Acquired antibodies to merozoite antigens in children from Uganda 1 with uncomplicated or severe Plasmodium falciparum malaria 2 3 Hodan Ahmed Ismaila, Ulf Ribackea, b, Linda Reilingc, Johan Normarka, Tom 4 Egwangd, Fred Kironded, James G. Beesonc, Mats Wahlgrena & Kristina E. M. 5 Perssona# 6 7
a. Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska 8 Institutet, Stockholm, Sweden 9
b. Department of Immunology and Infectious Diseases, Harvard School of Public 10 Health, Boston, MA 02115, USA 11
c. Burnet Institute for Medical Research and Public Health, Melbourne, Victoria, 12 Australia 13
d. Department of Biochemistry, Makerere University, Kampala, Uganda 14 15 16 17 Running title: Antibodies in uncomplicated and severe malaria 18 19 Corresponding author: Kristina E. M. Persson, Department of Microbiology, Tumor 20 and Cell Biology (MTC), Nobels väg 16, Karolinska Institutet, SE 17177 Stockholm, 21 Sweden. Phone +46-8-52484509 email [email protected] 22 23 24 25
Malaria can present itself as an uncomplicated or severe disease. We have here 28 studied the quantity and quality of antibody responses against merozoite antigens, as 29 well as the multiplicity of infection (MOI) in children from Uganda. We found higher 30 levels of IgG antibodies towards EBA181, MSP2-3D7/FC27 and AMA1 in patients 31 with uncomplicated malaria by ELISA, but no differences against EBA140, EBA175, 32 MSP1, Rh2 and Rh4 or for IgM against MSP2-3D7/FC27. Patients with 33 uncomplicated malaria were also shown to have higher antibody affinities for AMA1 34 by surface plasmon resonance (SPR). Decreased invasion of two clinical P. 35 falciparum isolates in presence of patient plasma correlated with lower initial 36 parasitemia in the patients, in contrast to comparisons of parasitemia to ELISA or 37 affinity that did not show any correlations. 38
Analysis of the heterogeneity of the infections revealed a higher MOI in 39 patients with uncomplicated disease, with MSP1-K1 and MSP2-3D7 being the most 40 discriminative allelic markers. Higher MOI also correlated positively with higher 41 antibody levels in several of the ELISAs. 42
In conclusion, certain antibody responses and MOI were associated with 43 differences between uncomplicated and severe malaria. When combining different 44 assays, some antibodies like those against AMA1 seemed particularly discriminative. 45 However, only decreased invasion correlated with initial parasitemia in the patient, 46 signaling the importance of functional assays in understanding development of 47 immunity against malaria and for evaluation of vaccine candidates. 48 49
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Introduction 50 51 Malaria is a parasitic disease caused by the intracellular protozoan Plasmodium. Most 52 of the deaths are caused by Plasmodium falciparum, and the majority of cases occur 53 in children and pregnant women in Sub-Saharan Africa (1, 2). P. falciparum invasion 54 of erythrocytes involves different invasion pathways with multiple interactions 55 between merozoite antigens and erythrocyte receptors (3). Two main protein families 56 involved in invasion are erythrocyte binding-like (EBL) proteins and reticulocyte 57 binding protein homologue (RBP/PfRh) proteins. The erythrocyte binding antigens 58 (EBAs) are part of the EBL family, and include EBA140, EBA175 and EBA181, 59 while PfRh1, PfRh2, PfRh4 and PfRh5 are among the PfRh proteins (4-6). Changes in 60 invasion pathways have been shown to influence the susceptibility of P. falciparum to 61 human invasion inhibitory antibodies (7). Other proteins that are central in the 62 invasion process include merozoite surface proteins (MSPs), such as MSP1 (8) and 63 MSP2 (9). The merozoite proteins are highly polymorphic, and MSP1 can be divided 64 into three (K1, MAD20, and RO33) and MSP2 into two allelic types (3D7 and FC27) 65 (10, 11). Apical membrane antigen 1 (AMA1) is a protein that has been described to 66 be essential for invasion, and in comparison to many other merozoite antigens, AMA1 67 is found in all Plasmodium species and it´s sequence is relatively conserved between 68 different parasite lines (12, 13), even though several polymorphisms have been 69 described in the ectodomain (14, 15). 70
Individuals living in malaria-endemic areas develop immunity, but only 71 slowly and after repeated exposure. Passive transfer of antibodies from immune 72 donors to individuals with P. falciparum infection has been shown to reduce 73 parasitemia and clinical symptoms (16-18). Immunity against severe malaria usually 74
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develops before total protection against disease is established (19), indicating either 75 that different P. falciparum antigens are important in protection from severe 76 compared to uncomplicated malaria, or that the quality of the antibodies in the two 77 groups is different. Antibodies against several merozoite antigens have been found to 78 be associated with protective immunity in prospective longitudinal studies (20-30). 79 However, very few studies have examined the functional properties of acquired 80 antibodies (31), or examined the role of antibodies to merozoite antigens in immunity 81 to severe malaria in young children. Invasion inhibition assays (IIA) and growth 82 inhibition assays (GIA) can be applied to study the function of antibodies in vitro. 83 Studies have described the invasion/growth inhibitory activities in sera from 84 individuals living in malaria endemic areas (7, 17, 32-37). These naturally acquired 85 inhibitory antibodies (causing reduced invasion of parasite isolates) are present in 86 many clinically immune individuals, but more seldom in susceptible individuals, and 87 are higher in areas with higher levels of malaria transmission (36). Some studies have 88 shown a protective effect of inhibitory antibodies (33, 38, 39), however, the 89 association between the inhibitory activity of antibodies and protection in malaria 90 remains unclear (35, 36, 40). 91
