Specificity of the IgG antibody response to Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale MSP119 subunit proteins in multiplexed serologic assays

Background Multiplex bead assays (MBA) that measure IgG antibodies to the carboxy-terminal 19-kDa sub-unit of the merozoite surface protein 1 (MSP119) are currently used to determine malaria seroprevalence in human populations living in areas with both stable and unstable transmission. However, the species specificities of the IgG antibody responses to the malaria MSP119 antigens have not been extensively characterized using MBA. Methods Recombinant Plasmodium falciparum (3D7), Plasmodium malariae (China I), Plasmodium ovale (Nigeria I), and Plasmodium vivax (Belem) MSP119 proteins were covalently coupled to beads for MBA. Threshold cut-off values for the assays were estimated using sera from US citizens with no history of foreign travel and by receiver operator characteristic curve analysis using diagnostic samples. Banked sera from experimentally infected chimpanzees, sera from humans from low transmission regions of Haiti and Cambodia (N = 12), and elutions from blood spots from humans selected from a high transmission region of Mozambique (N = 20) were used to develop an antigen competition MBA for antibody cross-reactivity studies. A sub-set of samples was further characterized using antibody capture/elution MBA, IgG subclass determination, and antibody avidity measurement. Results Total IgG antibody responses in experimentally infected chimpanzees were species specific and could be completely suppressed by homologous competitor protein at a concentration of 10 μg/ml. Eleven of 12 samples from the low transmission regions and 12 of 20 samples from the high transmission area had antibody responses that were completely species specific. For 7 additional samples, the P. falciparum MSP119 responses were species specific, but various levels of incomplete heterologous competition were observed for the non-P. falciparum assays. A pan-malaria MSP119 cross-reactive antibody response was observed in elutions of blood spots from two 20–30 years old Mozambique donors. The antibody response from one of these two donors had low avidity and skewed almost entirely to the IgG3 subclass. Conclusions Even when P. falciparum, P. malariae, P. ovale, and P. vivax are co-endemic in a high transmission setting, most antibody responses to MSP119 antigens are species-specific and are likely indicative of previous infection history. True pan-malaria cross-reactive responses were found to occur rarely. Electronic supplementary material The online version of this article (10.1186/s12936-018-2566-0) contains supplementary material, which is available to authorized users.


Background
Approximately 2.5 billion people, or one-third of the world's estimated 2018 population live in regions of stable or unstable malaria transmission, and are at risk for infection [1,2]. In sub-Saharan Africa, Plasmodium falciparum has been the major focus of treatment and intervention strategies because of the high mortality associated with infection. Three additional species of human malaria, two Plasmodium ovale sub-species and Plasmodium malariae, share much of the same geographic range in Africa yet are considered less important because prevalence estimates based on microscopic detection of parasites in blood films are generally low [3]. However, mounting clinical evidence suggests that malaria infection with species other than P. falciparum is not benign and that infection prevalence may be increasing in children, even in areas where anti-malarial drug therapy is regularly administered [4][5][6][7][8]. Similarly, the risk of Plasmodium vivax infection in sections of sub-Saharan and central Africa has been considered to be nil because large fractions of the populations in these regions lack the Duffy receptor used by the parasite for red blood cell invasion [9][10][11][12]. New evidence, however, suggests that low levels of P. vivax transmission in Africa may be occurring in susceptible Duffy-positive residents and that some level of infection is also occurring in Duffynegative individuals by another reticulocyte invasion mechanism [13][14][15][16]. Because these non-P. falciparum infections are frequently sub-patent and their symptoms may be masked by the overwhelming levels of P. falciparum parasitaemia, accurate mapping and the estimation of prevalence levels in this population are difficult using traditional microscopic or PCR methods.
Serologic assays that detect IgG antibodies to specific P. falciparum and P. vivax antigens have been used in multiple studies in many parts of the world to estimate infection incidence and immunity levels (reviewed in [17][18][19]). Antibody data from cross-sectional surveys can be used to calculate the community-level seroconversion rates [20][21][22][23][24][25][26][27], and longitudinal and cross-sectional data provide similar estimates of community seroconversion rates [28]. Serologic assays using species-specific antigens could identify individuals who either are currently infected or have been previously infected with different malaria species, even if the infections were sub-patent [29]. Advances in multiplex assay technology make serologic antibody assays for multiple malaria antigens more attractive because antibody responses to a range of malaria antigens can be detected in a single well from a small volume of blood or serum and because malariaspecific assays can be integrated with assays for other antibody responses of public health interest [30][31][32][33].
One target antigen frequently used in malaria serologic antibody studies is the 19-kDa carboxy-terminal sub-unit of the merozoite surface protein 1 (MSP1 19 ) [34][35][36], a glycosylphosphatidylinositol-anchored fragment of the larger MSP1 protein that is found in abundance on the parasite surface (reviewed in [37]). Although the MSP1 19 proteins from P. falciparum, P. malariae, P. ovale, and P. vivax share 48-58% identity at the amino acid level [38], many of the conserved residues are cysteines and other hydrophobic amino acids that are unlikely to be exposed to the immune system [39]. Despite the sequence similarity, Cook et al. [24] were able to demonstrate unique seroprevalence curves for the P. falciparum and P. vivax MSP1 19 antibody responses in areas of reduced transmission in Vanuatu. Similarly, Bousema et al. [40] did not observe any correlation between the P. falciparum and P. vivax MSP1 19 antibody responses in ELISA studies of sera from a population living in a Somalian region of low endemicity for both parasites. In a study of malaria antibody responses in adult Cambodian women, Priest et al. [33] found that 79% of sera from women who were positive for antibodies to malaria reacted with the MSP1 19 antigen from only one species. However, all of these three studies were conducted in regions of relatively low transmission, and it is important to determine whether the MSP1 19 antigen-based assays will be species specific in regions of high transmission with multiple circulating species of malaria parasite.
During a bed net intervention study in a high malaria transmission region of northern Mozambique [41], numerous samples from individuals were assayed and found to have very high IgG antibody responses to multiple malaria species MSP1 19 antigens, including the P. vivax antigen. These samples and samples from two low transmission regions in a multiplex assay format to expand on the MSP1 19 competition ELISA studies of Amanfo et al. [42].
between 1995 and 2011 for malaria diagnostic testing were used for the assessment of multiplex assay sensitivity. The panel included sera from patients having microscopically confirmed and/or PCR confirmed infections with P. falciparum (N = 33), P. malariae (N = 6), P. ovale (N = 7), or P. vivax (N = 35) [43]. The timing of sample collection relative to malaria infection or symptom development was not known. In addition to a pan-Plasmodium spp. immunofluorescence assay (IFA) positive serum pool (CDC Lot 8), mono-specific infection IFA serum controls were available for P. falciparum (CDC Lot 6) and P. malariae (CDC Lot 2).
Sera or dried blood spots previously identified by multiplex assay as having high levels of IgG antibodies to one or more MSP1 19 proteins were selected for the specificity studies. This set included: 3 anonymous, adult blood donor samples collected in 1998 from a region of Haiti with a low prevalence of P. falciparum infection [43]; 9 sera from an integrated serologic study of immune status to vaccine-preventable diseases and neglected tropical diseases conducted in 2012 among women 15-39 years of age in Cambodia [33,44,45]; and, 20 dried blood spots from participants (4-60 years of age) in a longlasting insecticide-treated bed net impact study conducted in 2013-2014 in a high malaria transmission province of northern Mozambique [41]. The sample set from Mozambique was biased towards individuals with a positive antibody response to the P. vivax, P. ovale and P. malariae antigens.

