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Running title: MAPPING B-CELL LINEAR EPITOPES ON DBPII 1

Mapping epitopes of the Plasmodium vivax Duffy binding protein with 2

naturally acquired inhibitory antibodies. 3

Patchanee Chootong1,2‡

, Francis B. Ntumngia1‡

, Kelley M. VanBuskirk3‡†

, Jia Xainli4, 4

Jennifer L. Cole-Tobian4, Christopher O. Campbell

1, Tresa S. Fraser

3, Christopher L. King

4 5

and John H. Adams1*

6

1 Global Health Infectious Disease Research Program, University of South Florida, Tampa, 7

Florida, USA 8

2 Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University, 9

Bangkok, Thailand 10

3 Department of Biological Sciences, University of Notre Dame, Notre Dame, IN. 11

4 Division of Geographic Medicine, Case Western Reserve University, Cleveland, Ohio, USA 12

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.01036-09 IAI Accepts, published online ahead of print on 14 December 2009

on June 22, 2020 by guesthttp://iai.asm

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MAPPING B-CELL LINEAR EPITOPES ON DBPII

2

Foot Notes:

Conflict of interest: Authors have no commercial or other association that pose a conflict of 13

interest. 14

* Correspondence: Dr. John H. Adams, 3720 Spectrum Blvd, Suite 304, Tampa, FL 33612. 15

Email: [email protected] 16

‡ Contributed equally to this manuscript. 17

†Current address: Seattle Biomedical Research Institute, Seattle, Washington, USA 18


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ABSTARCT 19

Plasmodium vivax Duffy binding protein (DBP) is a merozoite microneme ligand vital 20

for blood-stage infection, making it an important candidate vaccine for antibody-mediated 21

immunity against vivax malaria. A differential screen against a linear peptide array 22

compared the reactivity of non-inhibitory and inhibitory high titer human immune sera to 23

identify target epitopes associated with protective immunity. Naturally acquired anti-DBP-24

specific serologic responses observed in the residents of a region of Papua New Guinea 25

highly endemic for P. vivax had significant changes over time in DBP-specific titers. Anti-26

DBP functional inhibition for each serum ranged from complete to none even among high 27

titer responders to the DBP, indicating that epitope specificity is important. Inhibitory 28

immune human antibodies identified specific B-cell linear epitopes on the DBP (Sal 1) 29

ligand domain that showed significant correlation with inhibitory responses. Naturally 30

acquired antibodies affinity purified on these epitopes highly inhibited DBP erythrocyte 31

binding function confirming the protective value of specific epitopes. These results represent 32

an important advance in understanding a part of blood-stage immunity against P. vivax and 33

some of the specific targets for vaccine-elicited antibody protection. 34

35

Keywords: malaria; Plasmodium vivax; epitopes; DBP; ligand; immunity. 36


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INTRODUCTION 37

Plasmodium vivax is the major cause of malaria in most endemic regions outside of Africa, 38

causing substantial morbidity worldwide (16). Plasmodium microneme proteins, such as the 39

Duffy binding protein (DBP), have important roles in the merozoite invasion of reticulocytes 40

during the asexual blood-stage infection (1, 5). DBP is a member of the Duffy Binding-Like 41

Erythrocyte Binding Protein (DBL-EBP) family expressed in the micronemes and on the 42

surface of P. vivax merozoites and is associated with the decisive step of junction formation 43

during the invasion process (1). It is this critical interaction of DBP with its cognate receptor, 44

which makes DBP an important anti-malaria vaccine candidate. The critical erythrocyte 45

binding motif of DBP lies within a 330 amino acid cysteine-rich domain referred to as region 46

II (DBPII) or DBL domain, which is the minimal domain responsible for binding to Duffy 47

positive human erythrocytes (2, 6). The central portion of the DBP domain is hypervariable 48

relative to other DBP regions and polymorphisms are frequent at certain residues in a pattern 49

consistent with selection pressure on DBP, suggesting that allelic variation functions as a 50

mechanism of immune evasion (8, 14, 23). 51

Naturally acquired antibodies to DBP are prevalent in residents of areas highly endemic 52

for malaria, but individuals show significant quantitative and qualitative differences in their 53

anti-DBP serological responses (9, 11, 27, 28). Generally, serological responses to DBP and 54

the inhibition of DBP-erythrocyte binding activity increase with a person’s age, suggestive 55

of a boosting effect due to the repeated exposure through recurrent infections (12, 15, 17). 56