The lack of in vitro functional assays that correlate with protective immunity 92 in vivo has hampered the development of effective blood stage vaccines (1). There 93 have been inconsistencies in the correlations of antibody responses to recombinant 94 antigens and protection from malaria using ELISA (36). Trials that aim to improve the 95 value of ELISAs have included the use of ammonium thiocyanate (NH4SCN) ELISA 96 to estimate avidity of antibodies (41), but the introduction of surface plasmon 97 resonance (SPR) (42) has opened up new opportunities to measure affinity of 98 antibodies under flow, something that ought to be more similar to the physiological 99
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situation compared to static ELISAs. SPR is a method where association and 100 dissociation between antibody and antigen can be studied in real time and it has been 101 essential in vaccine development studies for other pathogens, such as HIV (42). In 102 malaria, SPR has mainly been used for studies of monoclonal antibodies, (43, 44) but 103 a recent study of naturally acquired polyclonal antibodies showed that individuals 104 with high affinity antibodies directed against MSP2-3D7 showed prolonged time to 105 developing clinical malaria (45), indicating that presence of high affinity antibodies 106 may be important in protection against malaria. The method used is probably of 107 importance, since another study using guanidine thiocyanate for evaluation of the 108 strength of antibody binding did not show any correlation to clinical malaria (46). 109
Whether a patient will develop uncomplicated or severe malaria, is not only 110 influenced by the immune responses of the host, but also by genetic differences in the 111 parasites, which might in turn be dependent on the transmission level in the area since 112 high transmission will cause more frequent recombination events in the mosquito. 113 Patients often harbor many variants of parasites at the same time (47-52), and 114 characterization of different clones of P. falciparum could be a useful tool to 115 understand the molecular epidemiology of malaria. However, the results in the 116 literature have been conflicting as to whether multiclonal infections are associated 117 with the outcome of the infection (53-60). A way of determining the importance of 118 the heterogeneity of infections is to measure multiplicity of infections (MOI), which 119 is an estimation of how many genetically different parasites the patient is harboring, 120 and investigate potential associations between MOIs and the outcomes of an infection. 121
In this study, we have examined antibody responses to merozoite antigens and 122 MOI among children with uncomplicated versus severe P. falciparum malaria. We 123 have studied quantitative and qualitative differences in antibody responses against 124
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merozoite antigens, including ELISA, IIA, NH4SCN-ELISA and SPR to identify 125 antibody responses that may play a role in protective immunity, and evaluate which 126 method(s) are predictive of immune differences between severe or uncomplicated 127 malaria. This will enable us to better understand how immunity in malaria is 128 developed and which antigens should be prioritized in future vaccine trials. 129 130
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Materials and Methods 131 132 Study site and population 133
A total of 85 children (6 months- 3 years of age) with active P. falciparum 134 infection were included in this study as described previously (61). They were 135 recruited from the district hospital in Apac, Northern Uganda in a holo-endemic area 136 and the patients were grouped according to WHO guidelines (62). This area has been 137 shown before to have a very high EIR of >1500 (63, 64). 46 patients were classified 138 as having severe malaria, and 39 patients were defined as having uncomplicated 139 malaria. Written informed consent to participate in the study was received from all 140 guardians, and ethical permissions were granted for the study from Makerere 141 University Faculty of Medicine Research and Ethics Committee in Uganda (nr MV 142 717), and the Stockholm Ethical Review Board (permission numbers 03-095). 143 144 Invasion inhibition assay 145
The method to study invasion inhibitory antibodies has been described 146 previously (7, 65). Two Ugandan P. falciparum isolates (UAM37, from a patient 147 with uncomplicated malaria, and UAS31, from a severe malaria patient) were chosen 148 as representative of isolates in this study group (61). Isolates were cultured in vitro in 149 AB+ non-immune Swedish serum and gassed with 90% NO2, 5% O2 and 5% CO2 150 and placed in a shaker incubator. In brief, parasites were synchronized (5% sorbitol, 151 v/w) before assay start, and at the day of the assay the majority of the parasites were 152 at late-pigmented trophozoite stage. 50 μl of parasite suspensions were cultured for 153 one cycle in 96 well plates. 5 μl of dialyzed test plasma was added to each well and 154 all samples were run in duplicate. Plates were incubated in a sealed, humidified, 155
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gassed box and put in an incubator for 48 hours at 37 °C. Parasitemia was estimated 156 using hydroethidine (10 ug/ml; Sigma Aldrich) in a flow cytometer (FACS Scan; 157 BD) after approximately 48 hours (determined by the parasite stage). Parasite 158 invasion for each sample was measured in comparison to controls (invasion in 159 presence of dialyzed Swedish plasma). If there was no invasion inhibitory activity in 160 the added plasma samples, the invasion of the parasites was 100%. 161
162 Recombinant proteins 163
