significantly associate with clinical malaria outcome (Fig. 2B, p = 0.17).
and bed-net use. The trends were similar to primary analysis and did not alter the interpretation of Table 1. The results are summarized in Fig. S6 and Table S2.
3.3. Identification of antibody responses associated with protection from clinical malaria
4. Discussion
Malaria morbidity was monitored in the study, which enabled us to analyze whether antibody levels to the PfEMP1 domains were associated with reduced risk of developing symptomatic disease. Based on Cox hazard regression model analysis, the DBL and CIDR domains were ranked according to their respective PPE (Table S1). We observed a significant association between high antibody levels to 18 (10 DBL and 8 CIDR) domains and reduced risk of having a symptomatic malaria episode (Hazard ratio (HR) < 1, p < 0.05; Fig. 3A and Table 1). For instance, a group B PfEMP1 PF3D7_0300100 CIDRb1 (Dm#21, Table 1), had an unadjusted PPE of 69.84% (CI 11.68–89.70%, p = 0.029). A DBLa of PF3D7_0733000 (Dm#150) with a seroprevalence of only 13%, showed unadjusted PPE of 100% (CI 30.3–100%, P = 0.025). Representative Kaplan and Meir plots are presented in Fig. S3. Generally, the evaluated domains displayed a negative significant correlation between seroprevalence and PPE (Spearman’s rho, CIDR -0.31, p < 001; DBL -0.44, p < 0.001; Fig. S4A and B). Although CIDR domains had significantly higher seroprevalence (Fig. 1A), when ranked according to the corresponding PPE (%), no selection bias was observed towards either CIDR or DBL domains. No antigen remained significant after adjustment for age and bed-net use (Fig. 3B). Randomly permuting the clinical outcome data resulted in similar associations to those in Fig. 3B. Of 1000 permutations the most frequent number of significant associates expected by chance was 0/271, with the median number being 2/271 (Supplementary Fig. S5). To test the robustness of this analysis, we subjected the same data to Modified Poisson regression analysis with malaria as a binary outcome, with and without adjustment for the effects of age
Residents of malaria endemic regions quickly develop immunity to life threatening malaria complications after repeated exposure [1]. The basis of this immunity appears to be, at least in part, due to acquisition of antibodies to polymorphic parasite antigens expressed on the surface of infected erythrocytes, referred to as variant surface antigens (VSA). Specifically, PfEMP1s have been proposed to play a key role in mediating this protective immunity [42]. However, due to the large number of antigens potentially expressed on the infected erythrocyte surface, it has been difficult to determine a pattern of PfEMP1-specific antibody responses and identify protein or domain targets of protective immunity. Therefore, in this study, we sought to comprehensively determine antibody responses to all DBL and CIDR domains derived from the P. falciparum 3D7 genome. We observed that individuals in this high-transmission region had achieved substantial levels of antibodies against all 271 PfEMP1 proteins. The high degree of immunoreactivity could at least be attributed to high quality proteins synthesized using the robust eukaryotic WGCFS. Although no structural analysis of the expressed antigens was conducted, WGCFS generated plasmodial proteins are comparatively more soluble, intact, biologically active and immunoreactive to human sera than those expressed by commonly used E. coli-based systems [24–27], suggesting the proteins possess significant levels of native conformations. In addition, application of AlphaScreen immunoscreening system offers a high-throughput platform for antibody quantification in a homogeneous aqueous environment with no antigen conjugation to a matrix or beads. This provides optimal interactions between recombinant PfEMP1 domains and human antibodies [36]. It is also
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Fig. 3. Associations between antibody levels and risk of P. falciparum clinical malaria. (A) Unadjusted association between antibody levels and risk of P. falciparum clinical malaria for each of the 271 antigens tested in the Ugandan cohort. Antigens are ranked – top to bottom – by the strength of their potential protective efficacy (PPE). PPE (percentage) for each antigen was derived from the hazard ratio (HR) calculated by the unadjusted Cox-regression hazard model analysis [comparing children with high (High Responders) versus low (Low Responders) antibody responses]. Black dots indicate the percentage protection, and the error bars indicate the 95% confidence interval. The red vertical line represents PPE of 0% (i.e. HR = 1). (B) After adjustment for age and bed-net use, none of the antigens remained significant responses. Complete list of the data for PPE (and 95% confidence interval) is provided in Table S1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 1 Association between antibody responses to PfEMP1 and protection from clinical malaria. Unadjusted PPEa Library ID
Gene ID
Age and Bed-net use adjusted PPE
Domain
PfEMP1 group
Seroprevalence (%)
PPE (%)
95% CI
p-value
PPE %
95% CI
p-value
CIDR domains Dm#21 PF3D7_0300100 Dm#119 PF3D7_0700100 Dm#239 PF3D7_1219300 Dm#102 PF3D7_0617400 Dm#158 PF3D7_0800100 Dm#49 PF3D7_0412900 Dm#17 PF3D7_0223500 Dm#15 PF3D7_0223500 Dm#71 PF3D7_0425800
CIDRb1 CIDRa3.1 CIDRa3.4 CIDRa2.1 CIDRb1 CIDRc2 CIDRb1 CIDRa3.4 CIDRa1.6
B B B C B C B B A
34.8 50.0 33.3 65.2 42.4 45.5 51.5 62.1 65.2
69.84 66.01 65.88 63.37 61.60 58.62 57.89 57.31 57.07
(11.68–89.70) (17.93–85.92) (0.12–88.35) (11.54–84.83) (3.16–84.77) (0.14–82.86) (1.53–81.99) (0.15–81.75) ( 0.37 to 81.63)
0.029 0.016 0.050 0.026 0.043 0.050 0.046 0.050 0.051
59.68 51.42 37.62 39.69 36.88 16.67 29.82 31.33 40.47
( ( ( ( ( ( ( ( (
29.00 to 87.40) 25.10 to 81.13) 117.28 to 82.09) 64.44 to 77.88) 83.87 to 78.33) 140.35 to 71.11) 81.68 to 72.89) 73.88 to 72.88) 44.24 to 75.43)
0.126 0.135 0.459 0.323 0.399 0.736 0.466 0.428 0.251
DBL domains Dm#150 Dm#54 Dm#72 Dm#266 Dm#242 Dm#73 Dm#169 Dm#137 Dm#110 Dm#118 Dm#157
DBLa0.16 DBLa0.1 DBLb3 DBLc12 DBLa0.7 DBLb3 DBLd1 DBLc9 DBLd4 DBLa0.1 DBLd1
B C A A B/C A B/A B/C B/A B B
13.6 28.8 28.8 34.8 33.3 57.6 57.6 39.4 66.7 39.4 63.6
100.00 71.56 70.69 68.12 66.66 66.01 65.53 65.46 65.10 64.96 57.08
(30.30–100.00) (4.60–91.52) (1.67–91.26) (6.65–89.11) (2.37–88.61) (17.93–85.92) (16.75–85.73) (7.42–87.11) (15.75–85.54) (6.09–86.93) ( 0.40–81.65)
0.025 0.042 0.047 0.037 0.045 0.016 0.018 0.035 0.019 0.037 0.051
100.00 51.80 58.23 40.01 43.57 37.28 57.51 44.38 44.74 47.13 29.55
( ( ( ( ( ( ( ( ( ( (
40 to 100.00) 73.85 to 86.64) 48.73 to 88.27) 99.67 to 81.98) 89.30 to 83.18) 71.23 to 77.03) 13.98 to 84.16) 66.18 to 81.39) 48.33 to 79.42) 53.23 to 81.76) 81.89 to 72.72)
0.119 0.265 0.178 0.405 0.354 0.363 0.089 0.293 0.239 0.240 0.469
PF3D7_0733000 PF3D7_0420700 PF3D7_0425800 PF3D7_1300300 PF3D7_1240300 PF3D7_0425800 PF3D7_0800300 PF3D7_0712400 PF3D7_0632500 PF3D7_0700100 PF3D7_0800100
In bold are p-values of antigens with responses that were statistically significant. a Potential Protective Efficacy (PPE) was calculated using hazard ratios (HR) generated by the Cox proportional hazards model comparing individuals with high versus low AlphaScreen Counts; (PPE% = (1 HR) 100%). After adjustments for confounders (age and bed-net use), no PPE remained statistically significant.
possible that the broad immunoreactivity was due to crossreactivity among the PfEMP1 domains. The majority of the domains exhibit some levels of amino-acid sequence similarity with others in the same group [5].