The initial antibody response to a single P. vivax infection occurs to conformational epitopes 57

and is not broadly protective while a strain transcending immunity develops only after 58

repeated exposure (9, 28). Such repeated exposure of residents of the P. vivax endemic areas 59


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of PNG was observed to correlate with development of antibodies reactive to linear epitopes 60

in the critical binding region of DBP. In this study, we compared reactivity of inhibitory 61

human immune sera to non-inhibitory immune sera to identify linear epitopes within DBPII 62

that may serve as a target for vaccine-induced protective humoral immunity. 63

64

MATERIALS AND METHODS. 65

Sample collection. Blood samples were collected March - July 2001 from 38 volunteers 66

selected from a previously surveyed population from Liksul in February 2002, a village 67

northwest of Madang, Papua New Guinea (27). Selected individuals ranged from 9 to 73 68

years of age and represented high-, low- and non-responder groups as classified in an earlier 69

study (17). Blood was collected by venipuncture into VacutainerTM

tubes without anti-70

coagulant. Approximately 8 ml were taken from each individual and kept at ambient 71

temperature (30-35 °C) for 30 min, then placed at 4 °C overnight. Serum was removed, 72

decomplemented at 56 °C for 30 min, and stored at –80 °C. Cryopreserved samples were 73

shipped to the United States for analysis. All human blood samples used in this study were 74

collected after obtaining consent from study participants under protocols approved by the 75

Ethical Review Board of the Cleveland Veteran’s Administration Medical Center, the Papua 76

New Guinea Medical Research Advisory Committee, and the University of Notre Dame 77

Institutional Review Board. 78

Measurement of serological responses to DBP. Anti-DBP responses were quantified 79

by ELISA using recombinant DBP regions II-IV (rDBPII-IV) as described previously (11, 17, 80

27). Briefly, rDBPII-IV was expressed as a GST fusion protein in E. coli, affinity purified on 81

glutathione and cleaved from GST with thrombin using standard methods (11, 18). Purified 82


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rDBPII-IV was added to 96-well plates at 2 µg/mL, incubated for 30 min at room temperature, 83

and washed three times with wash buffer (0.2% Tween-20 in PBS). Wells were incubated 84

with 200 µL block buffer (1% BSA in PBS) for 30 min, washed three times with wash buffer, 85

allowed to dry, and stored overnight at 4 °C. Serum diluted 1:400 in block buffer was added 86

to pre-wetted wells and incubated for 90 min at 37 °C. Plates were rinsed 3X in wash buffer, 87

incubated with 1:400 goat anti-human IgG-alkaline phosphatase, rinsed 3X, and substrate 88

added. Absorbance was recorded at 405 nm at 45 min after addition of developer reagent. 89

A baseline was established using control sera from non-exposed North Americans and this 90

control value was subtracted from the test OD values. ELISA data was classified into three 91

groups: High responders (OD=1.5–3.85); Low responders (OD=0.5–1.5); and Non-92

responders (OD < 0.5). 93

Antibody purification. Antibodies to B-cell epitopes were affinity purified from human 94

sera containing high titer DBPII inhibitory antibodies from P. vivax exposed individuals of 95

PNG or rabbit sera raised against peptides corresponding to selected B-cell epitopes. Pooled 96

human sera from a separate study in PNG were used for the affinity purification because of 97

the limited quantity of the experimental samples. According to the manufacturer’s 98

recommended protocol diluted serum was passed over an affinity column prepared by 99

coupling 3mg of each peptide to a Sulfur Link coupling resin (Thermo scientific, Rockford, 100

USA). After washing the column 3X with PBS, pH 7.4, the bound antibody was eluted with 101

0.1M Glycine-HCL pH 3.0 and immediately neutralized with 1M Tris-HCL, pH 8.5. 102

Antibodies were dialyzed against PBS before storage at -20 °C until needed. 103

COS7 cell expression of DBP and inhibition assays. COS 7 cells were transfected with 104

the plasmid (pEGFP-DBPII-Sal 1), which allows expression of DBPII as a fusion protein to 105