Ethics statement
Residual malaria diagnostic sera were made anonymous under a protocol approved by the CDC Institutional Review Board. Written informed consent was obtained prior to enrolment and participation in the Cambodian sero-survey, and the study protocol was reviewed and approved by the National Ethics Committee in Cambodia [33,44,45]. Written informed consent was obtained prior to enrolment and participation in the Mozambique bed net study and sero-survey, and the study was approved by the National Bioethics Committee in Mozambique. For both of these studies, CDC researchers had no access to personal identifiers, and CDC staff were not considered to be engaged with human research subjects.

Banked chimpanzee sera
Banked sera from malaria studies conducted in experimentally infected chimpanzees prior to 2000 were included in this report. Chimpanzees Bit and Klimatis were infected with the Uganda I strain of P. malariae [46,47], Alpert was infected with the Nigeria I strain of P. ovale [48], and Duff was infected with the Salvador I strain of P. vivax [49]. As previously described, all animals had been splenectomized before they were inoculated intravenously with heparinized, infected blood.

Antigens for multiplex assay
The cloning of the 3D7 strain P. falciparum MSP1 19 in pGEX 4T-2 plasmid (GE Healthcare, Piscataway, NJ, USA) as a fusion protein with Schistosoma japonicum glutathione-S-transferase (GST) and the purification of the MSP1 19 -GST fusion protein have been described elsewhere [50].
Using the protocol of Priest et al. [33] and a new reverse PCR primer, a P. vivax MSP1 19 clone lacking the carboxy-terminal, hydrophobic anchor sequence was generated in pGEX 4T-2 plasmid (GE Healthcare), and the MSP1 19 -GST fusion protein was purified. The target sequence was amplified from Belem strain DNA using a reverse deoxyoligonucleotide PCR primer with the following sequence: 5′-GCG GAA TTC TTA GCT GGA GGA GCT ACA GAA AAC TCC C-3′. The underlined sequence reverse primer identifies an EcoRI restriction endonuclease site used in directional cloning, and the italicized bases identify an introduced in-frame stop codon. All other cloning conditions remained as previously described [33]. The clone was sequenced using BigDye Terminator V3.1 chemistry (Applied Biosystems/ Thermo Fisher Scientific, Foster City, CA, USA).
Cloning, expression and purification of a P. ovale MSP1 19 -GST fusion protein from Nigeria I strain DNA was accomplished using the strategy described in Priest et al. [33] with the following deoxyoligonucleotide primers: forward, 5′-CGC GGA TCC TCT ATG GGA TCT AAA CAT AAA TGT-3′ and reverse, 5′-GCG GAA TTC TTA ACT TGA TGA GCC ACA GAA AAC ACC-3′. The underlined sequence in the forward primer identifies a BamHI restriction endonuclease site used in directional cloning. These primer sequences were based on the sequence of the Cameroon OMA1A P. ovale isolate sequence (GenBank accession number FJ824670) described by Birkenmeyer et al. [38].
Cloning of the P. malariae MSP1 19 coding sequence from China I strain DNA required two PCR reactions. The first reaction used long PCR primers (forward, 5′-AAT ATT AGC GCA AAA CAT GCA TGT ACC GAA ACA-3′; reverse, 5′-ACT TGA AGA ACC ACA GAA AAC ACC TTC AAA TAT AG-3′) and the amplification conditions previously described [33]. These primer sequences were based on the sequence of the Cameroon MM1A P. malariae isolate sequence (GenBank accession number FJ824669) described by Birkenmeyer et al. [38]. A total of 5% of the purified primary product (StrataPrep PCR purification kit, Stratagene, LaJolla, CA, USA) was used in a second amplification reaction with the following primers: forward, 5′-CGC GGA TTC AAT ATT AGC  GCA AAA CAT GCA TGT-3′; reverse, 5′-GCG GAA  TTC TTA ACT TGA AGA ACC ACA GAA AAC ACC-3′. This final PCR product was cloned in pGEX 4T-2  plasmid (GE Healthcare), and a GST fusion protein was expressed and purified using the protocol of Priest et al. [33].
Expression and purification of the control GST protein with no fusion partner has been described elsewhere [51]. A synthetic 20 amino acid peptide, (NANP) 5 -amide, corresponding to the carboxy-terminal repeat of the P. falciparum circumsporozoite protein (PfCSP peptide) [52,53] was cross-linked to GST using the glutaraldehyde protocol of Benitez et al. [54]. Tetanus toxoid antigen from Massachusetts Biologic Laboratories (Boston, MA, USA) was exchanged into buffer containing 10 mM Na 2 HPO 4 and 0.85% NaCl at pH 7.2 (PBS) [44].

