Background
Antibodies (Ab) play a critical role in immunity to
Plasmodium falciparum, primarily by blocking key epitopes on merozoites, preventing cytoadherence of infected erythrocytes, and enhancing phagocytosis. Antibody avidity (or functional affinity) is the net antigen-binding force of populations of Ab in sera [
1]. Thus, studying antibody avidity provides insight into the extent of somatic mutation of immunoglobulin hypervariable regions and subsequent clonal selection. Considering the importance of Ab in immunity to malaria, including to merozoite antigens, surprisingly little is known about antibody avidity in naturally-infected individuals. Early studies showed that Ab avidity to an extract of
P. falciparum-infected erythrocytes increased after a few infections in a low transmission region [
2,
3]. However, in high transmission areas, most studies have found little or no increase in antibody avidity with age to merozoite antigens, including MSP1, MSP2, MSP3 and EBA-175 [
4‐
9], although an increase with age for AMA1 has been reported [
4,
5]. Thus, there is much to be learned about the role of Ab avidity in immunity to malaria.
A variety of protocols have been used to study antibody avidity. Although a few investigators have employed plasma magnetic resonance or bilayer interferometry [
4,
7,
10], most studies have used the ELISA format with different concentrations of chaotropic agents [
2,
3,
5,
6,
8,
9,
11‐
17]. In these assays, results are often expressed as an Avidity Index (AI), defined as the amount (e.g., O.D.) of Ab remaining bound to an antigen in the presence of a chaotropic agent divided by amount of Ab bound in its absence multiplied by 100. Studies of avidity to merozoite antigens with plasma from naturally-infected humans have used a variety of chaotropes, including 0.5 M, 2 M, 4 M, or 5 M guanidine HCl (GdHCl) [
5,
6,
9,
11]; 8 M urea [
2,
14‐
17]; or 1 M or 2.4 M thiocyanate (SCN) [
3,
8]. Additional studies of other malarial antigens have used 3 M NH
4SCN for VAR2CSA [
18,
19] and 1 M NH
4SCN for volunteers immunized with the RTS/S vaccine [
12,
13]. A few studies, mainly laboratory-based, employed titration curves of urea or NH
4SCN to determine amount of chaotrope needed to release 50% of bound Ab, usually in animal models vaccinated with malaria antigens [
20,
21]. Prior avidity studies have usually considered only 1 to 2 antigens, have not explained why the chaotrope was selected, or have compared results among various chaotropes for the same or multiple antigens. The use of different methodologies makes it impossible to compare results between studies.
Today, bead-based multiplex immunoassays (MIA) are commonly used to measure Ab to combinations of malarial antigens [
22‐
26]. Currently, it is unclear if an avidity MIA with multiple malaria antigens can be developed that uses only one concentration of a chaotropic agent, since prior avidity studies of viral and bacterial antigens have found that different concentrations of salt were required to release 50% of bound Ab from different antigens [
1,
27].
This study explored the feasibility of an avidity MIA for 5 P. falciparum merozoite proteins (AMA1, EBA-175, MSP1-42, MSP2 and MSP3). The goal was to determine if accurate information could be obtained using a single concentration of only a single chaotrope. First, the most commonly used ELISA protocols for GdHCl, urea, and thiocyanate were adapted to the MIA format. These results allowed comparison of the amount of Ab released by the different chaotropes. Next, titration curves for GdHCl, urea and NH4SCN were examined; as well as, the length of incubation with different salt concentrations. Initial results revealed that 2 M NH4SCN consistently gave the widest range of AI values. Subsequent experiments determined (i) how 2 M NH4SCN compared with the amount of NH4SCN required to release 50% of Ab bound to the antigens, (ii) if the assay was appropriate for measuring avidity in infants with newly-developing malarial immunity as well as adults with high levels of immunity, and (iii) the reproducibility of the avidity MIA. Overall, the combined results lead to the conclusion that 2 M NH4SCN provides meaningful results in an avidity MIA for the 5 P. falciparum merozoite recombinant proteins evaluated.
Discussion
Considering the importance of Ab in immunity to malaria and the extensive search for correlates for protection, it is surprising that Ab avidity has received relatively little attention. The exclusion method used in this study is a simple approach for measuring Ab avidity, whereby Ab-antigen complexes are treated with a chaotrope and the amount of Ab release is determined. Although the exact mechanism is unknown, it is hypothesized that the gap between antigens and bound Ab with good complementarity is tight enough to exclude the denaturing agent, unlike the gap in low avidity Abs [
1]. As such, hydrophobic and ionic bonds stabilizing the complexes remain unbroken [
31‐
33]. Since merozoite antigens have multiple epitopes, each of which induces Ab with different binding-strengths (affinity), AI represent the overall percentage of Ab with sufficient complementarity to remain bound after exposure to a specific chaotrope. Accordingly, the term “high avidity antibodies” is defined by the concentration of chaotrope used. For example, in Fig.
1a, plasma from Individual #3 would be described as having 82% and 75% high avidity Ab to AMA1 if 4 M GdHCl and 8 M were used, but only 8% high avidity to AMA1 in assays using 3 M NH
4SCN. Likewise, in Fig.
2a, Individual #3 would be reported as having 95%, 30% and 8% high avidity Ab to AMA1 if 1 M, 2 M, or 3 M NH
4SCN were used. Since “High Avidity Ab” is defined by the concentration of chaotrope, it is difficult to compare results between studies and interpret results when different protocols are used.