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the N-terminus of EGFP used as a transfection marker as previously described (17). The 106

inhibition assay was performed 42 hr after initial transfection. Serum at 1:10 dilution or 107

different concentrations of the affinity purified antibodies were incubated 60 min with 108

transfected COS7 cells followed by incubation with Duffy positive human erythrocytes. 109

Unbound erythrocytes were removed by washing three times with PBS. Binding was 110

quantified by counting rosettes observed over thirty fields of view at 200X magnification. In 111

this assay rosettes were defined as COS7 cells covered by bound erythrocytes at 50% or 112

greater surface area. Percent inhibition was calculated for each serum sample relative to 113

binding in presence of non-exposed North American (NA) control serum. Each assay 114

included a duplicate test of each sample and results were determined from an average of 3 115

independent assays. 116

Synthesized peptide array. A peptide array consisting of 178 overlapping twelve-mer 117

peptides displaced by 2 amino acids, spanning the entire DBPII-Sal 1, was generated on 118

‘gears’ attached pins in a 96-well format (Chiron Mimotopes). Peptide purity was >90% as 119

determined by high performance liquid chromatography. Additional peptides were 120

synthesized that corresponded to the different variants for peptides 179 to 205. Sequence 121

variations were based on the most common alleles identified in the study population (10). 122

Assessment of antibody responses to DBPII peptides. Serological responses to each 123

peptide were measured using a modified ELISA method. Coating buffer (0.1M PBS, pH7.2) 124

was dispensed into each well of microtiter plate and the peptide gears were placed in the well 125

to incubate for 1 hr at room temperature. The gears were washed in 0.01 M PBS (pH 7.2) for 126

30 mins, incubated with 1:400 human sera overnight at 4 °C, and washed in washing buffer 127

for 10 mins. To detect primary antibody reactivity to each peptide secondary antibodies, goat 128


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anti-human IgG-peroxidase (KPL Inc, Maryland, USA), were incubated at RT for 1 hr with 129

agitation, washed, ABTS substrate was added to each well, incubated for 45 mins and the 130

optical density (OD) quantified at 405 nm. 131

Data analysis. Classification of high-, low-, and non-responders to DBP was based on 132

averaged OD values for three wells per individual; a baseline was created from non-exposed 133

North Americans (NA) and subtracted from test OD values to standardize the ELISA (11). 134

Cluster analysis was performed on the ELISA values using SYSTAT (version 6.0); 135

individual values clustered in these three distinct groups. High responders were defined as 136

having OD values > mean + 2 STD of the North American controls. Non-responder sera had 137

OD values < mean + 1 STD of the control serum. For the peptide scan, B-cell epitopes were 138

identified by comparative analysis of specific antibody reactivity (average OD values) 139

between inhibitory and non-inhibitory sera for each specific peptide. A baseline OD value 140

was established using serum from non-exposed North Americans. Peptide array data were 141

analyzed using SPSS (Version 11.5). Non-Parametric analysis (two independent samples; 142

Mann-Whitney test) was used for comparison of peptide reactivity between high and non-143

inhibitory sera. The results were considered statistically significant values at P< 0.05. 144


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RESULTS 145

Serological response to DBP. Naturally acquired anti-DBP responses of thirty-eight 146

residents living in a highly endemic area of Papua New Guinea were evaluated by ELISA at 147

the end of the 2001 high transmission season for malaria. None recalled experiencing a 148

clinical episode of malaria over the prior six months and none was positive for Plasmodium 149

parasitemia by blood smear at the time of collection. Antigen-specific titers were 150

surprisingly variable and could be assigned to three distinct antibody response groups: non-151

responders (N), low (L), or high (H) (Table1). Comparison of these responder 152

classifications to samples collected the previous year revealed that 50% of the volunteers 153

changed responder classification between the 2000 and 2001 transmission seasons (Table1). 154

Twelve of the original high responders switched to low responders and two changed to non-155

responders. The seven low responder individuals remain unchanged, but four of the non-156

responders moved into the low responder group and one non-responder switched to a high 157

responder. Changes in response category were not observed to correlate with either age or 158

gender of the individual. 159

Measurement of functional inhibition of DBP-erythrocyte binding. To evaluate the 160

correlation between anti-DBP titer and functional inhibition of the DBP-erythrocyte 161

interaction, serum samples were tested individually for in vitro inhibition of DBPII binding 162

to Duffy positive erythrocyte, in a manner similar to tests done in a previous study (17). 163