Comparison of Plasmodium malariae MSP1 19 sequences from other geographic locations
Ten nanograms of DNA from P. malariae strains Greece I, Guyana, and Uganda I were PCR amplified using the forward and reverse long deoxyoligonucleotides described above and the Expand High Fidelity PCR system (Roche Applied Science, Indianapolis, IN, USA). Cycle conditions were as follows: 94 °C for 5 min, 35 cycles of 95 °C for 30 s, 55 °C for 30 s, and 68 °C for 1 min, and a final extension step of 68 °C for 5 min. Products were purified (StrataPrep PCR purification kit, Stratagene) and sequenced as described above.
The multiplex bead assay protocol for total IgG has been described elsewhere [55,58]. Assays were run in duplicate wells, and each plate included a buffer only blank. The reported "median fluorescent intensity minus background" value (MFI-bg) is the average of the 2 median fluorescent intensity values minus background blank values from two replicate wells. Negative MFI-bg values were set to 0.

Assessment of coupling efficiency
To determine whether the Plasmodium spp. GST-MSP1 19 fusion proteins were coupled to the SeroMap beads with similar efficiencies, multiplex assays were run using a serial dilution of a goat anti-GST polyclonal IgG antibody (GE Healthcare) as the primary antibody to detect the fusion protein on the bead. The initial dilution of anti-GST antibody was 1:1000 in modified Buffer A lacking the E. coli extract (50 μl/well), and the final dilution was 1:1.0 × 10 7 . Bound anti-GST antibody was detected with 50 μl/well of a biotinylated rabbit anti-goat IgG secondary antibody (1:500 dilution in Buffer B; Invitrogen) and wells were developed with R-phycoerythrin-labelled streptavidin and read on a BioPlex 200 instrument (Bio-Rad) as described above.

MSP1 19 competition assays
Serial dilutions of purified MSP1 19 -GST competitor proteins were generated from a 0.5 mg/ml stock solution using PBS buffer at pH 7.2. A 96-well incubation plate (V-bottom, Fisher Scientific) was set up such that wells contained 3 μl of the competitor MSP1 19 -GST fusion protein dilution and 147 μl of serum dilution in Buffer A for 1:50 dilution of competitor protein and a negligible further dilution of the serum. Final competitor protein concentrations in the serum dilution ranged from 10 μg/ml to as low as 0.025 μg/ml. The plate was incubated at room temperature for 1 h, and then each well of the incubation plate was used to load duplicate multiplex bead assay wells at 50 μl each. The standard total IgG assay protocol was then followed as described above. The standard MSP1 19 competition assay used a final competitor protein concentration in diluted serum of 10 μg/ml, and a ≥ 30% reduction in multiplex assay signal was considered to be evidence of antibody cross-reactivity.

MSP1 19 -specific antibody binding and elution
Using the standard coupling protocol [55], individual MSP1 19 -GST fusion proteins were coupled to magnetic beads (region 14, Luminex) in 100 μl of MES/NaCl buffer at pH 5.0 at 4.5 μg for 1.25 × 10 6 microspheres (a 50% increase in protein compared to SeroMap bead amounts). Coupled beads were re-suspended in 120 μl of storage buffer with protease inhibitors [55]. A 1:200 dilution of serum in Buffer A or a 1:5 dilution of blood spot eluate in Buffer A (approximately 1:200 serum dilution) was incubated for 1 h at room temperature with 20 μl of coupled beads (washed 1× with 0.05% Tween-20 in PBS prior to use). Beads were collected by magnetic capture, the used serum or blood spot dilution was removed, and the beads were washed 4× with 200 μl 0.05% Tween-20 in PBS. To elute the bound antibodies, beads were resuspended for 10 min at room temperature in 100 μl of buffer containing 3 parts of 4 M MgCl 2 in 100 mM N-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) at pH 8.0 and 1 part ethylene glycol [59]. The beads were collected by magnetic capture, and the supernatant was removed and diluted into 0.9 ml of buffer containing 50 mM tris(hydroxymethyl)-aminomethane (Tris) at pH 7.5 and 0.85% NaCl. The antibody elution process was repeated once. The 2 ml of eluted antibody in Tris/NaCl buffer was concentrated to 50 μl using a Centricon-50 centrifugal filter device as directed by the manufacturer (Millipore, Bedford, MA, USA). The concentration procedure was repeated following a 2 ml Tris/ NaCl buffer dilution and again after a 1 ml PBS buffer dilution. The final 30-50 μl of concentrate was diluted with > 3 volumes of Buffer A, and duplicate multiplex assays were performed using half of the eluted antibody per well.