Antibodies to 18 (10 DBL and 8 CIDR) domains were associated with reduced risk to clinical malaria (Table 1), although these were not statistically significant after correction for age and bed net use. We observed a significant negative correlation between seropreva-
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lence and protective immunity. This was a surprising finding and may suggest; (i) that antibodies to commonly encountered domains were not as protective as antibodies to less commonly encountered domains, a potential product of selection pressure; (ii) the short-lived nature of protective antibodies or; (iii) the differential immunogenicity of the PfEMP1 domains. This further indicates the complexity of naturally acquired protective immunity and suggests that not all PfEMP1 proteins are targets of protective antibodies in this cohort. The identification of the 18 domains, that included previously well-evaluated domains that are currently under consideration as vaccine or anti-disease therapeutic targets [7], is very reassuring and brings into focus new domains that should be further investigated. For instance, higher antibody levels to PF3D7_0700100 CIDRa3.1 and DBLa0.1 domains (Fig. 4A), with prevalence of 50.0% and 39.4% respectively, were associated with lower risk of developing symptomatic malaria. This mirrored a similar observation in Ghanaian and Sudanese children where higher levels of antibodies to a recombinant peptide derived from a conserved epitope of the PF3D7_0700100 DBLa domain were associated with clinical protection [43,44]. One of the key findings in this paper is the observation that higher serum levels of antibodies to 4 EPCR-binding CIDRa domains (PF3D7_0700100, PF3D7_0425800, PF3D7_0223500 and PF3D7_0617400) were associated with lower risk of developing symptomatic malaria. This is relevant since it was previously observed that antibody to these (CIDRa) polymorphic cysteine-rich domains are associated with protective immunity in Tanzanian children [45]. Although it’s unclear how the protective immunity is attained, it was demonstrated that despite their huge sequence diversity, CIDRa1 are structurally and functionally conserved for binding to EPCR [10,11]; an interaction that could be blocked by antibodies in individuals exposed to natural infection in Tanzania [10]. It would be important to further understand
the potential role of these selected CIDR domains in complicated malaria. The data on PfEMP1 DBL domains reported here is consistent with that reported in Papua New Guinea, Tanzania and Kenya where antibody responses increased with age [46–48]. Furthermore, studies with PfEMP1 DBLa1.5 antibodies (variant HB3var6) or DBLa1.8 antibodies (variant TM284var1) showed broad crossreactivity against heterologous parasite strains with the same rosetting phenotype, including clinical isolates from four subSaharan African countries [23]. It therefore remains possible that, within the hypervariable regions, there exists antigenically conserved linear or structural epitopes shared by subsets of PfEMP1 types that are the targets of strain-transcending antibodies [10,23]. Analysis of multiple P. falciparum genomes has shown that several ICAM-1-binding PfEMP1s also interact with EPCR allowing infected erythrocytes to synergistically bind both receptors [6]. In our study, antibodies to a severe-malaria-associated DC4 family member, PF3D7_0425800 CIDRa1.6-DBLb3-DBLb3 domains (predicted as double ICAM-1 and EPCR binder) [7,49], significantly associated with clinical protection (Fig. 4B: i & ii). PF3D7_0425800 DC4 (DBLa1.4-CIDRa1.6-DBLb3-DBLb3) is preferentially expressed in plasmodium parasites associated with severe childhood malaria [7,49,50]. PF3D7_0425800 DBLb3 elicits adhesion-inhibitory antibodies that broadly inhibit ICAM-1 binding and are cross-reactive to DC4-containing PfEMP1 from genetically distant parasite isolates [51]. Specifically, the monoclonal antibodies to the ICAM-1 binding site of PF3D7_0425800 DBLb, convex surface of DBLb3_D4, specifically blocks interaction between ICAM-1 and DBLb3_D4 domains from genetically distant parasite isolates, suggesting that the monoclonal antibodies act by occluding the ICAM-1 binding surface [51]. Our study suggests that homologues of these DC4 domains are present in P. falciparum field isolates and that only a restricted set of specific domains are essential for
Fig. 4. Antibodies to tandem domains derived from PfEMP1 (A (i). PF3D7_0700100 and B (i). PF3D7_0425800) associated with clinical protection. The numbers below the primary structure represent p-values to PPE of each domain. Individuals who did not develop symptomatic malaria episode had significantly higher level of antibodies to A (ii). PF3D7_0700100 and B (ii). PF3D7_0425800, Statistical difference was assessed using the Mann-Whitney U test (p < 0.001).
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eliciting naturally acquired, protective immunity in malaria. In light of the current understanding on structurally conserved nature of some PfEMP1s [6,10,23,52], this work advances the selected domains for further evaluation as biomarkers of malaria immunity or vaccine targets. Indeed, a strain-specific PFEMP1 expressed in sporozoites has recently been identified and proposed as a vaccine target [53]. There is also a need to determine the mode of action of the protective antibodies to the selected PfEMP1 proteins, and identify the corresponding receptors on the human host cells. Considering the extensive genetic diversity that has been reported for PfEMP1, a major limitation is that we only assessed a well-established laboratory strain (3D7) in the assays. This parasite line may not recall all antibody response patterns of different parasite variants circulating in the population. Nevertheless, since none of the children in the study was likely to have been infected with 3D7, and yet many developed antibodies reacting with recombinant DBL and CIDR domains based on this genome, there must be widespread homologous - conserved - domains encoded from different genomes circulating in the population. In the future, we hope to expand this screening to cover larger sample sizes from multiple field sites and parasites expressing different PfEMP1 variants. In conclusion, our study demonstrates that WGCFS coupled with AlphaScreen is a powerful tool for mapping immune patterns induced by PfEMP1 proteins. We identified 18 DBL and CIDR domains associated with clinical protection. Identification of DC4 PF3D7_0425800 DBLb3 that were previously associated with severe childhood malaria and, in this study, with antibodies protective from symptomatic uncomplicated malaria, highlights the importance of specific PfEMP1s. This implies that, although PfEMP1s have been described as highly diverse, specific domains are central in naturally acquired protective immunity. Thus, the data supports the feasibility of developing a vaccine against malaria by sequentially identifying, characterizing and targeting to inhibit the function of these domains. We recommend PF3D7_0425800 DBLb3 as well as other selected domains to be extensively evaluated in larger population cohorts to assess their potential as alternative targets for malaria vaccine development. Conflict of interest statement The authors have no conflict of interest to declare. Acknowledgments We are grateful to Dr. Michael F. Duffy (University of Melbourne, Australia) for critical reading of the manuscript. We appreciate the study volunteers at Lira, Northern Uganda; the research teams from Med Biotech Laboratories, Lira Medical Center, Uganda, and Research Institute for Microbial Diseases, Osaka University for their technical assistance in obtaining the field samples. We thank adult volunteers in Thailand who participated in the study, and also acknowledge Dr. Rachanee Udomsangpetch for supporting the study. This work was supported in part by Strategic Promotion of International Cooperation to Accelerate Innovation in Africa by MEXT, MEXT KAKENHI (JP23117008) and JSPS KAKENHI (JP25460517, JP26253026, JP26670202, JP26860279, JP15H05276, JP16K15266, JP18H02651) in Japan. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.vaccine.2018.08. 058.
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References [1] Doolan DL, Dobano C, Baird JK. Acquired immunity to malaria. Clin Microbiol Rev 2009;22:13–36. [2] McGregor IA. Studies in the acquisition of immunity of Plasmodium falciparum infections in Africa. Trans R Soc Trop Med Hyg 1964;58:80–92. [3] Sabchareon A, Burnouf T, Ouattara D, Attanath P, Bouharoun-Tayoun H, Chantavanich P, et al. Parasitologic and clinical human response to immunoglobulin administration in falciparum malaria. Am J Trop Med Hyg 1991;45:297–308. [4] Chan JA, Fowkes FJ, Beeson JG. Surface antigens of Plasmodium falciparuminfected erythrocytes as immune targets and malaria vaccine candidates. Cell Mol Life Sci 2014;71:3633–57. [5] Rask TS, Hansen DA, Theander TG, Gorm Pedersen A, Lavstsen T. Plasmodium falciparum erythrocyte membrane protein 1 diversity in seven genomes– divide and conquer. PLoS Comput Biol 2010;6. [6] Lennartz F, Adams Y, Bengtsson A, Olsen RW, Turner L, Ndam NT, et al. Structure-guided identification of a family of dual receptor-binding PfEMP1 that is associated with cerebral malaria. Cell Host Microbe 2017;21:403–14. [7] Bengtsson A, Joergensen L, Rask TS, Olsen RW, Andersen MA, Turner L, et al. A novel domain cassette identifies Plasmodium falciparum PfEMP1 proteins binding ICAM-1 and is a target of cross-reactive, adhesion-inhibitory antibodies. J Immunol 2013;190:240–9. [8] Howell DP, Levin EA, Springer AL, Kraemer SM, Phippard DJ, Schief WR, et al. Mapping a common interaction site used by Plasmodium falciparum Duffy binding-like domains to bind diverse host receptors. Mol Microbiol 2008;67:78–87. [9] Janes JH, Wang CP, Levin-Edens E, Vigan-Womas I, Guillotte M, Melcher M, et al. Investigating the host binding signature on the Plasmodium falciparum PfEMP1 protein family. PLoS Pathog 2011;7:e1002032. [10] Lau CK, Turner L, Jespersen JS, Lowe ED, Petersen B, Wang CW, et al. Structural conservation despite huge sequence diversity allows EPCR binding by the PfEMP1 family implicated in severe childhood malaria. Cell Host Microbe 2015;17:118–29. [11] Turner L, Lavstsen T, Berger SS, Wang CW, Petersen JE, Avril M, et al. Severe malaria is associated with parasite binding to endothelial protein C receptor. Nature 2013;498:502–5. [12] Hsieh FL, Turner L, Bolla JR, Robinson CV, Lavstsen T, Higgins MK. The structural basis for CD36 binding by the malaria parasite. Nat Commun 2016;7:12837. [13] Robinson BA, Welch TL, Smith JD. Widespread functional specialization of Plasmodium falciparum erythrocyte membrane protein 1 family members to bind CD36 analysed across a parasite genome. Mol Microbiol 2003;47:1265–78. [14] Duffy MF, Noviyanti R, Tsuboi T, Feng ZP, Trianty L, Sebayang BF, et al. Differences in PfEMP1s recognized by antibodies from patients with uncomplicated or severe malaria. Malar J. 2016;15:258. [15] Petersen JE, Bouwens EA, Tamayo I, Turner L, Wang CW, Stins M, et al. Protein C system defects inflicted by the malaria parasite protein PfEMP1 can be overcome by a soluble EPCR variant. Thromb Haemost 2015;114:1038–48. [16] Mkumbaye SI, Wang CW, Lyimo E, Jespersen JS, Manjurano A, Mosha J, et al. The severity of Plasmodium falciparum infection is associated with transcript levels of var genes encoding endothelial protein C receptor-binding P. falciparum erythrocyte membrane protein 1. Infect Immun 2017:85. [17] Bull PC, Abdi AI. The role of PfEMP1 as targets of naturally acquired immunity to childhood malaria: prospects for a vaccine. Parasitology 2016;143:171–86. [18] Lavstsen T, Turner L, Saguti F, Magistrado P, Rask TS, Jespersen JS, et al. Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children. Proc Natl Acad Sci U S A 2012;109:E1791–800. [19] Salanti A, Dahlback M, Turner L, Nielsen MA, Barfod L, Magistrado P, et al. Evidence for the involvement of VAR2CSA in pregnancy-associated malaria. J Exp Med 2004;200:1197–203. [20] Travassos MA, Niangaly A, Bailey JA, Ouattara A, Coulibaly D, Laurens MB, et al. Seroreactivity to Plasmodium falciparum erythrocyte membrane protein 1 intracellular domain in malaria-exposed children and adults. J Infect Dis. 2013;208:1514–9. [21] Kanoi BN, Takashima E, Morita M, White MT, Palacpac NM, Ntege EH, et al. Antibody profiles to wheat germ cell-free system synthesized Plasmodium falciparum proteins correlate with protection from symptomatic malaria in Uganda. Vaccine. 2017;35:873–81. [22] Mayer C, Slater L, Erat MC, Konrat R, Vakonakis I. Structural analysis of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) intracellular domain reveals a conserved interaction epitope. J Biol Chem 2012;287:7182–9. [23] Ghumra A, Semblat JP, Ataide R, Kifude C, Adams Y, Claessens A, et al. Induction of strain-transcending antibodies against Group A PfEMP1 surface antigens from virulent malaria parasites. PLoS Pathog 2012;8:e1002665. [24] Arumugam TU, Ito D, Takashima E, Tachibana M, Ishino T, Torii M, et al. Application of wheat germ cell-free protein expression system for novel malaria vaccine candidate discovery. Expert Rev Vaccines 2014;13:75–85. [25] Tsuboi T, Takeo S, Iriko H, Jin L, Tsuchimochi M, Matsuda S, et al. Wheat germ cell-free system-based production of malaria proteins for discovery of novel vaccine candidates. Infect Immun 2008;76:1702–8.
