Dataset Information

Measuring antibody avidity to Plasmodium falciparum merozoite antigens using a multiplex immunoassay approach.

ABSTRACT:

Background

Antibodies (Ab) play a significant role in immunity to Plasmodium falciparum malaria. Usually, following repeated exposure to pathogens, affinity maturation and clonal selection take place, resulting in increased antibody avidity. However, some studies suggest affinity maturation may not occur to malaria antigens in endemic areas. Information on development of antibody avidity is confusing and conflicting, in part, because different techniques have been used to measure avidity. Today, bead-based multiplex immunoassays (MIA) are routinely used to simultaneously quantitate antibody levels to multiple antigens. This study evaluated the feasibility of developing an avidity MIA with 5 merozoite antigens (AMA1, EBA-175, MSP1-42, MSP2, MSP3) that uses a single chaotropic concentration.

Methods

The most common ELISA protocols that used the chaotropic reagents guanidine HCl (GdHCl), urea, and ammonium thiocyanate (NH₄SCN) were adapted to a multiplex MIA format. Then, different concentrations of chaotropes and incubation times were compared and results were expressed as an Avidity Index (AI), i.e., percentage of antibody remaining bound in the presence of chaotrope. Experiments were conducted to (i) identify the assay with the widest range of AI (discriminatory power), (ii) determine the amount of chaotrope needed to release 50% of bound Ab using plasma from adults and infants, and (iii) evaluate assay repeatability.

Results

Overall, 4 M GdHCl and 8 M urea were weaker chaotropes than 3 M NH₄SCN. For example, they failed to release significant amounts of Ab bound to MSP1-42 in adult plasma samples; whereas, a range of AI values was obtained with NH₄SCN. Titration of NH₄SCN revealed that 2 M NH₄SCN gave the widest range of AI for the 5 antigens. Binding studies using plasma from 40 adults and 57 1-year old infants in Cameroon showed that 2.1 M?±?0.32 (mean?±?SD) NH₄SCN (adults) and 1.8 M?±?0.23 M (infants) released 50% of bound Ab from the merozoite antigens.

Conclusions

An avidity MIA is feasible for the 5 merozoite antigens that uses a single concentration (2 M) of NH₄SCN. The assay provides a simple method to quickly obtain information about Ab quantity and quality in the acquisition of immunity to malaria in endemic populations.

SUBMITTER: Taylor DW

PROVIDER: S-EPMC7195780 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Publications

Measuring antibody avidity to Plasmodium falciparum merozoite antigens using a multiplex immunoassay approach.

Taylor Diane Wallace DW Bobbili Naveen N Kayatani Alex A Tassi Yunga Samuel S Kidima Winifrida W Leke Rose F G RFG

Malaria journal 20200501 1

<h4>Background</h4>Antibodies (Ab) play a significant role in immunity to Plasmodium falciparum malaria. Usually, following repeated exposure to pathogens, affinity maturation and clonal selection take place, resulting in increased antibody avidity. However, some studies suggest affinity maturation may not occur to malaria antigens in endemic areas. Information on development of antibody avidity is confusing and conflicting, in part, because different techniques have been used to measure avidity ...[more]