All recombinant proteins were expressed in E. coli, including the whole ecto-164 domain of AMA1-D10 (12), regions III-V of EBA140 (3D7; aa; 770-1064), EBA175 165 (3D7; aa 761-1298), and EBA181 (3D7; aa, 769-1365) (29), Rh2A9 (3D7; aa2027-166 2533), (66), and Rh4A3 (aa 1160-1370) (7, 67). The MSP2 corresponding to the 167 FC27 and 3D7 gene sequences (10) were expressed as described. MSP1-19 from the 168 3D7 sequence was amplified from LIEGKF-DGIFCS with flanking SacII and PstI 169 sites and ligated in pASK45+ from IBA, Germany. It was purified and refolded as 170 described (68). The recombinant proteins were GST tagged. 171 172 Antibodies against recombinant proteins by Enzyme-Linked Immunosorbent 173 Assays (ELISA) 174
ELISA: For MSP2-FC27, MSP2-3D7, and AMA1-D10, the ELISA was 175 performed as described (45). For MSP1, the EBAs and Rhs the method described by 176 Persson et al. was used (7). OD was measured at 405 nm. Test samples at dilution 177 1:100 were run in duplicate together with positive and negative controls to allow for 178 standardization; a pool of exposed adult individuals from Uganda, and 5-7 non-179
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immune Swedish donors. Antibody reactivity was considered positive when OD was 180 greater than mean +3 SDs of the non-immune Swedish donors. 181 Ammonium thiocyante (NH4SCN)-ELISA: To estimate the strength of the binding, 182 increasing concentrations of NH4SCN were added to ELISAs for the MSP2-3D7 and 183 MSP2-FC27 proteins as described (45). The affinity index was calculated from the 184 absorbance readings in the presence of increasing concentrations of NH4SCN, which 185 were converted to percentage of total bound antibody (in the absence of NH4SCN). 186 The index was calculated from the molar concentration of NH4SCN required to 187 reduce the initial absorbance by 50% (69). 188
189 190 Surface Plasmon Resonance 191
To estimate the affinity of antibodies in plasma binding to different antigens, 192 the recombinant MSP2-3D7, MSP2-FC27 and AMA1-D10 were bound to CM5 chips 193 in Surface Plasmon Resonance assays (Biacore 3000, Uppsala, Sweden) as described 194 (45). One lane was used as a control lane. Around 1000 response units of each 195 protein was bound to the chip. Plasma samples in at least two different dilutions 196 (used as “internal controls”, since the off rate kd should be the same independent of 197 concentration) were flowed over the antigen-coated surfaces, and the kd was 198 measured in real time. When antibody levels were very high, the samples were 199 diluted further until similar levels of response units were accomplished. For every 20 200 samples, a control sample (a pool of adult immune plasma) was run to check that 201 there was still enough protein bound on the chip to allow for measurement of 202 reproducible kd values, and this was also used as a control between different chips. 203 Swedish non-immune sera was used to determine the cut-off to be considered as 204
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background level (=100 RU). Data was analyzed using BIAevaluation 4.1 software 205 (Langmuir binding model). 206 207 Determination of P. falciparum genetic diversity 208
27 uncomplicated and 29 severe cases were randomly selected from the 209 collected samples for studies of genotypes. A nested PCR was used to study the 210 genetic markers for the MSP2 alleles FC27 and 3D7, for CSP, GLURP, and MSP1 211 K1, MAD20 and RO33 alleles. The primers and experimental conditions used has 212 been described previously (70, 71), with some modifications (72). 213 214 Statistical analysis 215
Data analyses were performed using GraphPad Prism Version 5.0a software 216 and SAS 9.2. To test for differences in mean absorbance and affinity to the 217 recombinant antigens, and in invasion, Mann-Whitney test was used. The Pearson 218 correlation coefficient was used to assess the association between two continuous 219 variables. Two-tailed P-values were considered significant if they were <0.05. 220 221 222 223 224
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Results 225 226 Patient information and initial parasitemias 227
In table 1, information about clinical status of the patients can be found. When 228 the initial clinical parasitemias in the patients were compared in uncomplicated and 229 severe malaria, a significant difference was found with higher parasitemias in the 230 severe malaria cases, as could be expected (Table 1, 2). 231 232 IgG and IgM antibody levels in uncomplicated and severe malaria estimated by 233 ELISA 234
IgG levels against the recombinant merozoite antigens EBA140, EBA175, 235 EBA181, PfRh2, PfRh4, MSP1, MSP-3D7, MSP2-FC27 and AMA1 were assessed 236 using ELISA. For EBA181, MSP2-3D7, MSP2-FC27 and AMA1 the IgG levels were 237 significantly higher in patients with uncomplicated compared to severe malaria (Table 238 2, Figure 1). As positive control, a pool of immune donors was used and usually gave 239 OD values around 0.8-1.0. Swedish non-immune plasma was used as negative 240 control, and resulted in OD values <0.07. 241
An extended analysis was performed for the two MSP2 proteins for which we 242 also performed IgM ELISAs, but no significant differences between uncomplicated 243 and severe malaria were observed (Table 2). 244
There was a high prevalence of antibodies in general against all antigens tested 245 (EBA140 88%, EBA175 94%, EBA181 87%, Rh2 92%, Rh4 80%, MSP1 98%, and 246 100% for AMA1, MSP2-FC27, MSP2-3D7). There were no major differences in 247 prevalence between uncomplicated and severe patients; The only difference that 248
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reached significance was EBA140 with a slightly lower prevalence in the mild cases, 249 78%, compared to 95% in severe cases, p=0.02. 250 251 Antibody affinity in uncomplicated and severe malaria 252