Unexpectedly, functional inhibition by these individual samples did not correlate with their 164

anti-DBP antibody titer level (Fig. 1). Only three of the ten high responder samples and one 165

of the twenty-three low responder samples (in a total of 38 samples) inhibited binding 166

completely or almost completely. Most high titer serum samples had a moderate to no 167


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inhibitory effect on DBP binding to erythrocytes. None of the highly inhibitory sera changed 168

responder classification between the 2000 and 2001 sample collections. Only sera with high 169

titer anti-DBPII antibodies completely inhibited DBPII binding but not all high titer sera 170

were inhibitory. This indicates that titer alone is not an indicator of functional inhibition, a 171

characteristic consistent with the predominant strain-specific responses observed in PNG 172

residents. The low levels of inhibition in the functional assays observed for the non-173

responder sera may reflect differences in epitope presentation on functional DBPII expressed 174

on COS cells, such as conformational epitopes not present on the recombinant DBPII used in 175

the ELISA. 176

Mapping of the dominant epitopes of DBP ligand domain. To identify potential 177

DBPII-neutralizing B-cell linear epitopes, we compared reactivity of inhibitory high titer 178

human sera (high inhibition (HI) group inhibited DBPII-erythrocyte binding) versus non-179

inhibitory high titer sera (no inhibition (NI) group did not inhibit DBPII-erythrocyte binding) 180

to 178 peptides, spanning the 330-residue ligand domain of DBP. The HI group included 181

three samples (1, 2, 3) that completely and one sample (11), that almost completely inhibited 182

DBPII-erythrocyte binding and the NI group included 22 of the remaining high and low titer 183

positive samples (Fig. 1). The average responses of non-exposed NA residents (N=6) served 184

as the control for background reactivity. A positive reaction to a peptide was defined as OD 185

reactivity > mean + 2 STD of NA controls (Fig. 2, grey profile). A peptide was considered 186

part of a potential epitope if there was significantly higher antibody reactivity of HI 187

antibodies versus NI group. Potential inhibitory B-cell epitopes were identified as the shared 188

sequence present in > 2 contiguous overlapping peptides with significantly more reactivity to 189

HI sera but not NI samples. An isolated positive peptide was not included as a B cell epitope. 190


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Comparative analysis between inhibitory and non inhibitory sera revealed a strong 191

differential binding to ten putative continuous B-cell epitopes, centered on peptides 22, 31, 192

46, 56, 63, 76, 83, 90, 98 and 109 (Table 2). Statistical analysis indicated some of these 193

presumptive linear targets of protective immune responses had significantly stronger 194

correlations with the inhibitory responses. Based on the significance of difference (P-value) 195

from the NI average reactivity, the inhibitory epitopes were categorized into groups high (H), 196

medium (M) and low (L) (Table 2). When mapped onto the 3D model structure of DBPII 197

most of these linear epitopes were localized in sub-domain II while two were present in sub-198

domain I of DBPII and no epitopes preferentially correlating with inhibitory sera were 199

identified in sub-domain III (Fig. 3, SI. 2). Of greatest interest as targets are the H epitopes, 200

especially the conserved H2 epitope that overlaps an area implicated as part of the DBPII 201

receptor recognition site associated with binding a putative sulfated tyrosine of DARC (7). 202

Allelic variation is concentrated in the inhibitory B-cell epitopes. Previous studies 203

identified that DBP polymorphisms are concentrated within DBPII sub-domain II, 204

suggesting positive changes driven by immune selection similar to other functional microbial 205

ligands. Consistent with this understanding, we find numerous polymorphisms in the 206

strongest B-cell epitopes identified in this study. These variant epitopes include four of the 207

most highly reactive peptides with the inhibitory immune sera, H1, H3, M2 and M3 (Table 208

2). These B-cell epitopes contain polymorphic residues in alleles identified in the PNG 209

population (26) and the variant residues tended to have a central location within the B-cell 210

epitopes as they are clustered on the 3D DBPII structure. 211

Antibodies to B-cell epitopes significantly reduce DBPII binding to erythrocytes. To 212

determine whether antibodies against the identified B-cell epitopes are inhibitory to DBPII 213