Today, bead-based MIA are commonly used to measure Ab levels to malarial antigens [
22‐
26]. The MIA format has many advantages, including the requirement for small amounts of antigen, speed, assaying > 100 replicates (beads) instead of only a few wells, and being internally controlled since all antigens and reagents are present within the same well (e.g., if Ab are not detected for one antigen, but are to others, then absence of Ab is not due to a technical error). For these reasons, having an avidity MIA using the exclusion method would allow researchers to quickly obtain quantitative data about Ab avidity. Based on the initial results, however, it was not clear if an avidity MIA using the 5 antigens and a single chaotrope was feasible.
Follow-up experiments sought to further characterize and refine the bead-based MIA using different concentrations of chaotropes (Fig.
2 and Additional file
3: Fig. S3) and different incubation periods (Fig.
3). Overall, 2 M NH
4SCN provided the widest range of AI for the 5 antigens. Since using a concentration of chaotrope that is in the center of the exclusion curve is desirable, 2 M NH
4SCN was compared with the actual molar amount of NH
4SCN needed to release 50% of bound Ab to each of the 5 antigens (Table
1). Using plasma from 40 adults living in a highly endemic malaria region, mean molar concentrations of NH
4SCN to release 50% of bound Ab ranged from 1.7 to 2.3 M, with an average of 2.1 ± 0.32 M for the 5 antigens (Table
1). Thus, 2 M NH
4SCN proved to be in the middle of the exclusion curves. Incubation with antigen-Ab complexes with 2 M NH
4SCN for 15 or 30 min didn’t alter AIs significantly (Fig.
3), but the longer incubation period proved to be practical when running multiple plates. Using the avidity MIA, over 100 samples can be screened against the 5 or more antigens in a single afternoon with good reproducibility (Fig.
4). Since the assay provides information on both the amount (MFI) and the proportion of high avidity Ab (AI), data on both Ab quantity and quality is obtained in a single experiment. Without further experimentation, it is unclear if the protocol can be used for other malarial antigens, but results from this study provide a starting point for development of future avidity assays for other antigens.
In repeat experiments, AI values were more consistent than MFI (Fig.
4). In other words, AIs are technically more error-proof than raw MFIs. Technical and instrumental differences, for example due to pipetting errors or mis-calibration, result in variation of MFI when the same sample is used in multiple assays. However, the AI values are less affected since they measure the proportion of high binding antibodies. The wide dynamic range of MIA (e.g., 500 MFI to 25,000 MFI in this study) helps explain the very strong association between AI and the coefficient of variation (r = 0.935) (Fig.
4). Thus, these data support the feasibility of a simple, useful, repeatable avidity MIA for merozoite antigens.
Initially, it was hoped that by comparing the most commonly-used protocols in the same experiment, one might gain insight to help make comparisons and interpret data from previous studies. A conscientious search of the literature revealed that the 4 studies using 4 M GdHCl focused on merozoite antigens, including AMA1 [
5,
6,
9,
11], MSP1 [
5,
6,
9,
11], MSP2 [
9], MSP3 [
5]. Whereas, the 5 studies that used 8 M urea measured avidity to a schizont extract [
2],
Plasmodium vivax MSP1 and
P. vivax Duffy Binding Protein [
14,
17], and Ab from individuals vaccinated with AMA1 and Pf25 [
15,
16]. In contrast, the 6 studies using SCN evaluated other malarial antigens including: a schizont-extract and 1 M NH
4SCN [
3], EBA-175 using 2.4 M NaSCN [
8], the RTS/S vaccine and 1 M NH
4SCN [
12,
13], and VAR2CSA with 3 M NH
4SCN [
18,
19]. Thus, it does not appear that any of these prior studies are similar enough to be directly compared with each other. Accordingly, comparisons can be made within the same study among different cohorts or treatment groups, but direct comparison between studies remains unfeasible. Variation in methodologies and antigens used in previous studies may explain why an inconsistent picture of changes in Ab avidity with age and the role of Ab avidity in protection from malaria exist.
Results from this study provide hints about maturation of the Ab response in individuals living in malaria endemic areas. First, individuals may have high AI to one antigen but low AI to another antigen. For example, in Fig.
2, using 2 M NH
4SCN, Individual #1 AI of 90 to MSP1-42, 85 to EBA-175, 25 to AMA1, but only 8% to MSP3. This result indicates that affinity maturation does not occur at the same rate (or reach the same level), for all antigens. Second, at the population level, Ab avidity tends to be greater for some antigens than others. For example, in Fig.
1, AI to MSP1-42 were high (> 90 with 3 M NH
4SCN), but low AI to MSP2 and MSP3 (AI < 15 for 3 M NH
4SCN). Thus, some antigens appear to induce affinity maturation better than others. Third, the amount of Ab was lower in infants than adults for the 5 merozoite antigens, requiring the use of 1:100 and 1:1000 dilutions of infant and adult plasma, respectively, for MFI to fall on the linear part of the binding curve. Interestingly, the amount of NH
4SCN needed to release 50% of bound Ab was quite similar for Ab-positive infants and adults (Table
1), although lower amounts of chaotrope were required for infants for AMA1 and EBA-175, slightly lower for MSP1-42, but similar for MSP2 and MSP3 (Table
1). Studies in high transmission areas have reported little or no increase in Ab avidity with age for EBA-175, MSP1, MSP2, and MSP3 [
4‐
9]. Plasma used in this study was from infants and adults living in a rural village with perennial transmission who received an estimated 257 infectious mosquito bites/person/year [
29,
30]. Clearly, it would be interesting to investigate affinity maturation with age in this high transmission setting to determine if and when affinity maturation occurs. Overall, the use of 2 M NH
4SCN in future studies evaluating maturation of immunity from infancy to adulthood seems appropriate; whereas, investigators studying acquisition of immunity in young children might consider using 1.5 M NH
4SCN as the lower concentration will give a wider range of AI values.
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