Please cite this article in press as: Kanoi BN et al. Comprehensive analysis of antibody responses to Plasmodium falciparum erythrocyte membrane protein 1 domains. Vaccine (2018), https://doi.org/10.1016/j.vaccine.2018.08.058
8
B.N. Kanoi et al. / Vaccine xxx (2018) xxx–xxx
[26] Ntege EH, Takashima E, Morita M, Nagaoka H, Ishino T, Tsuboi T. Blood-stage malaria vaccines: post-genome strategies for the identification of novel vaccine candidates. Expert Rev Vaccines 2017;16:769–79. [27] Rui E, Fernandez-Becerra C, Takeo S, Sanz S, Lacerda MV, Tsuboi T, et al. Plasmodium vivax: comparison of immunogenicity among proteins expressed in the cell-free systems of Escherichia coli and wheat germ by suspension array assays. Malar J 2011;10:192. [28] Takeda H, Ogasawara T, Ozawa T, Muraguchi A, Jih PJ, Morishita R, et al. Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay. Sci Rep 2015;5:11333. [29] Palacpac NM, Ntege E, Yeka A, Balikagala B, Suzuki N, Shirai H, et al. Phase 1b randomized trial and follow-up study in Uganda of the blood-stage malaria vaccine candidate BK-SE36. PLoS ONE 2013;8:e64073. [30] Egwang TG, Apio B, Riley E, Okello D. Plasmodium falciparum malariometric indices in Apac district, northern Uganda. East Afr Med J 2000;77:413–6. [31] Proietti C, Pettinato DD, Kanoi BN, Ntege E, Crisanti A, Riley EM, et al. Continuing intense malaria transmission in northern Uganda. Am J Trop Med Hyg 2011;84:830–7. [32] Yeka A, Gasasira A, Mpimbaza A, Achan J, Nankabirwa J, Nsobya S, et al. Malaria in Uganda: challenges to control on the long road to elimination: I. Epidemiology and current control efforts. Acta Trop 2012;121:184–95. [33] Okello PE, Van Bortel W, Byaruhanga AM, Correwyn A, Roelants P, Talisuna A, et al. Variation in malaria transmission intensity in seven sites throughout Uganda. Am J Trop Med Hyg 2006;75:219–25. [34] Osier FH, Mackinnon MJ, Crosnier C, Fegan G, Kamuyu G, Wanaguru M, et al. New antigens for a multicomponent blood-stage malaria vaccine. Sci Transl Med 2014;6. 247ra102. [35] Sakamoto H, Takeo S, Takashima E, Miura K, Kanoi BN, Kaneko T, et al. Identification of target proteins of clinical immunity to Plasmodium falciparum in a region of low malaria transmission. Parasitol Int 2018;67:203–8. [36] Matsuoka K, Komori H, Nose M, Endo Y, Sawasaki T. Simple screening method for autoantigen proteins using the N-terminal biotinylated protein library produced by wheat cell-free synthesis. J Proteome Res 2010;9:4264–73. [37] Longley RJ, White MT, Takashima E, Morita M, Kanoi BN, Li Wai Suen CSN, et al. Naturally acquired antibody responses to more than 300 Plasmodium vivax proteins in three geographic regions. PLoS Negl Trop Dis 2017;11:e0005888. [38] Osier FH, Fegan G, Polley SD, Murungi L, Verra F, Tetteh KK, et al. Breadth and magnitude of antibody responses to multiple Plasmodium falciparum merozoite antigens are associated with protection from clinical malaria. Infect Immun 2008;76:2240–8. [39] Rono J, Osier FH, Olsson D, Montgomery S, Mhoja L, Rooth I, et al. Breadth of anti-merozoite antibody responses is associated with the genetic diversity of asymptomatic Plasmodium falciparum infections and protection against clinical malaria. Clin Infect Dis 2013;57:1409–16. [40] Richards JS, Arumugam TU, Reiling L, Healer J, Hodder AN, Fowkes FJ, et al. Identification and prioritization of merozoite antigens as targets of protective
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52] [53]
human immunity to Plasmodium falciparum malaria for vaccine and biomarker development. J Immunol 2013;191:795–809. Volkman SK, Sabeti PC, DeCaprio D, Neafsey DE, Schaffner SF, Milner Jr DA, et al. A genome-wide map of diversity in Plasmodium falciparum. Nat Genet 2007;39:113–9. Bull PC, Lowe BS, Kortok M, Molyneux CS, Newbold CI, Marsh K. Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria. Nat Med 1998;4:358–60. Staalso T, Khalil EA, Elhassan IM, Zijlstra EE, Elhassan AM, Giha HA, et al. Antibody reactivity to conserved linear epitopes of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). Immunol Lett 1998;60:121–6. Dodoo D, Staalsoe T, Giha H, Kurtzhals JA, Akanmori BD, Koram K, et al. Antibodies to variant antigens on the surfaces of infected erythrocytes are associated with protection from malaria in Ghanaian children. Infect Immun 2001;69:3713–8. Magistrado PA, Lusingu J, Vestergaard LS, Lemnge M, Lavstsen T, Turner L, et al. Immunoglobulin G antibody reactivity to a group A Plasmodium falciparum erythrocyte membrane protein 1 and protection from P. falciparum malaria. Infect Immun 2007;75:2415–20. Cham GK, Turner L, Lusingu J, Vestergaard L, Mmbando BP, Kurtis JD, et al. Sequential, ordered acquisition of antibodies to Plasmodium falciparum erythrocyte membrane protein 1 domains. J Immunol 2009;183:3356–63. Dent AE, Nakajima R, Liang L, Baum E, Moormann AM, Sumba PO, et al. Plasmodium falciparum protein microarray antibody profiles correlate with protection from symptomatic malaria in Kenya. J Infect Dis 2015;212:1429–38. Barry AE, Trieu A, Fowkes FJ, Pablo J, Kalantari-Dehaghi M, Jasinskas A, et al. The stability and complexity of antibody responses to the major surface antigen of Plasmodium falciparum are associated with age in a malaria endemic area. Mol Cell Proteomics 2011;10(M111):008326. Tonkin-Hill GQ, Trianty L, Noviyanti R, Nguyen HHT, Sebayang BF, Lampah DA, et al. The Plasmodium falciparum transcriptome in severe malaria reveals altered expression of genes involved in important processes including surface antigen-encoding var genes. PLoS Biol 2018;16:e2004328. Jensen AT, Magistrado P, Sharp S, Joergensen L, Lavstsen T, Chiucchiuini A, et al. Plasmodium falciparum associated with severe childhood malaria preferentially expresses PfEMP1 encoded by group A var genes. J Exp Med 2004;199:1179–90. Lennartz F, Bengtsson A, Olsen RW, Joergensen L, Brown A, Remy L, et al. Mapping the binding site of a cross-reactive Plasmodium falciparum PfEMP1 monoclonal antibody inhibitory of ICAM-1 binding. J Immunol 2015;195:3273–83. Lennartz F, Thomas L, Higgins MK. Towards an anti-disease malaria vaccine. Emerg Top Life Sci 2017;1:539–45. Zanghi G, Vembar SS, Baumgarten S, Ding S, Guizetti J, Bryant JM, et al. A specific PfEMP1 is expressed in P. falciparum sporozoites and plays a role in hepatocyte infection. Cell Rep 2018;22:2951–63.