Antibody avidity determinations
To determine the avidity of IgG antibody binding, MSP1 19 -GST fusion protein coated SeroMap beads incubated for 1 h with 1:400 serum dilutions were immediately washed with 100 μl of 6 M urea in PBS for 5 min at room temperature [60]. The urea wash was repeated once followed by three 100 μl washes with 0.05% Tween-20 in PBS. The normal total IgG development protocol was then followed. An avidity index was calculated by dividing the 6 M urea-treated MFI-bg value by the untreated MFI-bg value.

Data analysis
Protein sequences were aligned using COBALT [61]. The means plus 3 standard deviations of the MBA responses from 88 adult US citizens with no history of foreign travel were used to define potential cutoffs for the MSP1 19 protein and CSP peptide assays. Receiver-operating characteristic (ROC) curves were also used to generate potential cut-offs for the MSP1 19 assays. The ROC analysis [62] and Spearman rank order correlation analysis were performed using SigmaPlot 13.0 (Systat Software, Inc., San Jose, CA, USA). The J-index [63] was calculated from the sensitivity and specificity values.

MSP1 19 sequences
The DNA sequence of the P. malariae China I strain MSP1 19 clone (deposited in GenBank as MH577182) differed from the Cameroon sequence of Birkenmeyer et al. [38] at 3 nucleotide base positions, leading to 2 amino acid substitutions in the deduced amino acid sequence: G41E and Q51K (numbering based on mature MSP1 19 protein sequence). As shown in Fig. 1, the deduced amino acid sequence of the China I strain was identical to that reported for the Brazil I11 strain [64] and was also identical to that of the Greece I strain (GenBank MH577183). Compared to the Cameroon strain, the Uganda I strain of P. malariae contained only a G41Q amino acid substitution (GenBank MH577184), while the Guyana strain contained only a G41E substitution (GenBank MH577185).
The DNA sequence of the P. ovale Nigeria I clone (GenBank MH577181) matched the GenBank sequence reported for the Cameroon OMA1A P. ovale isolate (FJ824670) by Birkenmeyer et al. [38]. The Nigeria I strain likely belongs to the newly identified Plasmodium ovale curtsi species as the MSP1 19 predicted protein sequence has a Ser at position 23 rather than a Pro [65]. The sequence of the P. vivax clone matched that found in GenBank (accession number AF435594.1) [66]. Alignment of the deduced MSP1 19 amino acid sequences of the P. malariae [38,64], P. falciparum 3D7 strain [67], P. vivax Belem strain [66,68], and P. ovale Nigeria I strain proteins in Fig. 1 showed conservation of 32 amino acids among the four species including 10 cysteines and 5 hydrophobic residues.

Assessment of coupling efficiency
The multiplex response titration curves using dilutions of the anti-GST antibody as the primary antibody in the multiplex reaction were similar for all 4 proteins (Fig. 2). In contrast, the multiplex response titration curves for the GST control bead (coupled at half the protein concentration of the MSP1 19 -GST reactions) and for the cross-linked P. falciparum CSP peptide-GST bead were  19 protein sequences using COBALT [61]. Residues in the P. malariae sequence that differ from the Cameroon sequence of Birkenmeyer et al. [38] are shaded. Predicted protein sequences resulting from the oligonucleotides used in PCR amplification are underlined. The positions of residues conserved among all the presented MSP1 19 protein sequences are indicated in the consensus with divergent residues indicated by a dot. GenBank accession numbers are MH577181, P. ovale Nigeria I strain; MH577182, P. malariae China I strain; MH577183, P. malariae Greece I strain; MH577184, P. malariae Uganda I strain; and MH577185, P. malariae Guyana strain indicative of lower amounts of bound target protein compared to the MSP1 19 beads.

Cut-off determinations
One outlier from the group of 88 US citizens with no history of foreign travel with a MBA response of 8690 MFI-bg units was censored from the P. vivax cut-off calculation, and one outlier with a MBA response of 10,377 MFI-bg units was censored from the P. falciparum CSP peptide calculation. The cut-offs in MFI-bg units were: P. falciparum CSP peptide, 1351; P. falciparum MSP1 19 , 313; P. malariae MSP1 19 , 397; P. ovale MSP1 19 , 65; and, P. vivax MSP1 19 , 86. In an analysis of the residual diagnostic serum panel that included sera from patients having microscopically confirmed and/or PCR confirmed infections, 26 of 33 (79%) of P. falciparum, 5 of 6 (83%) of P. malariae, 5 of 7 (71%) P. ovale, and 33 of 35 (94%) of P. vivax were positive by multiplex assay. The sensitivity of the P. falciparum CSP peptide assay was not determined. Specificities measured from the presumed negative US citizen panel were ≥ 96% for each assay.
Cut-offs determined from all MSP1 19 values (no outliers censored) using ROC curve analysis [62] were lower for P. falciparum and P. malariae (111 and 237 MFI-bg units, respectively) but higher for P. ovale and P. vivax (175 and 203 MFI-bg units, respectively). J-index analysis [63] yielded identical cut-off values. The ROC cut-off values had no impact on the sensitivities of the assays for P. malariae, P. ovale or P. vivax and either had no impact (P. malariae) or resulted in increases of 3% (P. ovale) or 2% (P. vivax) in specificity. Sensitivity and specificity for the P. falciparum assay using the ROC cut-offs were 88 and 94%, respectively.
In order to maximize specificity and to estimate seropositivity conservatively, the higher of the cut-off values from the various methods of analysis for the MSP1 19