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binding to Duffy positive erythrocytes, affinity purified antibodies against the H epitopes 214

(H1, H2, H3) and two L epitopes (L3 and L4), as well as a peptide NI 215

(CDGKINYTDKKVCKVP), from an epitope- recognized by the non-inhibitory sera 216

(between peptides 121-124, see Fig. 2), were affinity purified from a pool of high titer anti-217

DBPII inhibitory sera from vivax exposed individuals or rabbit sera raised against epitopes 218

H1 and H3. The purified antibodies were tested for their ability to inhibit DBPII-Sal 1 219

binding to human Duffy positive erythrocytes in a COS 7 cell assay. The antibodies showed 220

a dose dependent inhibitory response of DBPII binding to the erythrocytes (Fig. 4). At the 221

highest concentration of antibody tested (10ug/ml), we observed a 73-82% inhibition of 222

binding with the affinity purified anti-H epitope antibodies (IC50 = 2.5-5ug/ml), compared to 223

barely 42-43% with the purified anti-L epitope antibodies (Student’s t-test P < 0.0001) and 224

less than 18% with the purified anti-NI epitope antibody (Student’s t-test P < 0.0001). 225

Antibodies from similarly purified rabbit sera showed a lower inhibitory effect than the 226

naturally acquired human antibodies (see SI. 3a), while purified total IgG from the same-227

pool of inhibitory human sera when tested for inhibition showed a 50% binding inhibition 228

concentration at least 2-4 folds greater than that observed for the affinity purified antibodies 229

(data not shown). This indicates that multiple epitopes within the DBPII ligand domain 230

contribute to the overall induction of protective immunity to P. vivax infection. Control 231

experiments with total IgG from sera of non-exposed North Americans and total IgG from 232

pre-immune rabbit sera showed a binding inhibition ≤ 4% (data not shown). 233

Affinity purified antibody activity is inhibited by homologous peptide. To confirm 234

whether the purified antibodies against the synthetic linear peptides mediated the observed 235

inhibitory response observed in Fig. 4, 10ug/ml of the affinity purified human antibodies to 236


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epitopes H1, H3, L3, L4 and NI, were first incubated with the respective peptides at different 237

concentrations for 1hr and the binding experiment repeated. Pre-incubation of the purified 238

antibody with the matching peptides at a concentration of 20µg/ml greatly blocked the 239

functional inhibitory effect of the antibodies (SI. 3b), confirming that these antibodies are 240

highly enriched against the specific peptides. 241


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DISCUSSION 242

Plasmodium vivax depends upon the interaction of the DBP with its cognate receptor for 243

efficient invasion of human reticulocytes. The vital nature of this interaction makes DBP an 244

ideal target for vaccine development. Potential impediments to its use as an effective 245

vaccine has been attributed to the low frequency development of naturally acquired DBP 246

binding inhibitory antibodies, lack of knowledge of DBP contact residues necessary for 247

receptor recognition, and the polymorphic nature of the DBP ligand. Understanding the 248

specificity of the protective anti-DBP responses is critical for effective vaccine development. 249

Therefore, one purpose of this study is to obtain a clearer understanding of anti-DBP 250

serological responses from natural exposure and initiate identification of epitopes targeted by 251

functionally inhibitory anti-DBP antibodies. We present evidence identifying potential 252

inhibitory B-cell linear epitopes within the critical receptor-binding region of the DBP ligand. 253

When we screened residents of a highly endemic area of PNG we discovered that 254

naturally acquired antibodies that effectively inhibit DBPII-erythrocyte binding are 255

infrequent. There was great variability among these residents and a substantial proportion 256

(≈50%) changed significantly their titer from one transmission season to the next (Table1). 257

Such relatively rapid changes in serological responses is similar to a previous observation in 258

PNG that temporal variation was the primary source of variation in the IgG response to 259

Plasmodium antigens (21). The small number of high titer, highly inhibitory sera remained 260

relatively stable, which is consistent with another study in this endemic area (15), but low 261

responder individuals whose responses were mostly ineffective at functional inhibition of 262