Please cite this article in press as: Kanoi BN et al. Comprehensive analysis of antibody responses to Plasmodium falciparum erythrocyte membrane protein 1 domains. Vaccine (2018), https://doi.org/10.1016/j.vaccine.2018.08.058
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Comprehensive analysis of antibody responses to Plasmodium falciparum erythrocyte membrane protein 1 domains Bernard N. Kanoi a, Hikaru Nagaoka a, Masayuki Morita a, Michael T. White b, Nirianne M.Q. Palacpac c, Edward H. Ntege a, Betty Balikagala a, Adoke Yeka d,e, Thomas G. Egwang e, Toshihiro Horii c, Takafumi Tsuboi a,⇑, Eizo Takashima a,⇑ a
Division of Malaria Research, Proteo-Science Center, Ehime University, Matsuyama, Ehime 790-8577, Japan Unit of Malaria: Parasites and Hosts, Department of Parasites and Insect Vectors, Pasteur Institute, France Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, Suita 565-0871, Japan d Makerere University School of Public Health, Kampala, Uganda e Med Biotech Laboratories, Plot 4-6 Bell Close, Port Bell Road Luzira, Kampala, Uganda b c
a r t i c l e
i n f o
Article history: Received 2 May 2018 Received in revised form 3 August 2018 Accepted 22 August 2018 Available online xxxx Keywords: PfEMP1 Plasmodium falciparum Malaria Naturally acquired immunity Vaccine Uganda
a b s t r a c t Acquired antibodies directed towards antigens expressed on the surface of merozoites and infected erythrocytes play an important role in protective immunity to Plasmodium falciparum malaria. P. falciparum erythrocyte membrane protein 1 (PfEMP1), the major parasite component of the infected erythrocyte surface, has been implicated in malaria pathology, parasite sequestration and host immune evasion. However, the extent to which unique PfEMP1 domains interact with host immune response remains largely unknown. In this study, we sought to comprehensively understand the naturally acquired antibody responses targeting different Duffy binding-like (DBL), and Cysteine-rich interdomain region (CIDR) domains in a Ugandan cohort. Consequently, we created a protein library consisting of full-length DBL (n = 163) and CIDR (n = 108) domains derived from 62-var genes based on 3D7 genome. The proteins were expressed by a wheat germ cell-free system; a system that yields plasmodial proteins that are comparatively soluble, intact, biologically active and immunoreactive to human sera. Our findings suggest that all PfEMP1 DBL and CIDR domains, regardless of PfEMP1 group, are targets of naturally acquired immunity. The breadth of the immune response expands with children’s age. We concurrently identified 10 DBL and 8 CIDR domains whose antibody responses were associated with reduced risk to symptomatic malaria in the Ugandan children cohort. This study highlights that only a restricted set of specific domains are essential for eliciting naturally acquired protective immunity in malaria. In light of current data, tandem domains in PfEMP1s PF3D7_0700100 and PF3D7_0425800 (DC4) are recommended for extensive evaluation in larger population cohorts to further assess their potential as alternative targets for malaria vaccine development. Ó 2018 Elsevier Ltd. All rights reserved.
1. Introduction Naturally acquired immunity to Plasmodium falciparum malaria is characterized by a decrease in disease severity over several years after repeated infections. Antibodies directed against the variant surface antigens (VSAs) expressed on the surface of infected ery-
Abbreviations: WGCFS, wheat germ cell-free system; PPE, potential protective efficacy; DBL, duffy binding-like domain; CIDR, cysteine-rich interdomain regions; VSAs, variable surface antigens. ⇑ Corresponding authors. E-mail addresses: [email protected] (T. Tsuboi), takashima. [email protected] (E. Takashima).
throcytes have been suggested to be key targets of malaria immunity in people living in malaria-endemic regions [1–4] hence plausible targets of protective antibodies in malaria [4]. P. falciparum erythrocyte membrane protein 1 (PfEMP1) forms the major parasite component of the infected erythrocyte VSAs. PfEMP1s are encoded by 62 var genes per haploid genome and can be classified into groups A–E based on their chromosomal location [5]. The genes are mutually expressed at the individual parasite level [6]. Structurally, PfEMP1 has a large ectodomain consisting of multiple Duffy binding–like (DBL) and cysteine-rich interdomain regions (CIDR) that constitute the adhesive molecules. The DBL and CIDR extracellular domains are classified into several
https://doi.org/10.1016/j.vaccine.2018.08.058 0264-410X/Ó 2018 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kanoi BN et al. Comprehensive analysis of antibody responses to Plasmodium falciparum erythrocyte membrane protein 1 domains. Vaccine (2018), https://doi.org/10.1016/j.vaccine.2018.08.058
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major types (DBL; ɑ, b, c, d, e, f, and x, and CIDR; a, b, c, and d) and up to 147 subtypes [5]. The domains, which sometimes exist in combination as short tandem domain cassettes (DCs) of particular subtypes, mediate interactions with a variety of human cell surface receptors [5]. For instance, DBLb domains of PfEMP1 in groups A, B, and C bind intercellular adhesion molecule 1 (ICAM-1) [7–9]; group A PfEMP1 CIDRa domains bind endothelial protein C receptor (EPCR) [10,11]; and some group B and C PfEMP1 contain CIDRa domains that bind CD36 [12,13]. PfEMP1s extracellular domains are highly immunogenic and have been implicated in all forms of symptomatic and complicated malaria and in immune evasion. ICAM-1-binding DBLb domains found in PfEMP1s group A, B and C have been associated with risk of cerebral malaria [6] while individuals with uncomplicated malaria tend to have a wider breadth of antibodies against group A and B PfEMP1s [14]. Although CIDR domains are more complex with huge sequence diversity [10], expression of the EPCR binding subset of PfEMP1 CIDRa1 has been associated with severe malaria [6,15–17]. DC8 (DBLa2-CIDRa1.1-DBLb12-DBLc4/6) and DC13 (DBLa1.7-CIDRa1.4) found in group A PfEMP1 proteins have been implicated in the pathogenesis of cerebral malaria [18]. All these indicate that PfEMP1s have a defined role in malaria immunity and pathogenesis, providing a strong rationale that targeting these proteins may contribute to attenuating the disease. However, with the exception of var2csa in pregnancy malaria [19], the extent to which different individual PfEMP1 domains or DCs interact with host immune response and their potential as vaccine targets remain largely unknown. Several immuno-epidemiological studies examining the association between levels of antibodies and the risk of clinical malaria have pointed to an important role of P. falciparum antigens [6,20,21]. However, challenges associated with expression of the PfEMP1 cysteine-rich extracellular domains as well as complex Duffy binding–like folds [22] have made such screening for PfEMP1 a daunting task [20,23]. It has recently been demonstrated that wheat germ cell-free system (WGCFS), a robust eukaryotic alternative for expressing plasmodial proteins, overcomes limitations related to expression of functional recombinant plasmodial proteins in heterologous expression systems [reviewed in 24–27]. In this study, we leveraged this WGCFS platform to express a PfEMP1-domains-targeted genome-wide protein library that was assayed in an aqueous AlphaScreen system, a platform ideal for detection of protein-tertiary-structure-dependent antibody immunoreactions [28], to comprehensively characterize immunoreactivity of samples obtained from residents of a malaria hyperendemic region. We observed a high degree of antibody immunoreactivity to the recombinant proteins. Antibodies levels to several PfEMP1 domains were associated with reduced risk to symptomatic malaria in both children and young adults. The findings suggest that immune response to PfEMP1s is important in naturally acquired immunity and could be targeted as part of a strategy to prevent falciparum malaria infections.