To further study the different properties of antibodies in uncomplicated and 253 severe malaria, a subset of antigens were selected for studies of affinity of antibodies 254 using surface plasmon resonance (SPR). Two allelic variants of the highly 255 unstructured MSP2 protein, MSP2-FC27 and MSP2-3D7, were chosen together with 256 AMA1. These antigens were selected since we had access to two alleles of the highly 257 unstructured MSP2 proteins, and AMA1 was added because it is a globular protein 258 with a stable tertiary structure. Affinity was estimated using the dissociation rate, kd. 259 Antibodies against AMA1 showed the highest affinity (=lower kd values), while 260 antibodies against MSP2-3D7 had lower affinity and antibodies against MSP2-FC27 261 had the lowest affinity (Table 2, Figure 2). For all three antigens, there was trend of a 262 higher affinity among the uncomplicated malaria samples, but it only reached 263 statistical significance for AMA1. 264
Before, other studies have used NH4SCN to estimate affinity of antibodies, 265 and we therefore wanted to include this method in our studies for comparison with the 266 more precise SPR method. Increasing concentrations of NH4SCN were added after 267 incubation of MSP2-3D7 and MSP2-FC27 with total IgG. The affinity indexes were 268 not significantly different in uncomplicated compared to severe malaria (Table 2). 269 270 Invasion inhibitory antibodies in uncomplicated and severe malaria 271
We used two clinical P. falciparum isolates (derived from patients in the 272 study) to measure the invasion inhibitory activity of antibodies in patient plasma. If 273
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there was no invasion inhibitory activity in the added plasma samples, the invasion of 274 the parasites was 100%. One isolate was from a patient with uncomplicated malaria 275 (UAM37) and one was from a patient with severe malaria (UAS31). The plasma from 276 the patients harboring the parasites UAM37 and UAS31 showed decreased parasite 277 invasion in the presence of the plasma, with a range of 17-33% invasion (i.e. 83-67% 278 invasion inhibition), relative to control samples. The parasite UAM37 contained both 279 the Fc27 and 3D7 alleles of MSP2 and all 3 alleles tested for MSP1, while UAS 31 280 contained only 3D7 of MSP2 and RO33 of MSP1 (but not K1 or MAD20). When all 281 patient plasma samples were tested against UAM37 and UAS31, there was a trend of 282 decreased invasion (57.9%) when plasma from uncomplicated malaria patients was 283 used against UAS31, compared to severe malaria (70% invasion), but the difference 284 was not significant (Table 2). A subset of samples (4 from patients with 285 uncomplicated malaria and 4 from patients with severe malaria) was used to test for 286 whether the decreased parasite invasion was mainly due to an invasion inhibitory or 287 growth inhibitory effect. We performed invasion inhibition assays with analysis of 288 parasitemia using microscopy and flow cytometry at different time points (not 289 shown), and could see that a major part of the inhibition in our assay setup was found 290 within the first 8 hours, hence we chose to refer to our assay as “Invasion Inhibition 291 assay”, IIA. 292 293 Correlation between different assays for measuring antibodies, and initial 294 parasitemias 295
Both clinical P. falciparum isolates (UAM37 and UAS31) showed increased 296 invasion in the presence of plasma from individuals with high initial parasitemias 297
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(Figure 3), indicating a lack of inhibitory antibodies (UAS31; R2=0.22 p=0.0009; 298 UAM37; R2=0.16 p=0.006). 299
There was no correlation between initial parasitemias and the presence of 300 either IgG or IgM to any of the tested antigens as measured by ELISA, and no 301 correlation between initial parasitemias and affinity of antibodies against the antigens 302 tested. 303 304 Correlation between different aspects of measuring antibodies 305
The most extensive analysis for comparison of methods for measurement of 306 antibody responses in plasma was carried out for the MSP2-3D7 and MSP2-FC27 307 antigens, for which we had access to two different allelic families of the proteins. As 308 expected (since the two alleles partly contain the same sequences), IgG and IgM 309 ELISA results for both allelic families correlated well with each other (p-values for all 310 comparisons from 0.02 to <0.0001, R2 values 0.07-0.50, except for the comparison 311 IgM MSP2-3D7 – IgG MSP2-FC27 where the p-value was 0.1, R2=0.03). When 312 MSP2 NH4SCN results were compared to IgG ELISA results, significant positive 313 correlations could be seen both for MSP2-3D7 NH4SCN (R2=0.36, p=<0.0001 against 314 both MSP2-FC27 ELISA and MSP2-3D7 ELISA) and for MSP2-FC27 NH4SCN 315 (R2=0.3, p<0.0001 against MSP2-FC27 ELISA and R2=0.07, p=0.02 for MSP2-3D7 316 ELISA). 317
Between parasite invasion and presence of antibodies in IgG/IgM ELISAs, a 318 significant correlation could be seen for UAS31 between IIA and the IgG ELISA 319 response against MSP2-FC27 (R2= 0.20, p=0.0016), for UAM37 against EBA175 320 (R2=0.09 p= 0.03), and for both UAS31 and UAM37 against PfRh2 (UAS31 R2=0.16 321 p= 0.006; UAM37 R2=0.14, p=0.01) with higher levels of antibodies in samples with 322
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decreased parasite invasion. The best correlations were seen when IIA results were 323 compared to IgG ELISA for AMA1 for both isolates (UAS31 R2=0.25, p= 0.0003; 324 UAM37 R2=0.19, p=0.0023, Figure 4). For AMA1, we could also see a correlation 325 between high levels of IgG antibodies in ELISA and high affinity in SPR (R2=0.31, 326 p<0.0001) (Figure 5). 327
For the other recombinant proteins used in IgG ELISA (EBA140, EBA181, 328 PfRh4, MSP2-3D7 and MSP1), no significant correlations could be seen with IIA 329 results. 330