MSP1 19 multiplex assay specificity
If closely related antigens coupled on different beads share common epitopes and compete for the same pool of antibodies, the values from a multiplexed assay would be expected to be lower than the values from assays using a single bead only. To test this hypothesis, each Plasmodium spp. MSP1 19 was assayed in isolation (individual monoplex), and the results were compared to values obtained when all of the beads were included in the routine multiplex format. As shown in Table 1, responses from 2 defined sera (Pan Plasmodium spp. Lot 8 and P. malariae Lot 2), 3 elutions from individual Mozambique blood spots, and one high-titre elution from a combination of Mozambique blood spots were essentially identical regardless of the bead complexity of the assay (Spearman rank order correlation coefficient = 0.999; P < 0.001). The results from this limited panel of samples suggest that a response dilution effect in the multiplex assay format is not a universal feature of the MSP1 19 protein family and that the multiplex assay may be useful in infection species determinations.
That some MSP1 19 antibody responses are species specific can also be demonstrated using sera from experimentally infected chimpanzees (Table 2). Sera from chimpanzees Klimatis (P. malariae infection) and Duff (P. vivax infection) had high antibody response values to the corresponding species-specific MSP1 19 protein and had no responses to MSP1 19 antigens from other species. In contrast, other animals such as chimpanzees Alpert (P. ovale infection) and Bit (P. malariae infection) reacted strongly with the MSP1 19 antigen corresponding to the species of the infecting parasite, but they also had weak responses to P. vivax MSP1 19 and strong responses to P. falciparum MSP1 19 . The wild-caught chimpanzees used in the experimental infection studies were never exposed to P. falciparum sporozoites in the laboratory and were never experimentally infected with P. falciparum. Thus, the presence of a P. falciparum CSP peptide response suggests that the P. falciparum MSP1 19 response likely arose by natural infection with a closely related species of chimpanzee malaria, such as Plasmodium reichenowi [69], rather than with an experimentally-induced cross-reactivity. Similarly, the weak P. vivax antibody responses in these 2 chimpanzees may also reflect prior exposure to P. vivax-like parasites in the wild. These unexpected responses highlight the difficulty of differentiating historic infection from true antibody cross-reaction.

Specificity analysis using antigen competition
An alternative approach to assess the specificity of the MSP1 19 antibody response relies on the ability of soluble antigen to saturate the antibody in a pre-incubation step so as to prevent antibody binding to MSP1 19 coated beads during the multiplex assay. To determine the concentration of competitor protein necessary to prevent antibody binding to MSP1 19 coated beads, sera that had high antibody responses were incubated with 0.025-10 μg/ml of competitor protein prior to multiplex assay as described in "Methods". The GST control, PfCSP peptide-GST, and all 4 MSP1 19 -GST protein-coated beads were included in the multiplex assay, but only the homologous MSP1 19 was   Figure 3 shows that the multiplex responses to all 4 MSP1 19 proteins were reduced > 97% following pre-incubation with 2.5 μg/ml competitor protein, and all 4 MSP1 19 antibody responses were below their respective cut-off values when sera were pre-incubated with 5 μg/ml competitor protein.
Next, sera from chimpanzees Klimatis (P. malariae), Alpert (P. ovale), and Duff (P. vivax) (1:400 dilution of each serum) were combined, and the competitor titration assays were repeated. Figure 4 shows the multiplex responses in the presence of various concentrations of the 4 MSP1 19 competitor proteins and expressed as a percentage of the PBS control. In Panel A, addition of the P. falciparum MSP1 19 competitor protein had no effect on the P. malariae, P. ovale or P. vivax multiplex responses. Similarly, heterologous MSP1 19 competitor proteins had no effect on multiplex responses (Fig. 4b-d), while multiplex response curves for the homologous species of competitor MSP1 19 proteins in Fig. 4b-d resemble the individual curves previously shown in Fig. 3. The chimpanzee multiplex responses in the presence of homologous species competitor protein showed > 97% suppression at 2.5 μg/ml and were below the respective cut-off values at 5 μg/ml of competitor. Finally, combined human sera (pan-Plasmodium spp. positive serum pool and P. malariae mono-specific infection serum control, each at 1:400 dilution) competitor studies showed similar heterologous and homologous titration profiles except that the P. malariae response was reduced by approximately 27-29% at the 10 μg/ ml of heterologous MSP1 19 -GST competitor protein concentrations (Additional file 1). For the human multiplex responses in the presence of homologous species competitor protein, values were below the respective cut-off values at 2.5 μg/ml of competitor. Based on these studies, a competitor concentration of 10 μg/ml was selected to maximize suppression of antibody binding in the multiplex assay. Table 3 Representative MSP1 19 competition assay results using sera from low incidence settings a Multiplex bead assay responses that were completely suppressed by homologous protein competition are indicated in italics cells. These responses were below the respective cut-off values b Multiplex bead assay responses that, as a result of heterologous competition, decreased more than 30% compared to the PBS control but remained above the respective cut-off values are indicated in bold italics cells. These values indicate some level of cross-reactivity