2. Materials and methods 2.1. Study design and ethical statement Serum samples were collected in a prospective study of 66 nonvaccinated participants aged 6–20 years residing in Lira Municipality, Uganda; a region with perennial holoendemic malaria. The study, conducted in compliance with the International Conference on Harmonisation Good Clinical Practices, the Declaration of Helsinki, has been extensively described elsewhere [29–33]. For this study, a symptomatic malaria episode was defined as having fever >37.5 °C and asexual parasitemia of 2500/ml of blood [34] but
with no sign of complicated disease. All the individuals enrolled in this study were blood-smear negative at sampling [29]. Use of residual blood samples for this study was reviewed and approved by Lacor Hospital Institutional Research and Ethics Committee (LHIREC 023/09/13) and Uganda National Council for Science and Technology (HS1403) in Uganda. Use of 10 Thai malaria naïve sera from healthy adults in malaria-free Bangkok who had no history of travelling outside the region was approved by the Ethics Committee of the Thai Ministry of Public Health and the Institutional Review Board of Faculty of Medicine, Ramathibodi Hospital, Mahidol University (ID: 09-46-10) [35]. Written informed consent was obtained from all study participants and/or their parents or guardians. Approval to conduct the study in Japan was obtained from RIMD, Osaka University and Ehime University. 2.2. Production of P. falciparum parasite protein library We generated a protein library consisting of 271 PfEMP1 domains derived from 62 var genes based on 3D7 strain [5]. Specifically the library consisted of 163 DBL domains (DBLa (n = 60), DBLb (n = 14), DBLb2 (n = 3), DBLd (n = 50), DBLe (n = 14), DBLc (n = 14), DBLpam (n = 1), DBLpam2 (n = 1), DBLpam3 (n = 1), DBL2 (n = 4), DBLc2 (n = 1)) and 108 CIDR domains (CIDRa (n = 54), CIDRb (n = 35), CIDRd (n = 2), CIDRc (n = 16), CIDRpam (n = 1)) [5]. The transcription template was prepared in vitro as previously described [21]. In brief, the 271 domains were amplified from genomic DNA of P. falciparum 3D7 strain by polymerase chain reaction (PCR) using Primestar DNA polymerase (Takara Bio, Kusatsu, Japan) and domain specific primers (Table S1), and cloning into pEU vector (CellFree Sciences, Matsuyama, Japan) with In-Fusion HD Cloning Kit (Takara Bio, Kusatsu, Japan). The Nterminus mono-biotinylated recombinant proteins were expressed by the WGCFS as previously described [36]. Briefly, 1 ll (50 ng) WGCFS-expressed BirA was added to the translation lower layer and 500 nM D-biotin (Nacalai Tesque, Kyoto, Japan) added to both the translation and substrate upper layers. In vitro transcription and cell-free protein synthesis were carried out using GenDecoder1000 robotic protein synthesizer (CellFree Sciences). Successful expression of all domains as mono-biotinylated proteins was confirmed by Western blot analysis by probing with HRP conjugated streptavidin [36]. 2.3. Quantitation of PfEMP1-specific antibodies by AlphaScreen Human serum antibody levels to WGCFS-expressed PfEMP1 domains were quantified by AlphaScreen [36]. Briefly, the protocol, conducted in 384-well OptiPlate microtiter plates (PerkinElmer Life and Analytical Science, Boston, MA), was automated using JANUS Automated Workstation (PerkinElmer). The translation mixture (0.1 ml) containing the biotinylated recombinant P. falciparum proteins was diluted 50-fold (5 ml), mixed with 10 ll of 4000-fold diluted Ugandan sera in reaction buffer (100 mM TrisHCl [pH 8.0], 0.01% [v/v] Tween-20 and 0.1% [w/v] bovine serum albumin) and incubated for 30 min at 26 °C to form a proteinantibody (IgG) complex. Suspension of streptavidin-coated donor-beads (PerkinElmer) and acceptor-beads (PerkinElmer) conjugated with protein G (Thermo Scientific, Waltham, MA) in 10 ll reaction buffer was added to a final concentration of 12 lg/ml for both beads. The mixture was incubated in the dark for 1 h to allow optimal binding of the donor and acceptor-beads to biotinylated protein and human antibody, respectively. Upon excitation, the formed protein-antibody complex emitted a luminescence signal at 620 nm, detected by EnVision plate reader (PerkinElmer) and captured as raw AlphaScreen Counts (AlphaScreen-Countsraw). WGCFS translation mixture without mRNA template was included in each plate as a negative control. Each assay plate contained a
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standard curve of biotinylated rabbit IgG. This enabled standardisation between plates using a 5-parameter logistic standard curve. This standardised AlphaScreen-Countsraw was presented as AlphaScreen Counts. AlphaScreen assays were conducted in a randomised manner to avoid experimental bias. A seropositivity cut-off was set as half the lowest non-negative value from of the assayed samples [37]. Proteins were defined as reactive if more than 10% of the volunteers had levels above the seropositivity cut-off [37]. Immunoreactivity (AlphaScreen Counts) was compared between the 66 serum samples only for the same protein fragment, but not across the 271 protein fragments. 2.4. Statistical analysis Antibody associations with age and clinical outcome were examined by Mann–Whitney U test or Kruskal-Wallis with Bonferroni-Dunn as post hoc test. For analysis of effects of an individual antigen, individuals with AlphaScreen Counts above the population median for that antigen were categorized as ‘High Responders’ while the rest as ‘Low Responders’. Kaplan–Meier was used to generate curves for time-to-first clinical episode. Risk of acquiring clinical malaria was assessed using Cox proportional hazard model. Although some children had multiple episodes of parasitemia or symptomatic malaria, we considered only the time-to-first symptomatic episode. This was akin to previous approaches used in this type of studies [21,38–40]; and analysis based on multiple episodes was precluded because of low incidence. Potential protective efficacy (PPE) was computed as 1- hazard ratio (PPE% = (1 HR) 100%). Multivariate survival analysis while adjusting for confounders focused on bed-net use and age as a categorical variable (6–10 years, 11–15 years and 16– 20 years). For sensitivity analysis, the data was also analyzed with modified Poisson Regression Model for prospective study with binary outcome associating High Responders vs. Low Responders to asymptomatic or symptomatic malaria. Analysis while adjusting for different antibody responses as covariates was limited by their highly correlated nature. p-value < 0.05 was considered statistically significant without correction for multiple comparisons. Because of the large number of antigens tested (n = 271), the clinical outcome data was randomly permutated 1000 times amongst
3
all participants to assess the number of associations that expected to be significant whilst still accounting for the correlated structure of the data. All analysis was by R software (Version 3.2.3; R Foundation for Statistical Computing) or Prism 6 (GraphPad Software Inc., La Jolla, CA). 3. Results 3.1. WGCFS expresses quality PfEMP1 proteins In this study, we established a PfEMP1 library consisting of 271 domains derived from 3D7-strain sequence, whose genome sequence is a broad representative of West African isolates [41]. All 163 DBL and 108 CIDR domains were successfully expressed by WGCFS [24]. The DBL and CIDR domains showed strong immunoreactivity with sera from malaria-exposed volunteers compared to the pooled sera from 10 malaria-naïve individuals (Fig. S1). Immunoreactivity to sera was observed in all PfEMP1 domains (Fig. 1A). Seroprevalence varied widely between DBL domains (12–100%) and CIDR domains (22–92%) (Mann-Whitney test, p = 0.022; Fig. 1A), with no difference among PfEMP1 groups (Fig. S2). This could be due to high level of exposure to these abundantly expressed proteins or cross-reactivity among the conserved regions of the domains [4]. 3.2. Anti-PfEMP1 antibody repertoire increased with age The probability of an individual responding to a specific domain increased with age for both CIDR and DBL with individuals aged 16–20-year-olds, but not 6–10 or 11–15-year-olds, responding significantly more to CIDR than DBL domains (Fig. 1B). Magnitude of antibody responses increased with age except for 16.6% (45/271) of the domains that did not associate with age and seems to have been acquired early in life and remaining stable with age (Table S1). The overall breadth of immune response associated with age, with serum samples from young adults (16 years) recognizing more PfEMP1 domains (Fig. 2A, p < 0.001) potentially due to increasing yearly exposure to P. falciparum antigens hence expanding the antibody repertoire. The breadth of immune
Fig. 1. Seroprevalence of antibodies to 271 PfEMP1 domains in Uganda. (A) Antibodies immunoreactivity to CIDR (n = 108) are represented in green while DBL (n = 163) are in blue. The dashed line indicates the protein immunoreactivity cut-off point, set at 10% of volunteers. The horizontal line in each group denotes the overall median per group (p = 0.022). (B) Proportion of individuals according to age groups (n = 22 per age group) whose antibodies recognized a given CIDR domain (green columns) or DBL domains (blue columns). A total of 163 DBL and 108 CIDR domains were tested. Box plots illustrate medians with 25th and 75th and whiskers for 10th and 90th percentiles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Kanoi BN et al. Comprehensive analysis of antibody responses to Plasmodium falciparum erythrocyte membrane protein 1 domains. Vaccine (2018), https://doi.org/10.1016/j.vaccine.2018.08.058
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Fig. 2. Breadth of the antibody response per person at the time of enrolment. (A) The number of seropositive antigens in each individual is shown, with box-plots indicating the median and ranges for each age cohort. Seropositivity was set at the limit of detection of the assay, equal to half the lowest non-negative AlphaScreen Counts value. Statistical difference was assessed using the Kruskal-Wallis test (p < 0.001); with Dunn’s test as post hoc test, p < 0.01 between age 6–10 and other age groups (11–15, and 16–20 years). (B) There was no statistical difference between individuals who developed symptomatic malaria and those who did not experience symptomatic malaria.
response did not significantly associate with clinical malaria outcome (Fig. 2B, p = 0.17).
and bed-net use. The trends were similar to primary analysis and did not alter the interpretation of Table 1. The results are summarized in Fig. S6 and Table S2.