When IgG/IgM ELISA results were compared to each other, many of the IgG 331 results correlated to each other. For example, AMA1 ELISA results correlated with 332 ELISA results for MSP2-FC27, MSP2-3D7, EBA175, EBA181, and Rh2; and 333 EBA181 ELISA results correlated with ELISA results for MSP2-FC27, MSP2-3D7, 334 AMA1, EBA175, Rh2, and Rh4. 335
In conclusion, many of the ELISA results correlated with each other and with 336 NH4SCN ELISA, but when ELISA was compared to IIA and affinity, AMA1 stood 337 out as showing more correlations between methods than for other proteins. 338 339 Genotypes present in Ugandan clinical isolates and correlations with antibody 340 levels in the same blood samples 341
The majority of analyzed samples were multi-clonal, including presence of 342 both MSP2-FC27 and MSP2-3D7 alleles (Table 3). When CSP, GLURP, MSP1 (K1, 343 MAD20, and RO33) and MSP2 (FC27 and 3D7) were analyzed separately there were 344 significantly larger heterogeneity in patients with uncomplicated compared to severe 345 malaria for MSP1-K1 (p=0.003) and MSP2-3D7 (p=0.04). When the total MOI was 346 calculated according to Snounou (70), uncomplicated cases (3.5) showed a significant 347
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difference compared to severe cases (2.6), p=0.0095. There was no significant change 348 in total MOI over the time as the samples were collected (not shown). 349
When the number of clones calculated as total MOI was correlated to the 350 antibody responses in the same blood samples (ELISA, invasion), significant positive 351 correlations could be found for MOI against IgG ELISA for MSP2-FC27 (R2=0.16, 352 p=0.001), IgG ELISA AMA1 (R2=0.18, p=0.0008) IgG ELISA EBA175 (R2=0.14, 353 p=0.004), IgG ELISA EBA-181 (R2=0.09, p=0.02), and IgG ELISA Rh2 (R2=0.12, 354 p=0.006). A higher MOI correlated weakly with lower affinity of antibodies in SPR 355 for MSP2-FC27 (R2=0.1, p=0.01) and MSP2-3D7 (R2=0.15, p=0.01). 356 357 Variation with age 358
We observed a positive correlation between age and the presence of IgM 359 antibodies against MSP2-FC27 (R2=0.03, p=0.009), and between age and IgG 360 antibodies against MSP2-FC27 (R2=0.09, p=0.007) and AMA1 (R2=0.12, p=0.001), 361 meaning that older children had higher levels of antibodies. Older children also 362 showed higher affinity of antibodies against the MSP2 antigens as measured by 363 NH4SCN ELISAs (MSP2-FC27; R2=0.17, p=0.0002 and MSP2-3D7; R2=0.15, 364 p=0.0004), but not when using SPR. No correlations were observed between age and 365 parasite invasion, or with the number of clones present in the blood. 366 367 368
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Discussion 369 370
In this study, antibody responses in plasma from Ugandan children with 371 uncomplicated or severe P. falciparum malaria were tested against a panel of 372 merozoite antigens. In efforts to try and find a vaccine against malaria, the goal is to 373 achieve sterile immunity against malaria, but if this can’t be accomplished (something 374 that so far has been difficult in spite of numerous trials), protection against severe 375 malaria would be a great achievement in reducing mortality due to malaria. Indeed, it 376 has been estimated that immunity against severe disease can be reached after a small 377 number of infections (31, 73), indicating that reduction of the severe malaria burden 378 by a vaccine is an achievable goal. However, to know which antigens that are 379 protective, we have to know which methods to use. A first step is to compare 380 uncomplicated and severe patients, to discern differences in antibody responses, and 381 we have in this paper used a selection of methods including ELISA, IIA, SPR and 382 NH4SCN-ELISA, in combination with studies of the parasites in the blood, since no 383 single method yet is strongly predictive of immunity (12, 17, 33, 34, 41, 43-45). 384 ELISA has been used for many years, but it is a static assay and may not be reflective 385 of the function of the antibodies. SPR can offer studies of protein-protein interactions 386 under flow, which ought to be more physiological than ELISA. By using a 387 combination of assays we aimed to increase the understanding of the immunological 388 response, with a bearing towards better prioritization of vaccine candidates. 389
We compared children with uncomplicated and severe malaria, and found 390 higher IgG levels in ELISA in patients with uncomplicated malaria against a selection 391 of merozoite antigens; EBA181, MSP2-3D7, MSP2-FC27 and AMA1. Earlier studies 392 have shown associations between high levels of antibodies against several different 393
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merozoite antigens and protection against symptomatic malaria in general (even 394 though severe malaria has usually not been studied specifically), while other studies 395 consider high antibody levels more as a marker of exposure (22, 25, 28, 40, 73-76). 396
In our study, all patients lived in the same village, and the average age of the 397 two groups (uncomplicated and severe malaria) were not significantly different, 398 indicating similar exposure between the two groups. Many of the genetic host factors 399 were probably also similar. The area is highly endemic with an EIR of >1,500 400 infective bites per person per year (63, 64), and most of the children had probably 401 been exposed to malaria many times before they were included in the study, and no 402 severe malaria cases occurred in children over 3 years of age during our study period. 403 Despite the extreme transmission rate and presumed high levels of protection among 404 patients in general, we could still detect some differences in antibodies between the 405 two groups of patients. 406