Assay specificity in low malaria incidence settings
Representative competition assay results for a serum sample set from two regions of low malaria incidence (Haiti and Cambodia) are presented in Table 3. Additional results from this sample set can be found in Additional file 2. Most of the samples chosen from these areas had positive antibody responses to only one or two MSP1 19 proteins, and only one person (Cambodia 5) reacted with MSP1 19 proteins from three malaria species. Antibody responses to the P. falciparum CSP peptide were mostly negative, and, when present, were < 4000 MFI-bg units (median = 64.5; range 14-3930). Addition of GST control protein to the competition assay at a concentration of 10 μg/ml had no effect on any of the antibody responses. One person had an antibody response to the GST coupled control bead, but the response was not inhibited by pre-incubation with soluble GST protein.
This response was probably unrelated to the presence of the GST protein as the P. ovale MSP1 19 -GST response was consistently < 50 MFI-bg units.
In 9 of the 12 serum samples tested, all of the malaria MSP1 19 antibody responses appeared to be species specific as only homologous competitor MSP1 19 protein completely eliminated the antibody response (highlighted in italics in Table 3 and Additional file 2). For two of the tested sera (Table 3) multiplex assay response values to the P. vivax antigen demonstrated a weak heterologous competition effect with P. falciparum MSP1 19 competitor protein, but the effect did not meet the 30% threshold definition (approximately 25% reduction; Cambodia 4 and 9). Interestingly, the sample from Cambodia donor 4 had no antibodies to either P. falciparum antigen by MBA. Only one donor had a heterologous competition assay response reduction of > 30%: for Cambodia 5 (indicated in bolditalics in Table 3), addition of P. falciparum MSP1 19 competitor protein reduced a weak P. malariae response by 41% while completely eliminating a strong homologous P. falciparum MSP1 19 response (> 28,000 MFI-bg units). The MFI-bg value for the heterologous competition assay remained above the respective P. malariae cut-off.

Assay specificity in a high malaria incidence setting
Representative competition assay results for a sample set from a high malaria incidence region of Mozambique are presented in Table 4 with additional values shown in Additional file 3. These 20 samples were selected from the parent study [41] because they exhibited high IgG antibody responses to one or more MSP1 19 antigens by multiplex bead assay, and the selection was biased towards samples that had a strong positive responses to the P. vivax, P. ovale or P. malariae proteins. Historically, rates of vivax malaria have been expected to be low in East African populations lacking the Duffy antigen [12], and an antibody response to the P. vivax MSP1 19 might be indicative of antibody crossreactivity. In contrast to the samples from low prevalence areas described above, eluted blood spot samples from Mozambique often had strong antibody responses to the P. falciparum CSP peptide (median = 23,407; range = 2833-27,033).
Species-specific anti-MSP1 19 antibody responses, as indicated by the presence of complete homologous competition and the absence of heterologous competitor effects, were observed in 10 of the 20 samples tested (Table 4 and Additional file 3). In one additional sample (Mozambique 20 in Table 4), species-specific responses were observed, but the suppression of the P. malariae MSP1 19 -specific antibody responses was incomplete: values remained above the 397 MFI-bg assay cut-off threshold in the presence of 10 μg/ml P. malariae competitor protein. Given the very high P. malariae MSP1 19 control antibody responses for these samples (> 27,000 MFI-bg units), it is possible that the competitor protein concentration was insufficient for complete antibody blocking. As previously demonstrated, addition of GST control protein to the competition assay at a concentration of 10 μg/ml had no effect on the antibody responses.
One sample (Mozambique 1, Table 4) demonstrated a partial loss (40% reduction) of anti-P. malariae MSP1 19 antibody response in the presence of P. falciparum competitor protein, but antibodies to the other 3 species antigens were specific. Four samples, represented by Mozambique 16 in Table 4, demonstrated some combination of incomplete homologous response suppression and heterologous assay inhibition. In the case of Mozambique 16, the P. falciparum competitor protein partially inhibited the heterologous P. malariae MSP1 19 antibody response (68% reduction), and the P. vivax competitor protein had a major impact on the P. ovale heterologous response (99% reduction), but, in the same reaction, this competitor did not completely block the homologous P. vivax MSP1 19 antibody response (90% reduction). Reciprocal heterologous competition was only observed between P. ovale and P. vivax MSP1 19 antigens and only in two donors represented by Mozambique 19 (Table 4). Heterologous competition assays leading to response values below the cut-off were observed for both P. malariae and P. vivax MSP1 19 proteins and the P. ovale antigen partially reduced the P. vivax antibody response.
As shown in Table 5, what appeared to be a true, panmalaria MSP1 19 antibody response was observed in blood spot elutions from two 20-30 years old Mozambique donors, numbers 3 and 12. In contrast to assays described above, control response values to MSP1 19 proteins for 3 of the malaria species (P. malariae, P. ovale, P. vivax) were Table 4 Representative MSP1 19 competition assay results using sera from a high incidence setting a Multiplex bead assay responses that were completely suppressed by homologous protein competition are indicated in italics cells. These responses were below the respective cut-off values b Multiplex bead assay responses that, as a result of heterologous competition, decreased more than 30% relative to the PBS control but remained above the respective cut-off values are indicated in bold italics cells. These values indicate some level of cross-reactivity c Multiplex bead assay responses that were only partially reduced by homologous protein competition are indicated in underlined cells. These responses remained above the respective cut-off values and likely represent incomplete antibody blocking d Multiplex bead assay responses that were completely suppressed by heterologous protein competition are indicated in underlined italics cells. These responses were below the respective cut-off values reduced by about 50% upon addition of GST control protein. The reason for the observed signal suppression by GST is not understood, but it was probably not related to the presence of the GST component of the MSP1 19 -GST fusion proteins since no antibody bound to the GST control bead and there was no decrease in the PfCSP peptide-GST response. For both donors, the response to the P. falciparum MSP1 19 antibody response was less affected by the addition of the GST control protein, and residual P. falciparum MSP1 19 antibody signal (6-9% of control) was observed in the presence of each of the heterologous competitor proteins. Antibody responses to the other 3 MSP1 19 proteins in the presence of heterologous competitor proteins were either below or very near the cut-off values for the respective assays (Table 5).