3.3. Identification of antibody responses associated with protection from clinical malaria
4. Discussion
Malaria morbidity was monitored in the study, which enabled us to analyze whether antibody levels to the PfEMP1 domains were associated with reduced risk of developing symptomatic disease. Based on Cox hazard regression model analysis, the DBL and CIDR domains were ranked according to their respective PPE (Table S1). We observed a significant association between high antibody levels to 18 (10 DBL and 8 CIDR) domains and reduced risk of having a symptomatic malaria episode (Hazard ratio (HR) < 1, p < 0.05; Fig. 3A and Table 1). For instance, a group B PfEMP1 PF3D7_0300100 CIDRb1 (Dm#21, Table 1), had an unadjusted PPE of 69.84% (CI 11.68–89.70%, p = 0.029). A DBLa of PF3D7_0733000 (Dm#150) with a seroprevalence of only 13%, showed unadjusted PPE of 100% (CI 30.3–100%, P = 0.025). Representative Kaplan and Meir plots are presented in Fig. S3. Generally, the evaluated domains displayed a negative significant correlation between seroprevalence and PPE (Spearman’s rho, CIDR -0.31, p < 001; DBL -0.44, p < 0.001; Fig. S4A and B). Although CIDR domains had significantly higher seroprevalence (Fig. 1A), when ranked according to the corresponding PPE (%), no selection bias was observed towards either CIDR or DBL domains. No antigen remained significant after adjustment for age and bed-net use (Fig. 3B). Randomly permuting the clinical outcome data resulted in similar associations to those in Fig. 3B. Of 1000 permutations the most frequent number of significant associates expected by chance was 0/271, with the median number being 2/271 (Supplementary Fig. S5). To test the robustness of this analysis, we subjected the same data to Modified Poisson regression analysis with malaria as a binary outcome, with and without adjustment for the effects of age
Residents of malaria endemic regions quickly develop immunity to life threatening malaria complications after repeated exposure [1]. The basis of this immunity appears to be, at least in part, due to acquisition of antibodies to polymorphic parasite antigens expressed on the surface of infected erythrocytes, referred to as variant surface antigens (VSA). Specifically, PfEMP1s have been proposed to play a key role in mediating this protective immunity [42]. However, due to the large number of antigens potentially expressed on the infected erythrocyte surface, it has been difficult to determine a pattern of PfEMP1-specific antibody responses and identify protein or domain targets of protective immunity. Therefore, in this study, we sought to comprehensively determine antibody responses to all DBL and CIDR domains derived from the P. falciparum 3D7 genome. We observed that individuals in this high-transmission region had achieved substantial levels of antibodies against all 271 PfEMP1 proteins. The high degree of immunoreactivity could at least be attributed to high quality proteins synthesized using the robust eukaryotic WGCFS. Although no structural analysis of the expressed antigens was conducted, WGCFS generated plasmodial proteins are comparatively more soluble, intact, biologically active and immunoreactive to human sera than those expressed by commonly used E. coli-based systems [24–27], suggesting the proteins possess significant levels of native conformations. In addition, application of AlphaScreen immunoscreening system offers a high-throughput platform for antibody quantification in a homogeneous aqueous environment with no antigen conjugation to a matrix or beads. This provides optimal interactions between recombinant PfEMP1 domains and human antibodies [36]. It is also
Please cite this article in press as: Kanoi BN et al. Comprehensive analysis of antibody responses to Plasmodium falciparum erythrocyte membrane protein 1 domains. Vaccine (2018), https://doi.org/10.1016/j.vaccine.2018.08.058
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Fig. 3. Associations between antibody levels and risk of P. falciparum clinical malaria. (A) Unadjusted association between antibody levels and risk of P. falciparum clinical malaria for each of the 271 antigens tested in the Ugandan cohort. Antigens are ranked – top to bottom – by the strength of their potential protective efficacy (PPE). PPE (percentage) for each antigen was derived from the hazard ratio (HR) calculated by the unadjusted Cox-regression hazard model analysis [comparing children with high (High Responders) versus low (Low Responders) antibody responses]. Black dots indicate the percentage protection, and the error bars indicate the 95% confidence interval. The red vertical line represents PPE of 0% (i.e. HR = 1). (B) After adjustment for age and bed-net use, none of the antigens remained significant responses. Complete list of the data for PPE (and 95% confidence interval) is provided in Table S1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 1 Association between antibody responses to PfEMP1 and protection from clinical malaria. Unadjusted PPEa Library ID
Gene ID
Age and Bed-net use adjusted PPE
Domain
PfEMP1 group
Seroprevalence (%)
PPE (%)
95% CI
p-value
PPE %
95% CI
p-value
CIDR domains Dm#21 PF3D7_0300100 Dm#119 PF3D7_0700100 Dm#239 PF3D7_1219300 Dm#102 PF3D7_0617400 Dm#158 PF3D7_0800100 Dm#49 PF3D7_0412900 Dm#17 PF3D7_0223500 Dm#15 PF3D7_0223500 Dm#71 PF3D7_0425800
CIDRb1 CIDRa3.1 CIDRa3.4 CIDRa2.1 CIDRb1 CIDRc2 CIDRb1 CIDRa3.4 CIDRa1.6
B B B C B C B B A
34.8 50.0 33.3 65.2 42.4 45.5 51.5 62.1 65.2
69.84 66.01 65.88 63.37 61.60 58.62 57.89 57.31 57.07
(11.68–89.70) (17.93–85.92) (0.12–88.35) (11.54–84.83) (3.16–84.77) (0.14–82.86) (1.53–81.99) (0.15–81.75) ( 0.37 to 81.63)
0.029 0.016 0.050 0.026 0.043 0.050 0.046 0.050 0.051
59.68 51.42 37.62 39.69 36.88 16.67 29.82 31.33 40.47
( ( ( ( ( ( ( ( (
29.00 to 87.40) 25.10 to 81.13) 117.28 to 82.09) 64.44 to 77.88) 83.87 to 78.33) 140.35 to 71.11) 81.68 to 72.89) 73.88 to 72.88) 44.24 to 75.43)
0.126 0.135 0.459 0.323 0.399 0.736 0.466 0.428 0.251
DBL domains Dm#150 Dm#54 Dm#72 Dm#266 Dm#242 Dm#73 Dm#169 Dm#137 Dm#110 Dm#118 Dm#157
DBLa0.16 DBLa0.1 DBLb3 DBLc12 DBLa0.7 DBLb3 DBLd1 DBLc9 DBLd4 DBLa0.1 DBLd1
B C A A B/C A B/A B/C B/A B B
13.6 28.8 28.8 34.8 33.3 57.6 57.6 39.4 66.7 39.4 63.6
100.00 71.56 70.69 68.12 66.66 66.01 65.53 65.46 65.10 64.96 57.08
(30.30–100.00) (4.60–91.52) (1.67–91.26) (6.65–89.11) (2.37–88.61) (17.93–85.92) (16.75–85.73) (7.42–87.11) (15.75–85.54) (6.09–86.93) ( 0.40–81.65)
0.025 0.042 0.047 0.037 0.045 0.016 0.018 0.035 0.019 0.037 0.051
100.00 51.80 58.23 40.01 43.57 37.28 57.51 44.38 44.74 47.13 29.55
( ( ( ( ( ( ( ( ( ( (
40 to 100.00) 73.85 to 86.64) 48.73 to 88.27) 99.67 to 81.98) 89.30 to 83.18) 71.23 to 77.03) 13.98 to 84.16) 66.18 to 81.39) 48.33 to 79.42) 53.23 to 81.76) 81.89 to 72.72)
0.119 0.265 0.178 0.405 0.354 0.363 0.089 0.293 0.239 0.240 0.469
PF3D7_0733000 PF3D7_0420700 PF3D7_0425800 PF3D7_1300300 PF3D7_1240300 PF3D7_0425800 PF3D7_0800300 PF3D7_0712400 PF3D7_0632500 PF3D7_0700100 PF3D7_0800100
In bold are p-values of antigens with responses that were statistically significant. a Potential Protective Efficacy (PPE) was calculated using hazard ratios (HR) generated by the Cox proportional hazards model comparing individuals with high versus low AlphaScreen Counts; (PPE% = (1 HR) 100%). After adjustments for confounders (age and bed-net use), no PPE remained statistically significant.
possible that the broad immunoreactivity was due to crossreactivity among the PfEMP1 domains. The majority of the domains exhibit some levels of amino-acid sequence similarity with others in the same group [5].