DBP binding to RBCs also maintained a poor quality antibody response across the sampling 263

periods. 264


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In the high titer group only three individuals of 10 effectively inhibited DBP function 265

while only one of the 23 low responders achieved a strong level of inhibition. Such marked 266

individual differences in titers of the naturally acquired anti-DBP responses and their 267

functional inhibition of DBP-erythrocyte binding, indicated that epitope specificity as well as 268

antibody avidity are critically important for inhibitory efficacy. In addition, our results 269

indicate that DBP inhibitory antibodies are relatively infrequent and most antibody responses 270

are relatively unstable. These discoveries for anti-DBP serological responses contrast with 271

acquisition of immunity to the homologous VAR2CSA DBL domain where there is a better 272

correlation between development of protective immunity and repeated exposure (20). 273

However, a longitudinal study to closely monitor the development of antibodies and B-cell 274

memory to DBP will be necessary to adequately determine the stability of naturally acquired 275

anti-DBP antibodies. 276

Comparing the epitope specificity of the highly inhibitory anti-DBP sera with the non-277

inhibitory immune sera identified potential targets associated with protective anti-DBP 278

immunity. Nearly all of the putative inhibitory B-cell linear peptides identified in this study 279

are localized in the central region of the DBP ligand domain. This is similar to naturally 280

acquired antibodies to the VAR2CSA-DBL that are directed towards surface-exposed 281

epitopes within the S1/S2 domains, a region homologous to the central region of the DBPII 282

and the F1/F2 domains of EBA-175 (3). This central region is known to be important for 283

receptor recognition, but precisely how the Duffy receptor, (DARC), interacts with this 284

region is not yet understood. Mutagenesis studies have identified numerous residues 285

throughout this region as important or essential for receptor recognition, including many 286

residues absent from the 3D model based on the crystal structure of the P. knowlesi DBPα 287


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(13, 19, 24, 25). Much attention is focused on a receptor-binding pocket that is proposed to 288

recognize a sulfated tyrosine thought to be a part of the N-terminal domain of the DARC 289

receptor similar to CXC-chemokine receptors (7, 19). This DARC binding site lies on the 290

opposite surface from the residues on the homologous DBL domains of P. falciparum EBA-291

175 identified as important for interacting with its receptor, glycophorin A. Even though 292

these homologous domains are structurally conserved, this highlights a significant difference 293

reported for how these ligands bind their cognate receptors, which for EBA-175 is a 294

“handshake” dimerization of the tandem F1/F2 DBL to form central channels that bind 295

glycophorin A (22) compared to a proposed monomeric interaction for DBP (19). 296

The most significant neutralizing epitopes identified in our study are split between two 297

areas of the DBL domain that are potentially important for receptor recognition (H1, H3) and 298

binding the DARC sulfated tyrosine (H2). Epitopes H1 and H3 lie within regions containing 299

variant residues with radical substitutions while H2 positioned has minimal variation. 300

Epitopes designated as moderately inhibitory (M1, M2, M3) occupy a region parallel to the 301

H2 epitope flanking the other side of the putative tyrosine recognition motif. The location of 302

multiple putative epitopes (H2, M1-3) as targets of protective immunity does not support the 303

‘’just in time’’ release model of immune evasion (6, 19), since identification of multiple 304

targets for neutralizing antibodies in the area flanking the DARC sulfated tyrosine-binding 305

site shows that this site is accessible to inhibitory antibodies. The relative lack of variability 306

in this area may be a consequence of other functional requirements necessary for receptor 307

recognition and a somewhat weaker effect of immune selection on these epitopes. 308

Antibody reactivity to the H1, H2 and H3 epitopes, which possess clusters of 309

polymorphic residues, had a significant correlation with inhibition of DBPII-erythrocyte 310


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binding. For that reason, these epitopes do not appear to be simply associated with a non-311

protective immune evasion mechanism misdirecting antibodies away from crucial functional 312

sites elsewhere on the ligand domain. Instead the pattern of polymorphisms of DBPII is 313

similar to the pattern of variability observed for the P. falciparum vaccine candidate AMA1 314

where polymorphisms are concentrated adjacent to the putative receptor binding site (4). It 315

appears likely that the receptor-binding site of DBPII is larger than just that identified so far 316

for the DARC sulfated tyrosine. 317

Our results identify critical DBP epitopes that are the targets of inhibitory antibodies 318

naturally acquired in P. vivax infection and represent an important advance in understanding 319

a part of blood-stage immunity against P. vivax. As targets of functionally inhibitory 320

antibody, recognition of these linear epitopes may represent potential correlates of immunity 321

for a DBP vaccine, although further characterization is necessary to confirm their relative 322

importance to other epitopes (e.g. conformational epitopes). Identification of specific 323

epitope targets of inhibitory immunity against DBP opens the way to optimize DBP 324

immunogenicity for protection against diverse P. vivax strains. 325


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ACKNOWLEDGMENTS 326

We thank Dr. Ren Chen, Biostatistics Core Research, College of Medicine, University of 327