When comparing different ways of measuring antibodies, the presence of 407 antibodies against some antigens showed some interesting correlations with other 408 properties as parasite invasion and affinity. For example, presence of antibodies 409 against Rh2 and AMA1 in ELISA correlated to invasion for both tested clinical 410 isolates, and antibodies against AMA1 in ELISA also correlated to affinity SPR 411 results, indicating that this protein might be targeted by functionally important, high 412 affinity antibodies that could contribute to protection from severe malaria. Our 413 findings are consistent with recent studies from Uganda (77, 78) that showed a strong 414 association between antibodies against AMA1 and protection against malaria. Other 415 antigens, such as MSP1, showed no difference in antibody levels between 416 uncomplicated and severe malaria and did not show any correlations with invasion. 417 Invasion of the merozoite into red blood cells is a process that takes only a few 418
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minutes, and antibodies probably need to be of high affinity to be able to exert their 419 function. Antibodies against an antigen like AMA1 might therefore turn out to be 420 more important compared to other merozoite antigens in correlations with functional 421 invasion assays. Presence of antibodies against AMA1 has been shown before to be 422 associated with reduced incidence of malaria (22, 28), and it has also been shown that 423 individuals living in malaria endemic areas have increased levels of anti-AMA1 424 antibodies, which can be strongly inhibitory (12, 79, 80). A general problem for many 425 vaccine candidates is polymorphism, and this might be a hurdle also for AMA1. 426 AMA1 has been shown to be a target of allele specific immune responses (28, 81, 82) 427 and positive selection could have diversified conformational epitopes to avoid 428 recognition by antibodies. However, studies have shown that both conserved and 429 strain-specific epitopes are targets of inhibitory antibodies (14, 81, 83, 84), and when 430 multi-allele immunization was used in vaccine studies, the cross-reactivity of induced 431 antibodies could be increased (85), which points out a way to overcome the problem 432 of polymorphism. It can also make a difference which form of protein is chosen to use 433 in vaccine studies. When 262 individuals in Papua New Guinea were tested for 434 presence of antibodies against different allelic forms of AMA1, it was found that D10 435 contained most of the epitopes (14). This was the protein used in our studies. We have 436 not tested whether the parasites in the study region contain D10, but since 100% of 437 the individuals were positive in ELISA we assume that they have at some stage been 438 exposed to this parasite. 439
MSP2 can be grouped into two main allelic families; either IC1/3D7 or FC27 440 (11, 86, 87). In our studies, we mainly used recombinant proteins from the 3D7 441 parasite for most of the antigens, but for MSP2 we included both 3D7 and FC27. 442 When the parasites in the blood of the patients were analyzed with PCR, we could see 443
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that most patients had presence of both MSP2-3D7 and MSP2-FC27 allelic variants. 444 Absence or low levels of antibodies in some patients can therefore not be explained 445 by people in general not being exposed to the parasites in this area. Presence of both 446 alleles in Uganda has also been shown before (88, 89). 447
Not many studies have investigated differences between uncomplicated and 448 severe malaria for merozoite antigens; most of the studies have so far focused on 449 surface antigens of the infected red blood cell such as PfEMP1, or have used schizont 450 extract (that includes both merozoite and infected red cell antigens) in these kinds of 451 investigations (90-92). In one study using merozoite antigens comparing 452 uncomplicated and severe malaria, it was found that antibodies against MSP1 was 453 higher in those that recently had uncomplicated malaria, whereas antibodies against 454 specific peptides of MSP1 were higher in patients that had severe malaria (93). For 455 MSP1 it has also been shown that there are antibodies that can block cleavage of the 456 protein, but there is also presence in human plasma of antibodies that can block the 457 processing-inhibiting antibodies (94). This further shows the importance of using 458 more functional assays to evaluate the total effect of different antibodies together. 459
In our study group, the levels of IgG antibodies against both MSP2-FC27 and 460 MSP2-3D7 were significantly higher in uncomplicated malaria compared to severe 461 malaria. The IgG ELISA results correlated very well with NH4SCN-ELISA, which is 462 not surprising since both methods are very similar. MSP2 (full-length recombinant 463 protein used in the assays) contains both conserved and variable domains, and there 464 might be some overlap in antibody responses between the two allelic variants due to 465 antibodies reacting against the same epitopes. We could not see any major 466 correlations in affinity of antibodies when comparing SPR and NH4SCN-ELISA, 467 which could be explained by the NH4SCN-ELISA being a static method, while SPR 468
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measures the interaction between antibody and antigen in real time under flow. Other 469 antibodies might be of importance and higher affinity might be needed to stay bound 470 when there is a constant flow in the system. Earlier studies have shown correlations 471 between low levels of antibodies against MSP2 and increased risk of malaria (27). It 472 has also been shown that antibodies against MSP2-3D7, but not against MSP2-FC27, 473 have been associated with protection against malaria (95). In line with this, it is 474 interesting to note that we consistently found higher affinity of antibodies against the 475 3D7 allelic variant (compared to FC27) when SPR was used. Part of the reason for 476 this difference might be that the 3D7 allelic variant of the protein can form a structure 477 that is more prone to forming high affinity antibodies. MSP2 is considered to be an 478 