Antibodies to 18 (10 DBL and 8 CIDR) domains were associated with reduced risk to clinical malaria (Table 1), although these were not statistically significant after correction for age and bed net use. We observed a significant negative correlation between seropreva-
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lence and protective immunity. This was a surprising finding and may suggest; (i) that antibodies to commonly encountered domains were not as protective as antibodies to less commonly encountered domains, a potential product of selection pressure; (ii) the short-lived nature of protective antibodies or; (iii) the differential immunogenicity of the PfEMP1 domains. This further indicates the complexity of naturally acquired protective immunity and suggests that not all PfEMP1 proteins are targets of protective antibodies in this cohort. The identification of the 18 domains, that included previously well-evaluated domains that are currently under consideration as vaccine or anti-disease therapeutic targets [7], is very reassuring and brings into focus new domains that should be further investigated. For instance, higher antibody levels to PF3D7_0700100 CIDRa3.1 and DBLa0.1 domains (Fig. 4A), with prevalence of 50.0% and 39.4% respectively, were associated with lower risk of developing symptomatic malaria. This mirrored a similar observation in Ghanaian and Sudanese children where higher levels of antibodies to a recombinant peptide derived from a conserved epitope of the PF3D7_0700100 DBLa domain were associated with clinical protection [43,44]. One of the key findings in this paper is the observation that higher serum levels of antibodies to 4 EPCR-binding CIDRa domains (PF3D7_0700100, PF3D7_0425800, PF3D7_0223500 and PF3D7_0617400) were associated with lower risk of developing symptomatic malaria. This is relevant since it was previously observed that antibody to these (CIDRa) polymorphic cysteine-rich domains are associated with protective immunity in Tanzanian children [45]. Although it’s unclear how the protective immunity is attained, it was demonstrated that despite their huge sequence diversity, CIDRa1 are structurally and functionally conserved for binding to EPCR [10,11]; an interaction that could be blocked by antibodies in individuals exposed to natural infection in Tanzania [10]. It would be important to further understand
the potential role of these selected CIDR domains in complicated malaria. The data on PfEMP1 DBL domains reported here is consistent with that reported in Papua New Guinea, Tanzania and Kenya where antibody responses increased with age [46–48]. Furthermore, studies with PfEMP1 DBLa1.5 antibodies (variant HB3var6) or DBLa1.8 antibodies (variant TM284var1) showed broad crossreactivity against heterologous parasite strains with the same rosetting phenotype, including clinical isolates from four subSaharan African countries [23]. It therefore remains possible that, within the hypervariable regions, there exists antigenically conserved linear or structural epitopes shared by subsets of PfEMP1 types that are the targets of strain-transcending antibodies [10,23]. Analysis of multiple P. falciparum genomes has shown that several ICAM-1-binding PfEMP1s also interact with EPCR allowing infected erythrocytes to synergistically bind both receptors [6]. In our study, antibodies to a severe-malaria-associated DC4 family member, PF3D7_0425800 CIDRa1.6-DBLb3-DBLb3 domains (predicted as double ICAM-1 and EPCR binder) [7,49], significantly associated with clinical protection (Fig. 4B: i & ii). PF3D7_0425800 DC4 (DBLa1.4-CIDRa1.6-DBLb3-DBLb3) is preferentially expressed in plasmodium parasites associated with severe childhood malaria [7,49,50]. PF3D7_0425800 DBLb3 elicits adhesion-inhibitory antibodies that broadly inhibit ICAM-1 binding and are cross-reactive to DC4-containing PfEMP1 from genetically distant parasite isolates [51]. Specifically, the monoclonal antibodies to the ICAM-1 binding site of PF3D7_0425800 DBLb, convex surface of DBLb3_D4, specifically blocks interaction between ICAM-1 and DBLb3_D4 domains from genetically distant parasite isolates, suggesting that the monoclonal antibodies act by occluding the ICAM-1 binding surface [51]. Our study suggests that homologues of these DC4 domains are present in P. falciparum field isolates and that only a restricted set of specific domains are essential for
Fig. 4. Antibodies to tandem domains derived from PfEMP1 (A (i). PF3D7_0700100 and B (i). PF3D7_0425800) associated with clinical protection. The numbers below the primary structure represent p-values to PPE of each domain. Individuals who did not develop symptomatic malaria episode had significantly higher level of antibodies to A (ii). PF3D7_0700100 and B (ii). PF3D7_0425800, Statistical difference was assessed using the Mann-Whitney U test (p < 0.001).
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eliciting naturally acquired, protective immunity in malaria. In light of the current understanding on structurally conserved nature of some PfEMP1s [6,10,23,52], this work advances the selected domains for further evaluation as biomarkers of malaria immunity or vaccine targets. Indeed, a strain-specific PFEMP1 expressed in sporozoites has recently been identified and proposed as a vaccine target [53]. There is also a need to determine the mode of action of the protective antibodies to the selected PfEMP1 proteins, and identify the corresponding receptors on the human host cells. Considering the extensive genetic diversity that has been reported for PfEMP1, a major limitation is that we only assessed a well-established laboratory strain (3D7) in the assays. This parasite line may not recall all antibody response patterns of different parasite variants circulating in the population. Nevertheless, since none of the children in the study was likely to have been infected with 3D7, and yet many developed antibodies reacting with recombinant DBL and CIDR domains based on this genome, there must be widespread homologous - conserved - domains encoded from different genomes circulating in the population. In the future, we hope to expand this screening to cover larger sample sizes from multiple field sites and parasites expressing different PfEMP1 variants. In conclusion, our study demonstrates that WGCFS coupled with AlphaScreen is a powerful tool for mapping immune patterns induced by PfEMP1 proteins. We identified 18 DBL and CIDR domains associated with clinical protection. Identification of DC4 PF3D7_0425800 DBLb3 that were previously associated with severe childhood malaria and, in this study, with antibodies protective from symptomatic uncomplicated malaria, highlights the importance of specific PfEMP1s. This implies that, although PfEMP1s have been described as highly diverse, specific domains are central in naturally acquired protective immunity. Thus, the data supports the feasibility of developing a vaccine against malaria by sequentially identifying, characterizing and targeting to inhibit the function of these domains. We recommend PF3D7_0425800 DBLb3 as well as other selected domains to be extensively evaluated in larger population cohorts to assess their potential as alternative targets for malaria vaccine development. Conflict of interest statement The authors have no conflict of interest to declare. Acknowledgments We are grateful to Dr. Michael F. Duffy (University of Melbourne, Australia) for critical reading of the manuscript. We appreciate the study volunteers at Lira, Northern Uganda; the research teams from Med Biotech Laboratories, Lira Medical Center, Uganda, and Research Institute for Microbial Diseases, Osaka University for their technical assistance in obtaining the field samples. We thank adult volunteers in Thailand who participated in the study, and also acknowledge Dr. Rachanee Udomsangpetch for supporting the study. This work was supported in part by Strategic Promotion of International Cooperation to Accelerate Innovation in Africa by MEXT, MEXT KAKENHI (JP23117008) and JSPS KAKENHI (JP25460517, JP26253026, JP26670202, JP26860279, JP15H05276, JP16K15266, JP18H02651) in Japan. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.vaccine.2018.08. 058.
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References [1] Doolan DL, Dobano C, Baird JK. Acquired immunity to malaria. Clin Microbiol Rev 2009;22:13–36. [2] McGregor IA. Studies in the acquisition of immunity of Plasmodium falciparum infections in Africa. Trans R Soc Trop Med Hyg 1964;58:80–92. [3] Sabchareon A, Burnouf T, Ouattara D, Attanath P, Bouharoun-Tayoun H, Chantavanich P, et al. Parasitologic and clinical human response to immunoglobulin administration in falciparum malaria. Am J Trop Med Hyg 1991;45:297–308. [4] Chan JA, Fowkes FJ, Beeson JG. Surface antigens of Plasmodium falciparuminfected erythrocytes as immune targets and malaria vaccine candidates. Cell Mol Life Sci 2014;71:3633–57. [5] Rask TS, Hansen DA, Theander TG, Gorm Pedersen A, Lavstsen T. Plasmodium falciparum erythrocyte membrane protein 1 diversity in seven genomes– divide and conquer. PLoS Comput Biol 2010;6. [6] Lennartz F, Adams Y, Bengtsson A, Olsen RW, Turner L, Ndam NT, et al. Structure-guided identification of a family of dual receptor-binding PfEMP1 that is associated with cerebral malaria. Cell Host Microbe 2017;21:403–14. [7] Bengtsson A, Joergensen L, Rask TS, Olsen RW, Andersen MA, Turner L, et al. A novel domain cassette identifies Plasmodium falciparum PfEMP1 proteins binding ICAM-1 and is a target of cross-reactive, adhesion-inhibitory antibodies. J Immunol 2013;190:240–9. [8] Howell DP, Levin EA, Springer AL, Kraemer SM, Phippard DJ, Schief WR, et al. Mapping a common interaction site used by Plasmodium falciparum Duffy binding-like domains to bind diverse host receptors. Mol Microbiol 2008;67:78–87. [9] Janes JH, Wang CP, Levin-Edens E, Vigan-Womas I, Guillotte M, Melcher M, et al. Investigating the host binding signature on the Plasmodium falciparum PfEMP1 protein family. PLoS Pathog 2011;7:e1002032. [10] Lau CK, Turner L, Jespersen JS, Lowe ED, Petersen B, Wang CW, et al. Structural conservation despite huge sequence diversity allows EPCR binding by the PfEMP1 family implicated in severe childhood malaria. Cell Host Microbe 2015;17:118–29. [11] Turner L, Lavstsen T, Berger SS, Wang CW, Petersen JE, Avril M, et al. Severe malaria is associated with parasite binding to endothelial protein C receptor. Nature 2013;498:502–5. [12] Hsieh FL, Turner L, Bolla JR, Robinson CV, Lavstsen T, Higgins MK. The structural basis for CD36 binding by the malaria parasite. Nat Commun 2016;7:12837. [13] Robinson BA, Welch TL, Smith JD. Widespread functional specialization of Plasmodium falciparum erythrocyte membrane protein 1 family members to bind CD36 analysed across a parasite genome. Mol Microbiol 2003;47:1265–78. [14] Duffy MF, Noviyanti R, Tsuboi T, Feng ZP, Trianty L, Sebayang BF, et al. Differences in PfEMP1s recognized by antibodies from patients with uncomplicated or severe malaria. Malar J. 2016;15:258. [15] Petersen JE, Bouwens EA, Tamayo I, Turner L, Wang CW, Stins M, et al. Protein C system defects inflicted by the malaria parasite protein PfEMP1 can be overcome by a soluble EPCR variant. Thromb Haemost 2015;114:1038–48. [16] Mkumbaye SI, Wang CW, Lyimo E, Jespersen JS, Manjurano A, Mosha J, et al. The severity of Plasmodium falciparum infection is associated with transcript levels of var genes encoding endothelial protein C receptor-binding P. falciparum erythrocyte membrane protein 1. Infect Immun 2017:85. [17] Bull PC, Abdi AI. The role of PfEMP1 as targets of naturally acquired immunity to childhood malaria: prospects for a vaccine. Parasitology 2016;143:171–86. [18] Lavstsen T, Turner L, Saguti F, Magistrado P, Rask TS, Jespersen JS, et al. Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children. Proc Natl Acad Sci U S A 2012;109:E1791–800. [19] Salanti A, Dahlback M, Turner L, Nielsen MA, Barfod L, Magistrado P, et al. Evidence for the involvement of VAR2CSA in pregnancy-associated malaria. J Exp Med 2004;200:1197–203. [20] Travassos MA, Niangaly A, Bailey JA, Ouattara A, Coulibaly D, Laurens MB, et al. Seroreactivity to Plasmodium falciparum erythrocyte membrane protein 1 intracellular domain in malaria-exposed children and adults. J Infect Dis. 2013;208:1514–9. [21] Kanoi BN, Takashima E, Morita M, White MT, Palacpac NM, Ntege EH, et al. Antibody profiles to wheat germ cell-free system synthesized Plasmodium falciparum proteins correlate with protection from symptomatic malaria in Uganda. Vaccine. 2017;35:873–81. [22] Mayer C, Slater L, Erat MC, Konrat R, Vakonakis I. Structural analysis of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) intracellular domain reveals a conserved interaction epitope. J Biol Chem 2012;287:7182–9. [23] Ghumra A, Semblat JP, Ataide R, Kifude C, Adams Y, Claessens A, et al. Induction of strain-transcending antibodies against Group A PfEMP1 surface antigens from virulent malaria parasites. PLoS Pathog 2012;8:e1002665. [24] Arumugam TU, Ito D, Takashima E, Tachibana M, Ishino T, Torii M, et al. Application of wheat germ cell-free protein expression system for novel malaria vaccine candidate discovery. Expert Rev Vaccines 2014;13:75–85. [25] Tsuboi T, Takeo S, Iriko H, Jin L, Tsuchimochi M, Matsuda S, et al. Wheat germ cell-free system-based production of malaria proteins for discovery of novel vaccine candidates. Infect Immun 2008;76:1702–8.