South Florida and Samantha Jones for help with statistical analyses. We also thank Amy M. 328

McHenry for critical discussion. Finally we are grateful to participants in Papua New 329

Guinea. This study is supported by the National Institutes of Health, grant R01AI064478 330

(JHA). 331


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Figures, Tables and Legends

Table 1. Naturally acquired anti-DBP serum antibody levels of residents in an area of Papua New Guinea

highly endemic for vivax malaria during two transmission seasons.

425

426

427

428

429

430

431

432

433

434

435

436

437

438

439

440

441

442

443

444

445

446

447

448

449

450

451

452

453

454

High responder (H), Low responder (L) and Non-responder (N) as defined in the text. 455

____________________


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Table 2. The potential inhibitory B-cell epitopes in DBPII ligand domain. 457

459 a Mean ± SD values.

b P-values obtained by comparison between average values HI and NI in 460

non-parametric Mann-Whitney test. The results were considered statistically significant 461

values at P< 0.05. Highly inhibitory B-cell epitopes (H1-H3), moderately inhibitory B-cell 462

epitopes (M1-M3) and Low inhibitory B-cell epitopes (L1-L4). 463


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Figure 1. Inhibition of DBPII binding to Duffy positive erythrocytes by human sera from 464

Papua New Guinea. Individual sera were tested at a dilution of 1:10 for inhibition of binding 465

in an in vitro assay (COS7 cell assay). Sera are color coded by category of response to 466

recombinant DBP as determined by ELISA: high responders (black), low responders (grey) 467

and non-responders (light grey). Sera showed marked differences in inhibitory efficacy, and 468

these differences in inhibition did not correlate with antibody titers. Classifications for 469

binding inhibitory effect: major effect ≥80% inhibition; moderate effect is 40-80% inhibition; 470

and minor or no effect – 0-40% inhibition. 471

NB: subject ID used is not the same as in table 1. 472

Serum samples

Pe

rce

nta

ge r

ela

tive b

ind

ing

High responders Low responders Non-responders


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Figure 2. Reactivity of human sera to overlapping peptide array of DBPII. The average OD 473

values of each peptide to high inhibitory sera (n=4) and non-inhibitory sera (n=22) are 474

shown in red and green respectively, while gray represents the average OD values of the 475

non-exposed North American sera (n=6) that served as background control. An epitope is 476

considered a B-cell epitope when the difference in OD value between the high inhibitory sera 477

(HI) and non-inhibitory sera (NI) is greater than the mean + 2 STD of the North American 478

control sera. 479

Op

tica

l de

nsity

Peptides


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Figure 3: 3D model structure of DBPII. The structure is depicted as a space-filling molecule 480

showing localization of B-cell epitopes. The structure is rotated in each representation by 481

90 o in the horizontal axis. (A) front: 0

0, (B) top: 90

0, (C) back: 180

0, (D) 270

0. High (H1-H3), 482

medium (M1-M3) and Low (L1-L4) inhibitory B-cell epitopes are shown in red, yellow and 483

green respectively against a gray background, while residues of predicted DARC binding site 484

are shown in dark blue. 485


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Figure 4. Inhibition of DBPII binding to human erythrocytes. Human antibodies specific to 486

peptides H1, H2, H3, L3, L4 and NI were tested for ability to inhibit DBP binding to Duffy 487

positive erythrocytes. Different concentrations of the antibodies were pre-incubated with 488

transfected COS 7 cells prior to addition of erythrocytes and number of rosettes counted per 489

30 fields of the microscope at X200. The bars indicate the mean percentage binding of three 490

independent experiments (± STD) relative to control experiment with 0µg/ml antibody. At 491

10ug/ml antibody concentration P < 0.0001 for H vs L; H vs NI and L vs NI. P-values were 492

adjusted for multiple comparisons. 493


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