Affinity purification of MSP1 19 antibodies
Another potential method to detect antibody crossreactivity is to affinity purify antibody using an MSP1 19 protein from a single malaria species and then assess the reactivity of the eluted antibody using MSP1 19 coated beads from all 4 species. Tetanus toxoid, a protein lacking GST, was included in the multiplex panel as an additional assay control. Plasmodium vivax MSP1 19 -GST-coated magnetic beads were used to affinity purify antibodies from two Mozambique samples: sample 13, previously shown to have specific responses to all 4 MSP1 19 proteins; and sample 16, previously shown to have significant cross-reactivity between the P. vivax protein and the P. malariae antibody response (Table 4). In the case of Mozambique sample 13, only the antibody response to the P. vivax MSP1 19 decreased in the serum dilution after exposure to the magnetic beads (Table 6), and antibodies eluted from the magnetic bead only reacted with the P. vivax bead in the multiplex assay. In a separate experiment with sample 13, captured P. malariae antibodies were eluted from beads coated with the homologous antigen (Additional file 4). For sample 16, antibody responses to both the P. ovale and the P. vivax MSP1 19 proteins decreased upon exposure of the serum dilution to P. vivax protein-coated magnetic beads, and eluted antibodies reacted to both P. vivax and P. ovale beads in the multiplex assay. Thus, the cross-reactivity previously observed in the competition assays was confirmed for this sample.
Plasmodium falciparum MSP1 19 -GST-coated magnetic beads were used to affinity purify antibodies from two additional Mozambique samples: sample 15, previously shown to have specific responses to all 4 MSP1 19 proteins (Table 4); and sample 12, previously shown to have a pan-malaria cross-reactive response ( Table 5). As expected for a species-specific antibody response, only the P. falciparum MSP1 19 antibody response decreased in the serum dilution following exposure to magnetic beads, and the elution only contained antibodies that recognized the P. falciparum protein in the multiplex assay ( Table 6). Incubation of Mozambique sample 12 with the P. falciparum MSP1 19 -GST-coated magnetic beads drastically decreased the antibody responses to proteins from all 4 species in the post-treatment serum dilution, but  19 antibody responses a Multiplex bead assay responses that were completely suppressed by homologous protein competition are indicated in italics cells. These responses were below the respective cut-off values b Multiplex bead assay responses that, as a result of heterologous competition, decreased more than 30% relative to the PBS control but remained above the respective cut-off values are indicated in bold italics cells. These values indicate some level of cross-reactivity c Multiplex bead assay responses that were completely suppressed by heterologous protein competition are indicated in underlined cells. These responses were below the respective cut-off values positive responses were not observed in the MBA analysis of the eluted antibodies. Plasmodium ovale MSP1 19 -GST-coated magnetic beads were also used for antibody capture from sample 12 with results similar to those described above (Additional file 4).

Sub-class and avidity studies
The inability to affinity purify and recover antibodies from a highly cross-reactive sample suggested that the antibody response in Mozambique 12 might have some unique features relative to responses that were species specific or weakly cross-reactive.  Table 6.
The pan-malaria cross-reactive response of Mozambique 12 was completely different. The response to MSP1 19 proteins from all four malaria species was almost exclusively of the IgG 3 sub-class, and the entire IgG response was low avidity with avidity index values of 0.01-0.03 (Table 7). The donor was clearly capable of making high avidity IgG antibodies of other sub-classes as evidenced by the responses to the P. falciparum CSP and tetanus toxoid (Table 7). Unfortunately, this observation could not be confirmed using the other highly cross-reactive sample (Mozambique 3) as no additional antibody eluate was available for testing.