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[26] Ntege EH, Takashima E, Morita M, Nagaoka H, Ishino T, Tsuboi T. Blood-stage malaria vaccines: post-genome strategies for the identification of novel vaccine candidates. Expert Rev Vaccines 2017;16:769–79. [27] Rui E, Fernandez-Becerra C, Takeo S, Sanz S, Lacerda MV, Tsuboi T, et al. Plasmodium vivax: comparison of immunogenicity among proteins expressed in the cell-free systems of Escherichia coli and wheat germ by suspension array assays. Malar J 2011;10:192. [28] Takeda H, Ogasawara T, Ozawa T, Muraguchi A, Jih PJ, Morishita R, et al. Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay. Sci Rep 2015;5:11333. [29] Palacpac NM, Ntege E, Yeka A, Balikagala B, Suzuki N, Shirai H, et al. Phase 1b randomized trial and follow-up study in Uganda of the blood-stage malaria vaccine candidate BK-SE36. PLoS ONE 2013;8:e64073. [30] Egwang TG, Apio B, Riley E, Okello D. Plasmodium falciparum malariometric indices in Apac district, northern Uganda. East Afr Med J 2000;77:413–6. [31] Proietti C, Pettinato DD, Kanoi BN, Ntege E, Crisanti A, Riley EM, et al. Continuing intense malaria transmission in northern Uganda. Am J Trop Med Hyg 2011;84:830–7. [32] Yeka A, Gasasira A, Mpimbaza A, Achan J, Nankabirwa J, Nsobya S, et al. Malaria in Uganda: challenges to control on the long road to elimination: I. Epidemiology and current control efforts. Acta Trop 2012;121:184–95. [33] Okello PE, Van Bortel W, Byaruhanga AM, Correwyn A, Roelants P, Talisuna A, et al. Variation in malaria transmission intensity in seven sites throughout Uganda. Am J Trop Med Hyg 2006;75:219–25. [34] Osier FH, Mackinnon MJ, Crosnier C, Fegan G, Kamuyu G, Wanaguru M, et al. New antigens for a multicomponent blood-stage malaria vaccine. Sci Transl Med 2014;6. 247ra102. [35] Sakamoto H, Takeo S, Takashima E, Miura K, Kanoi BN, Kaneko T, et al. Identification of target proteins of clinical immunity to Plasmodium falciparum in a region of low malaria transmission. Parasitol Int 2018;67:203–8. [36] Matsuoka K, Komori H, Nose M, Endo Y, Sawasaki T. Simple screening method for autoantigen proteins using the N-terminal biotinylated protein library produced by wheat cell-free synthesis. J Proteome Res 2010;9:4264–73. [37] Longley RJ, White MT, Takashima E, Morita M, Kanoi BN, Li Wai Suen CSN, et al. Naturally acquired antibody responses to more than 300 Plasmodium vivax proteins in three geographic regions. PLoS Negl Trop Dis 2017;11:e0005888. [38] Osier FH, Fegan G, Polley SD, Murungi L, Verra F, Tetteh KK, et al. Breadth and magnitude of antibody responses to multiple Plasmodium falciparum merozoite antigens are associated with protection from clinical malaria. Infect Immun 2008;76:2240–8. [39] Rono J, Osier FH, Olsson D, Montgomery S, Mhoja L, Rooth I, et al. Breadth of anti-merozoite antibody responses is associated with the genetic diversity of asymptomatic Plasmodium falciparum infections and protection against clinical malaria. Clin Infect Dis 2013;57:1409–16. [40] Richards JS, Arumugam TU, Reiling L, Healer J, Hodder AN, Fowkes FJ, et al. Identification and prioritization of merozoite antigens as targets of protective
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52] [53]
human immunity to Plasmodium falciparum malaria for vaccine and biomarker development. J Immunol 2013;191:795–809. Volkman SK, Sabeti PC, DeCaprio D, Neafsey DE, Schaffner SF, Milner Jr DA, et al. A genome-wide map of diversity in Plasmodium falciparum. Nat Genet 2007;39:113–9. Bull PC, Lowe BS, Kortok M, Molyneux CS, Newbold CI, Marsh K. Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria. Nat Med 1998;4:358–60. Staalso T, Khalil EA, Elhassan IM, Zijlstra EE, Elhassan AM, Giha HA, et al. Antibody reactivity to conserved linear epitopes of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). Immunol Lett 1998;60:121–6. Dodoo D, Staalsoe T, Giha H, Kurtzhals JA, Akanmori BD, Koram K, et al. Antibodies to variant antigens on the surfaces of infected erythrocytes are associated with protection from malaria in Ghanaian children. Infect Immun 2001;69:3713–8. Magistrado PA, Lusingu J, Vestergaard LS, Lemnge M, Lavstsen T, Turner L, et al. Immunoglobulin G antibody reactivity to a group A Plasmodium falciparum erythrocyte membrane protein 1 and protection from P. falciparum malaria. Infect Immun 2007;75:2415–20. Cham GK, Turner L, Lusingu J, Vestergaard L, Mmbando BP, Kurtis JD, et al. Sequential, ordered acquisition of antibodies to Plasmodium falciparum erythrocyte membrane protein 1 domains. J Immunol 2009;183:3356–63. Dent AE, Nakajima R, Liang L, Baum E, Moormann AM, Sumba PO, et al. Plasmodium falciparum protein microarray antibody profiles correlate with protection from symptomatic malaria in Kenya. J Infect Dis 2015;212:1429–38. Barry AE, Trieu A, Fowkes FJ, Pablo J, Kalantari-Dehaghi M, Jasinskas A, et al. The stability and complexity of antibody responses to the major surface antigen of Plasmodium falciparum are associated with age in a malaria endemic area. Mol Cell Proteomics 2011;10(M111):008326. Tonkin-Hill GQ, Trianty L, Noviyanti R, Nguyen HHT, Sebayang BF, Lampah DA, et al. The Plasmodium falciparum transcriptome in severe malaria reveals altered expression of genes involved in important processes including surface antigen-encoding var genes. PLoS Biol 2018;16:e2004328. Jensen AT, Magistrado P, Sharp S, Joergensen L, Lavstsen T, Chiucchiuini A, et al. Plasmodium falciparum associated with severe childhood malaria preferentially expresses PfEMP1 encoded by group A var genes. J Exp Med 2004;199:1179–90. Lennartz F, Bengtsson A, Olsen RW, Joergensen L, Brown A, Remy L, et al. Mapping the binding site of a cross-reactive Plasmodium falciparum PfEMP1 monoclonal antibody inhibitory of ICAM-1 binding. J Immunol 2015;195:3273–83. Lennartz F, Thomas L, Higgins MK. Towards an anti-disease malaria vaccine. Emerg Top Life Sci 2017;1:539–45. Zanghi G, Vembar SS, Baumgarten S, Ding S, Guizetti J, Bryant JM, et al. A specific PfEMP1 is expressed in P. falciparum sporozoites and plays a role in hepatocyte infection. Cell Rep 2018;22:2951–63.
Please cite this article in press as: Kanoi BN et al. Comprehensive analysis of antibody responses to Plasmodium falciparum erythrocyte membrane protein 1 domains. Vaccine (2018), https://doi.org/10.1016/j.vaccine.2018.08.058