PMID: 32357882

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Project description:BackgroundAntibodies against merozoite antigens are key components of malaria immunity. The naturally acquired antibody response to these antigens is generally considered short-lived; however, the underlying mechanisms remain unclear. Prospective studies of travellers with different levels of prior exposure, returning to malaria-free countries with Plasmodium infection, offer a unique opportunity to investigate the kinetics and composition of the antibody response after natural infection.MethodsAdults diagnosed with P. falciparum malaria in Stockholm, Sweden (20 likely malaria naïve and 41 with repeated previous exposure during residency in sub-Saharan Africa) were sampled at diagnosis and 10 days and 1, 3, 6, and 12 months after treatment. Total and subclass-specific IgG responses to P. falciparum merozoite antigens (AMA-1, MSP-119, MSP-2, MSP-3, and RH5) and tetanus toxoid were measured by multiplex bead-based immunoassays and ELISA. Mathematical modelling was used to estimate the exposure-dependent longevity of antibodies and antibody-secreting cells (ASCs).ResultsA majority of individuals mounted detectable antibody responses towards P. falciparum merozoite antigens at diagnosis; however, the magnitude and breadth were greater in individuals with prior exposure. In both exposure groups, antibody levels increased rapidly for 2 weeks and decayed thereafter. Previously exposed individuals maintained two- to ninefold greater antibody levels throughout the 1-year follow-up. The half-lives of malaria-specific long-lived ASCs, responsible for maintaining circulating antibodies, ranged from 1.8 to 3.7 years for merozoite antigens and were considerably short compared to tetanus-specific ASCs. Primary infected individuals did acquire a long-lived component of the antibody response; however, the total proportion of long-lived ASCs generated in response to infection was estimated not to exceed 10%. In contrast, previously exposed individuals maintained substantially larger numbers of long-lived ASCs (10-56% of total ASCs).ConclusionThe short-lived nature of the naturally acquired antibody response, to all tested merozoite antigens, following primary malaria infection can be attributed to a combination of a poor acquisition and short half-life of long-lived ASCs. Greater longevity is acquired with repeated infections and can be explained by the maintenance of larger numbers of long-lived ASCs. These insights advance our understanding of naturally acquired malaria immunity and will guide strategies for further development of both vaccines and serological tools to monitor exposure.

Project description:Background: Antibody responses have been used to characterise transmission and exposure history in malaria-endemic settings for over a decade. Such studies have typically been conducted on well-standardised enzyme-linked immunosorbent assays (ELISAs). However, recently developed quantitative suspension array technologies (qSAT) are now capable of high-throughput and multiplexed screening of up to hundreds of analytes at a time. This study presents a customised protocol for the Luminex MAGPIX © qSAT using a diverse set of malaria antigens. The aim is to develop a standardised assay for routine serological surveillance that is implementable across laboratories and epidemiological settings. Methods: A panel of eight Plasmodium falciparum recombinant antigens, associated with long- and short-lived antibody responses, was designed for the Luminex MAGPIX © platform. The assay was optimised for key steps in the protocol: antigen-bead coupling concentration, buffer composition, serum sample dilution, and bead storage conditions. Quality control procedures and data normalisation methods were developed to address high-throughput assay processing. Antigen-specific limits of quantification (LOQs) were also estimated using both in-house and WHO reference serum as positive controls. Results: Antigen-specific bead coupling was optimised across five serum dilutions and two positive controls, resulting in concentrations operational within stable analytical ranges. Coupled beads were stable after storage at room temperature (22?C) for up to eight weeks. High sensitivity and specificity for distinguishing positive and negative controls at serum sample dilutions of 1:500 (AUC 0.94 95%CI 0.91-0.96) and 1:1000 (AUC 0.96 95%CI 0.94-0.98) were observed. LOQs were also successfully estimated for all analytes but varied by antigen and positive control. Conclusions: This study demonstrates that developing a standardised malaria-specific qSAT protocol for a diverse set of antigens is achievable, though further optimisations may be required. Quality control and data standardisation methods may also be useful for future analysis of large sero-epidemiological surveys.