Discussion
Serologic antibody responses to malaria MSP1 19 antigens are increasingly used to map geographic distributions and transmission intensities of malaria infection, but questions about the specificity of the responses remain incompletely explored [17][18][19]. Genomic sequence analysis demonstrates limited allelic variability within species (often only 2-3 amino acids), but considerable sequence heterogeneity between species (this work [35,38,65,66,67]). In a recent serologic IgG antibody survey of two antigen was observed in a population that is expected to be ≥ 95% negative for the Duffy marker used for RBC invasion [10][11][12]70]. The current study was undertaken to determine whether these unexpected responses represented antibody cross-reactivity resulting from the transmission of P. malariae and P. ovale in the context of intense P. falciparum infection or whether they represented true P. vivax infections [41]. First, monoplex bead assays using a single MSP1 19 antigen were compared to multiplex bead assays that included beads coated with antigens from all 4 species as well as GST control and PfCSP peptide coupled to GST. The monoplex versus multiplex results for all 4 MSP1 19 antigens using a panel of 2 sera and 4 blood spot elutions with a range of antibody response values were virtually identical, and no response dilution effect was detected. Similar results were previously reported by Kerkhof et al. [31] using the P. falciparum and P. vivax MSP1 19 antigens and 3 different positive control serum dilutions. However, the observation that a two-fold increase in the number of beads used per assay well had only marginal effects on the measured P. falciparum and P. vivax MSP1 19 antibody responses [31] suggests that this technique may not be a sensitive method for identifying partial cross-reactivity.
Second, banked sera from chimpanzees infected with a single species of malaria in a controlled laboratory setting were tested by MBA. While all 4 animals had homologous antibody responses to the laboratory-administered  [36]. The absence of preexposure baseline sera for the chimpanzees meant that it was impossible to discriminate between cross-reactive responses resulting from the laboratory infections and pre-existing responses from infections acquired in the wild prior to capture. Suppression of antibody binding by pre-absorption with excess heterologous or homologous MSP1 19 antigen should be a sensitive method for the identification of cross-reactive antibody responses in MBA. Amanfo et al. [42] used 2 sera with a competition ELISA technique to demonstrate a lack of cross-reactivity between P. falciparum, P. ovale and P. malariae MSP1 19 antigens (the P. vivax antigen was not included in their analysis). In the third part of this current study, 12 samples from low transmission areas in Haiti and Cambodia and 20 samples from a high transmission area in Mozambique were used to assess cross-reactivity by antigen competition MBA. Eleven of 12 sera from residents of the low transmission areas had MSP1 19 antibody responses that were completely species specific. Only one individual had a heterologous competition response decrease that met the > 30% reduction definition. In the Mozambique sample set, antibody responses for 12 of the 20 samples tested were totally species specific, and 6 of these 12 samples were positive for antibodies to all 4 malaria parasite species. For 6 additional Mozambique samples, the P. falciparum MSP1 19 responses were species specific, but various levels of incomplete heterologous competition were observed for the non-P. falciparum assays ranging from a 31 to 99% response reduction. The high specificity of the P. falciparum assay may reflect the affinity maturation of the immune response upon repeated infection with P. falciparum in the high intensity transmission setting of Mozambique. Most heterologous competition was nonreciprocal, suggesting that infection with one malaria species elicited both specific and cross-reactive antibodies while infection with the other malaria species resulted in only specific antibodies. Most commonly, P. malariae responses cross-reacted with P. falciparum antigen. Two examples of reciprocal heterologous competition were also identified, and both of these involved P. vivax and P. ovale responses. Whether higher concentrations of competitor MSP1 19 -GST protein (> 10 μg/ml) might have resulted in more complete heterologous competition of these responses was not determined.
Two individuals were identified who had very high responses to all 4 MSP1 19 antigens (> 9000 MFI-bg units) and who appeared to have pan-malaria MSP1 19 antibody responses by antigen competition MBA. However, perhaps because of the very high levels of antibodies generated by intense levels of P. falciparum transmission, heterologous antigens could only partially suppress the P. falciparum antibody response. These 2 samples represent only 15% of the 13 samples that were positive for antibodies to all four malaria species in the high transmission area sample set, and it should be noted that the sample set was not randomly selected from the overall Mozambique bed net study population. In fact, samples with high responses to the non-P. falciparum MSP1 19 antigens were intentionally chosen in an attempt to identify those 'worst case scenario' samples where cross-reactivity might be observed. Because only 40 samples from the overall Mozambique bed net study set (N = 2408) had responses above the cut-off values to all 4 MSP1 19 antibodies [41], the number of potential pan-malaria reactive individuals in Mozambique is likely quite low (< 0.3%).
Finally, an antibody capture/elution technique was used with the MBA to confirm the results of the competition assays described above. While species specific and partially cross-reactive MSP1 19 antibodies could be eluted from capture beads, appreciable quantities of captured antibodies could not be recovered from the pan-malaria responsive DBS elution despite repeated attempts with multiple capture antigens. Further analysis of the samples from the species specific and partially cross-reactive donors revealed IgG responses of varying avidity dominated by the IgG 1 and IgG 3 sub-classes. Others have reported that exposure to P. falciparum MSP1 19 elicits a mixed pattern of IgG 1 and IgG 3 antibodies and that repeated infection causes a shift towards an IgG 1 -dominated response [35,[71][72][73][74][75][76][77][78]. The species specific and partially cross-reactive results presented here are consistent with those reports. In contrast, the panmalaria response from Mozambique sample 12 exhibited very low avidity binding to MSP1 19 antigens from all 4 malaria species and was skewed entirely to the IgG 3 sub-class. Low avidity responses to the P. falciparum MSP1 19 are relatively rare [74], and only one previously reported example of a mixed IgG 1 /IgG 3 response that skewed almost entirely to an IgG 3 response upon repeat infection with P. falciparum was found in the literature [78]. At present, it cannot be determined whether these observations result from host-specific factors or are a universal feature of pan-malaria responses, nor can any definitive conclusions be drawn about the impact of such responses on malaria immunity or potential malaria pathology.
Previous studies on allele-specific antibody responses to P. falciparum MSP1 19 and apical membrane antigen 1 (AMA1) suggested that children develop allele-specific responses upon primary infection and that the prevalence of cross-reactive antibodies to conserved epitopes increases with age and increasing experience of infection [79,80]. The number of samples in this study was too small to definitively address the issue of age as a proxy for infection experience and the development of cross-reactive antibody responses. However, two of the partially cross-reactive samples from Mozambique were from 5-years old donors, and 5 of the donors with specific antibody responses to all 4 malaria species were > 50 years of age. Simultaneous infection with multiple malaria species, which is known to occur in Mozambique [81], might play a larger role in the development of antibody responses against shared MPS1 19 epitopes than total infection experience [82]. Thus, even in a high transmission setting with multiple co-endemic malaria species, most antibody responses to P. falciparum, P. malariae, P. ovale, and P. vivax MSP1 19 antigens are likely indicative of previous infection history with those parasite species.

Conclusions
Globally, most areas of malaria transmission are seldom mono-specific. In sub-Saharan Africa, P. falciparum is the most prevalent infection with the highest intensity of transmission, but significant transmission attributable to P. malariae, P. ovale, and, in some areas, P. vivax occurs. In South and Central America, P. malariae, P. falciparum, and P. vivax are transmitted endemically whereas in Asia all four human malaria species can be transmitted. MSP-1 19 is a major antigen recognized by the IgG antibody response of a majority of exposed individuals in an endemic population. Malaria control efforts would likely benefit from being able to rapidly and easily monitor immune responses not only for the main targeted species such as P. falciparum and P. vivax, but also the lesser species, P. malariae and P. ovale. The analyses presented in this work indicate that the multi-species MSP-1 19 multiplex bead assay will be a useful tool in future malaria epidemiologic surveillance and control program studies.