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, 4M GdHCl and 8M urea were weaker chaotropes than 3M 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 2M NH4SCN gave the widest range of AI for the 5 antigens. Binding studies using plasma from 40 adults and 57 one-year old infants in Cameroon showed that 2.1M ± 0.32 (mean ± SD) NH4SCN (adults) and 1.8M ± 0.23M (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 (2M) 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.

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Measuring Antibody Avidity to Plasmodium Falciparum Merozoite Antigens Using a Multiplex Immunoassay Approach

10.21203/rs.2.23647/v2 ◽

2020 ◽

Author(s):

Diane Wallace Taylor ◽

Naveen Bobbili ◽

Alexander K Kayatani ◽

Samuel Tassi-Yunga ◽

Winifrida Kidima ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Clonal Selection ◽

Ammonium Thiocyanate ◽

Affinity Maturation ◽

Binding Studies ◽

Simple Method ◽

Antibody Avidity ◽

Merozoite Antigens ◽

Adult Plasma ◽

One Year

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, 4M GdHCl and 8M urea were weaker chaotropes than 3M 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 2M NH4SCN gave the widest range of AI for the 5 antigens. Binding studies using plasma from 40 adults and 57 one-year old infants in Cameroon showed that 2.1M ± 0.32 (mean ± SD) NH4SCN (adults) and 1.8M ± 0.23M (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 (2M) 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.

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Measuring Antibody Avidity To Plasmodium Falciparum Merozoite Antigens Using A Multiplex Immunoassay Approach

10.21203/rs.2.23647/v1 ◽

2020 ◽

Author(s):

Diane Wallace Taylor ◽

Naveen Bobbili ◽

Alexander K Kayatani ◽

Samuel Tassi-Yunga ◽

Rose FG Leke

Keyword(s):

Plasmodium Falciparum ◽

Clonal Selection ◽

Ammonium Thiocyanate ◽

Plasmodium Falciparum Malaria ◽

Affinity Maturation ◽

Binding Studies ◽

Simple Method ◽

Antibody Avidity ◽

Merozoite Antigens ◽

One Year

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 take place 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 used 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, 4M GdHCl and 8M urea were weaker chaotropes than 3M 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 2M urea gave the widest range of AI for the 5 antigens. Binding studies using plasma from 40 adults and 57 one-year old infants in Cameroon showed that 2.1M ± 0.32 (mean ± SD) NH4SCN (adults) and 1.8M ± 0.23M released 50% of bound Ab from the merozoite antigens. The final avidity multiplex assay was highly repeatable. Conclusions. An avidity MIA is feasible for the 5 merozoite antigens that uses a single concentration (2M) 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.

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

Malaria Journal ◽

10.1186/s12936-020-03243-3 ◽

2020 ◽

Vol 19 (1) ◽

Author(s):

Diane Wallace Taylor ◽

Naveen Bobbili ◽

Alex Kayatani ◽

Samuel Tassi Yunga ◽

Winifrida Kidima ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Antibody Avidity ◽

Multiplex Immunoassay ◽

Falciparum Merozoite ◽

Merozoite Antigens

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Does Antibody Avidity to Plasmodium falciparum Merozoite Antigens increase with Age in Individuals living in Malaria-Endemic Areas?

Infection and Immunity ◽

10.1128/iai.00522-20 ◽

2021 ◽

Author(s):

Samuel Tassi Yunga ◽

Naveen Bobbili ◽

Yukie M. Lloyd ◽

Jovikka Antallan ◽

Masako Matsunaga ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Affinity Maturation ◽

High Avidity ◽

Plasma Samples ◽

Avidity Index ◽

Hyperendemic Area ◽

Age Related ◽

Falciparum Merozoite ◽

Merozoite Antigens ◽

Strength Of Association

Introduction: High avidity antibodies (Abs) are acquired after a few Plasmodium falciparum infections in low transmission areas, but it remains unclear if Ab avidity to different merozoite antigens increases with age in individuals with persistent antigenemia and if so, when a fully mature Ab response occurs. Methods: The study used plasma samples collected between 1996 and 1998 from 566 individuals aged 4-84 years in Simbok, Cameroon where residents received an estimated 1.6 infectious mosquito bites/person/night. Plasma samples were examined for Ab levels (median fluorescence intensity, MFI) and Ab avidity index (AI = [MFI after treatment with 2M NH4SCN/MFI without salt] x 100) using a bead-based multiplex immunoassay for recombinant AMA1, EBA-175, MSP1-42 (3D7, FVO), MSP2 (3D7, Fc27), and MSP3. Results: Blood-smear positivity for P. falciparum declined with age from 54.3% at 4-5 years to 18% at 16-40 years and <11% at >40 years of age, although most individuals had submicroscopic parasitemia. Ab affinity maturation, based on age-related patterns of median AI, percent of individuals with AI ≥50 and strength of association between MFI and AI, occurred at different rates among the antigens: developing rapidly before age 4 years for AMA1, increasing gradually with age for EBA-175 and MSP1 until ∼16-25 years, but occurring negligibly for MSP2 and MSP3. Conclusion: In a hyperendemic area with perennial transmission, affinity maturation resulting in an increase in the proportion of high avidity Abs occurred for some merozoite antigens, in parallel with a decline in malaria slide passivity, but not for others.

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Memory B-Cell Responses Against Merozoite Antigens After Acute Plasmodium falciparum Malaria, Assessed Over One Year Using a Novel Multiplexed FluoroSpot Assay

Frontiers in Immunology ◽

10.3389/fimmu.2020.619398 ◽

2021 ◽

Vol 11 ◽

Author(s):

Peter Jahnmatz ◽

Christopher Sundling ◽

Victor Yman ◽

Linnea Widman ◽

Muhammad Asghar ◽

...

Keyword(s):

Plasmodium Falciparum ◽

B Cell ◽

Plasmodium Falciparum Malaria ◽

Sub Saharan Africa ◽

Single Infection ◽

Cell Responses ◽

Merozoite Antigens ◽

Sub Saharan ◽

One Year ◽

Kinetics Of

Memory B cells (MBCs) are believed to be important for the maintenance of immunity to malaria, and these cells need to be explored in the context of different parasite antigens and their breadth and kinetics after natural infections. However, frequencies of antigen-specific MBCs are low in peripheral blood, limiting the number of antigens that can be studied, especially when small blood volumes are available. Here, we developed a multiplexed reversed B-cell FluoroSpot assay capable of simultaneously detecting MBCs specific for the four Plasmodium falciparum blood-stage antigens, MSP-119, MSP-2, MSP-3 and AMA-1. We used the assay to study the kinetics of the MBC response after an acute episode of malaria and up to one year following treatment in travelers returning to Sweden from sub-Saharan Africa. We show that the FluoroSpot assay can detect MBCs to all four merozoite antigens in the same well, and that the breadth and kinetics varied between individuals. We further found that individuals experiencing a primary infection could mount and maintain parasite-specific MBCs to a similar extent as previously exposed adults, already after a single infection. We conclude that the multiplexed B-cell FluoroSpot is a powerful tool for assessing antigen-specific MBC responses to several antigens simultaneously, and that the kinetics of MBC responses against merozoite surface antigens differ over the course of one year. These findings contribute to the understanding of acquisition and maintenance of immune responses to malaria.

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