unstructured protein, and might therefore adopt different conformations when 479 different antibodies bind to the protein (10, 96). Another reason could be that the 3D7 480 allelic variant has a unique epitope that can trigger production of high affinity 481 antibodies. Other studies have also pointed towards MSP2-3D7 as being more 482 important in protection against severe malaria (97, 98). When the parasites in the 483 blood of the patients in this study was analyzed, more genotypes were found in 484 uncomplicated malaria cases of MSP2-3D7 compared to in severe cases, but no 485 significant differences were seen for MSP2-FC27. This might also point towards the 486 3D7 allele as being more important. For MSP1, it was only the K1 allele that reached 487 significant differences, but the values for MAD20 (equivalent to 3D7) reached close 488 to significant differences between uncomplicated and severe cases. It might be that 489 once a good immune response has been mounted against the 3D7 allele, it is easier to 490 survive the next episode of malaria. When the total MOI of the parasites was 491 considered, uncomplicated malaria cases had higher number of genotypes. This has 492 been shown before in highly endemic settings (57) and is important information for 493
22
vaccine trials, where it might be better to include several variants of a protein to 494 achieve the best possible response. An alternative interpretation of the in general 495 higher MOI for uncomplicated malaria cases is that tolerance to multiclonal infections 496 is just simply a measure of exposure, but since the MOI values did not correlate with 497 age we do not believe that this is the case. 498
To study the function of antibodies, we used IIA. In previous studies where 499 this method has been applied, mainly laboratory strains have been used, and it has 500 been reported that antibodies that inhibit merozoite invasion could contribute to 501 acquisition of natural immunity but maybe not necessarily confer definitive protective 502 immunity against malaria, and there are conflicting results as to whether the inhibitory 503 effect of antibodies is increasing or decreasing with age (31, 33, 35, 36, 38, 39, 99). In 504 our study, we used two clinical P. falciparum isolates, UAM37 and UAS31, to 505 investigate whether there is decreased invasion in presence of plasma from 506 uncomplicated and severe malaria patients. For UAS31 we found a difference with 507 less invasion in the presence of plasma from uncomplicated cases of malaria, but this 508 difference did not reach significance. This might be explained by the limited number 509 of patients included in the study. However, there was more invasion in both clinical 510 isolates in the presence of plasma from patients with high initial clinical parasitemia 511 (severe malaria patients), suggesting the possible importance of functional assays in 512 evaluation of antibody responses. We also believe that usage of fresh clinical isolates 513 in the IIA might have added a substantial improvement to these assays, as laboratory 514 isolates are known to change after long-term growth in vitro (100). We could not see 515 any correlation of IIA with age, which might be explained by the limited age group of 516 the children (all were under 3 years of age). 517
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In conclusion, this study showed that both occurrence of certain allelic 518 variants of parasites, a higher MOI as well as presence of elevated levels of antibodies 519 with high affinity, especially against AMA1 but possibly also against MSP2, might be 520 protective against developing severe malaria. It is of special interest to note that the 521 only assay that presented a major correlation with initial parasitemia in the patients 522 was IIA using clinical isolates, which highlights the potential importance of using 523 functional assays. Based on our results we think that for future studies, ELISA is a 524 good start to establish that antibodies are formed against a particular antigen, but apart 525 from ELISA it is important to select a combination of functional assays, such as IIA 526 and/or SPR, to better understand how immunity against malaria is developed, and to 527 be able to prioritize antigens for vaccine trials. When considering merozoite antigens 528 as vaccine candidates, differences between uncomplicated and severe malaria should 529 be considered in the evaluations, as vaccines that reduce severe disease and mortality 530 would be of major public health value. 531 532 Acknowledgements 533
We thank the patients for their participation in the study, Robin Anders and 534 Jack Richards for providing recombinant proteins. This work was supported by 535 Vinnova, Sida, VR, EvimalaR, MSB and Svenska Läkaresällskapet, the National 536 Health and Medical Research Council of Australia, the Australia Research Council, 537 and Victorian State Government Operational Infrastructure grant. 538
539 540 541 542
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93. Kohler C, Tebo AE, Dubois B, Deloron P, Kremsner PG, Luty AJ. 2003. 897 Temporal variations in immune responses to conserved regions of 898 Plasmodium falciparum merozoite surface proteins related to the severity of a 899 prior malaria episode in Gabonese children. Trans R Soc Trop Med Hyg 900 97:455-461. 901
94. Guevara Patino JA, Holder AA, McBride JS, Blackman MJ. 1997. 902 Antibodies that inhibit malaria merozoite surface protein-1 processing and 903 erythrocyte invasion are blocked by naturally acquired human antibodies. J 904 Exp Med 86:1689-1699. 905
95. Al-Yaman F, Genton B, Reeder JC, Mokela D, Anders RF, Alpers MP. 906 1997. Humoral response to defined Plasmodium falciparum antigens in 907 cerebral and uncomplicated malaria and their relationship to parasite genotype. 908 Am J Trop Med Hyg 56:430-435. 909
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931 932