Project description:BACKGROUND: Antibodies have an essential role in the acquired immune response against blood stage P. falciparum infection. Although several antigens have been identified as important antibody targets, it is still elusive which antigens have to be recognized for clinical protection. Herein, we analyzed antibodies from plasmas from symptomatic or asymptomatic individuals living in the same geographic area in the Western Amazon, measuring their recognition of multiple merozoite antigens. METHODS: Specific fragments of genes encoding merozoite proteins AMA1 and members of MSP and EBL families from circulating P. falciparum field isolates present in asymptomatic and symptomatic patients were amplified by PCR. After cloning and expression of different versions of the antigens as recombinant GST-fusion peptides, we tested the reactivity of patients' plasmas by ELISA and the presence of IgG subclasses in the most reactive plasmas. RESULTS: 11 out of 24 recombinant antigens were recognized by plasmas from either symptomatic or asymptomatic infections. Antibodies to MSP9 (X2(DF=1)?=?9.26/p?=?0.0047) and MSP5 (X2(DF=1)?=?8.29/p?=?0.0069) were more prevalent in asymptomatic individuals whereas the opposite was observed for MSP1 block 2-MAD20 (X2(DF=1)?=?6.41/p?=?0.0206, Fisher's exact test). Plasmas from asymptomatic individuals reacted more intensely against MSP4 (U?=?210.5, p?<?0.03), MSP5 (U?=?212, p?<?0.004), MSP9 (U?=?189.5, p?<?0.002) and EBA175 (U?=?197, p?<?0.014, Mann-Whitney's U test). IgG1 and IgG3 were predominant for all antigens, but some patients also presented with IgG2 and IgG4. The recognition of MSP5 (OR?=?0.112, IC95%?=?0.021-0.585) and MSP9 (OR?=?0.125, IC95%?=?0.030-0.529, cross tab analysis) predicted 8.9 and 8 times less chances, respectively, to present symptoms. Higher antibody levels against MSP5 and EBA175 were associated by odds ratios of 9.4 (IC95%?=?1.29-69.25) and 5.7 (IC95%?=?1.12-29.62, logistic regression), respectively, with an asymptomatic status. CONCLUSIONS: Merozoite antigens were targets of cytophilic antibodies and antibodies against MSP5, MSP9 and EBA175 were independently associated with decreased symptoms.

Project description:Concerted efforts to fight malaria have caused significant reductions in global malaria cases and mortality. Sustaining this will be critical to avoid rebound and outbreaks of seasonal malaria. Identifying predictive attributes that define clinical malaria will be key to guide development of second-generation tools to fight malaria. Broadly reactive antibodies against variable surface antigens that are expressed on the surface of infected erythrocytes and merozoites stage antigens are targets of naturally acquired immunity and prime candidates for anti-malaria therapeutics and vaccines. However, predicting the relationship between the antigen-specific antibodies and protection from clinical malaria remains unresolved. Here, we used new datasets and multiple approaches combined with re-analysis of our previous data to assess the multi-dimensional and complex relationship between antibody responses and clinical malaria outcomes. We observed 22 antigens (17 PfEMP1 domains, 3 RIFIN family members, merozoite surface protein 3 (PF3D7_1035400), and merozoites-associated armadillo repeats protein (PF3D7_1035900) that were selected across three different clinical malaria definitions (1,000/2,500/5,000 parasites/µl plus fever). In addition, Principal Components Analysis (PCA) indicated that the first three components (Dim1, Dim2 and Dim3 with eigenvalues of 306, 48, and 29, respectively) accounted for 66.1% of the total variations seen. Specifically, the Dim1, Dim2 and Dim3 explained 52.8%, 8.2% and 5% of variability, respectively. We further observed a significant relationship between the first component scores and age with antibodies to PfEMP1 domains being the key contributing variables. This is consistent with a recent proposal suggesting that there is an ordered acquisition of antibodies targeting PfEMP1 proteins. Thus, although limited, and further work on the significance of the selected antigens will be required, these approaches may provide insights for identification of drivers of naturally acquired protective immunity as well as guide development of additional tools for malaria elimination and eradication.

Dataset Information

Measuring antibody avidity to Plasmodium falciparum merozoite antigens using a multiplex immunoassay approach.

Background

Methods

Results

Conclusions

Publications

Measuring antibody avidity to Plasmodium falciparum merozoite antigens using a multiplex immunoassay approach.

Similar Datasets

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