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Tables and Figures 933 Table 1. Classification and parasitemia of all patients included in the study. 934
Uncomplicated malaria (n=39) Patient Sex Age (months) Initial parasitemia
(%)Disease state group
CO 05 F 12 1 Mild malaria CO 06 M 36 2 Mild malaria CO 09 F 4 2.4 Mild malaria CO11 M 14 2.8 Mild malaria CO 13 F 5 1 Mild malaria CO 14 F 35 1.7 Mild malaria CO 15 M 24 1.4 Mild malaria CO 16 F 9 1.6 Mild malaria CO 17 M 9 1 Mild malaria CO 18 M 27 2 Mild malaria CO 19 F 24 1 Mild malaria CO 20 M 27 1 Mild malaria CO 21 M 6 3.2 Mild malaria CO 22 M 11 4 Mild malariaCO 24 F 5 6.7 Mild malaria CO 25 F 11 4.2 Mild malaria CO 27 F 15 1.5 Mild malaria CO 28 M 24 2.3 Mild malaria CO 29 F 12 2.5 Mild malaria CO 31 F 9 1.9 Mild malaria CO 32 F 20 1 Mild malaria CO 33 F 7 3.8 Mild malaria CO 34 M 7 2 Mild malaria CO 35 F 10 1.2 Mild malariaCO 36 M 12 4.8 Mild malaria CO 38 M 8 1.8 Mild malaria CO 39 F 7 1.5 Mild malaria CO 40 F 24 5.1 Mild malaria CO 41 M 18 1.1 Mild malariaCO 42 M 9 5.9 Mild malaria CO 43 M 8 4.1 Mild malaria CO 44 M 6 2.3 Mild malaria CO 45 F 29 1.8 Mild malaria CO 46 F 30 3 Mild malaria CO 47 F 12 1.3 Mild malaria CO 48 M 19 2.8 Mild malaria CO 50 F 20 3.6 Mild malaria CO 51 F 6 1.6 Mild malaria CO 52 M 6 2 Mild malariaMean: 14.8 2.5 935 936 937 938 939 940 941
42
942 943 944 945 946 947 948 949 950
Severe malaria (n=46) Patient Sex Age (months) Initial parasitemia (%) Disease state group
SE 01 M 8 Severe malaria NUD SE 02 F 8 6 Severe malaria NUD SE 04 M 24 3 Respiratory distress SE 05 F 24 1 Severe malaria NUD SE 07 F 11 9 Respiratory distress SE 08 F 16 14.8 Respiratory distress SE 09 M 17 15 Respiratory distress SE 11 F 4 46 Severe malaria NUD SE 12 F 12 1 Severe malaria NUD SE 13 M 14 5 Respiratory distress SE 14 F 14 10 Severe malaria NUD SE 15 M 12 8 Respiratory distress SE 16 F 6 5 Severe malaria NUD SE 17 F 12 9 Circulatory collapse SE 18 F 5 1.4 Respiratory distress SE 19 F 14 1.1 Respiratory distress SE 20 M 6 1.9 Severe malaria NUD SE 21 M 8 9 Respiratory distress SE 22 M 17 21.8 Respiratory distress SE 23 M 20 4 Cerebral malaria SE 24 F 15 5 Respiratory distress SE 25 M 8 5.2 Respiratory distress SE 26 F 6 3.1 Respiratory distress SE 27 M 32 2 Respiratory distress SE 28 F 5 6.1 Respiratory distress SE 29 F 12 3.9 Respiratory distress SE 30 M 19 2 Severe malaria NUD SE 31 M 30 7.6 Severe malaria NUD SE 32 M 9 6.2 Cerebral malaria SE 33 F 14 2.8 Respiratory distress SE 34 F 8 10 Respiratory distress SE 35 M 30 8.3 Respiratory distress
SE 36 F 5 9.3 Respiratory distress
SE 37 F 14 5 Respiratory distress
SE 38 M 30 9.5 Severe malaria NUD
SE 39 M 6 9.3 Respiratory distress
SE 40 M 9 5.5 Respiratory distress
SE 41 F 11 7.9 Circulatory collapse
SE 42 M 9 2.2 Respiratory distress
SE 43 F 14 5.6 Respiratory distress
SE 44 M 5 4.5 Respiratory distress
SE 45 F 5 2.9 Respiratory distress
SE 46 M 9 1.8 Respiratory distress SE 47 F 7 1.3 Severe anemia SE 48 M 15 7.3 Respiratory distress
SE 49 M 9 5.7 Respiratory distress Mean: 12.8 6.9
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Table 2. Comparison of antibody responses in plasma from patients with 951 uncomplicated or severe malaria. Total IgM/IgG levels to different recombinant 952 antigens were estimated using ELISA, and an extended analysis of MSP2 antigens 953 was performed for estimation of affinity using NH4SCN ELISA. kd (s-1) values were 954 measured by SPR. Invasion was determined for two clinical isolates (UAS31 and 955 UAM37). Initial clinical parasitemia in the patients were determined by counting 956 Giemsa slides. Differences between means were determined by Mann Whitney’s t-957 test. One standard deviation is shown in parenthesis, significant p-values in bold. 958 959
Figure 1. Comparison of levels of IgG in plasma (ELISA) between patients with 972 uncomplicated (UM) and severe (SE) malaria for a selection of recombinant antigens. 973 Box plots show distribution of optical density (OD) at 405 nm among uncomplicated 974 and severe malaria cases. Horizontal bars in the middle of each box indicate the mean 975 percentage of OD. The top and bottom of each box represent the upper and lower 976 quartile, respectively. The whiskers show the 5th and 95th percentiles. There were 977 significant differences between uncomplicated and severe malaria for all antigens 978 shown (* = p<0.05; ** = p<0.01; *** = p<0.001). 979 980 Figure 2. Antibody affinity in plasma from patients with uncomplicated (UM) and 981 severe (SE) malaria (measured as kd by SPR). Box plots as in Fig 1. Differences in kd 982 (s-1) values between antigens were all significant (p<0.001) with AMA1 showing the 983 highest affinity and MSP2-FC27 the lowest affinity. This was significant whether 984 analyzed as uncomplicated and severe malaria separately, or for all cases together. 985 Within each antigen, when uncomplicated and severe malaria cases were compared, 986 only AMA1 showed a significant difference in kd (s-1) values (p=0.02). 987 988 Figure 3. The ability of two clinical P. falciparum isolates (UAS31, bottom line, and 989 UAM37, top line) to invade red blood cells in vitro in the presence of plasma from 990 patients showed a positive correlation with initial parasitemia (%) in the blood of the 991 same patients (UAS31; R2=0.22 p=0.0009; UAM37; R2=0.16 p=0.006). 992
47
993 Figure 4. The correlation of parasite invasion (%) of two clinical P. falciparum 994 isolates (UAS31, bottom line, and UAM37, top line) in the presence of plasma from 995 patients in relation to anti-AMA1-IgG (measured by ELISA). Significant correlations 996 were found both for UAS31 (R2=0.26, p= 0.0003) and for UAM37 (R2=0.19, 997 p=0.002). 998 999 Figure 5. The correlation of antibody affinity (measured as kd (s-1) by SPR) in 1000 relation to anti-AMA1-IgG (measured by ELISA) in plasma from patients with 1001 uncomplicated or severe malaria (R2=0.31, p<0.0001). 1002 1003
