Novel antibody competition binding assay identifies distinct serological profiles associated with protection

Abstract

Introduction: Pre-erythrocytic malaria vaccines hold the promise of inducing sterile protection thereby preventing the morbidity and mortality associated with Plasmodium infection. The main surface antigen of P. falciparum sporozoites, i.e., the circumsporozoite protein (CSP), has been extensively explored as a target of such vaccines with significant success in recent years. Systematic adjuvant selection, refinements of the immunization regimen, and physical properties of the antigen may all contribute to the potential of increasing the efficacy of CSP-based vaccines. Protection appears to be dependent in large part on CSP antibodies. However due to a knowledge gap related to the exact correlates of immunity, there is a critical need to improve our ability to down select candidates preclinically before entering clinical trials including with controlled human malaria infections (CHMI).

Methods: We developed a novel multiplex competition assay based on well-characterized monoclonal antibodies (mAbs) that target crucial epitopes across the CSP molecule. This new tool assesses both, quality and epitope-specific concentrations of vaccine-induced antibodies by measuring their equivalency with a panel of well-characterized, CSP-epitope-specific mAbs.

Results: Applying this method to RTS,S-immune sera from a CHMI trial demonstrated a quantitative epitope-specificity profile of antibody responses that can differentiate between protected vs. nonprotected individuals. Aligning vaccine efficacy with quantitation of the epitope fine specificity results of this equivalency assay reveals the importance of epitope specificity.

Discussion: The newly developed serological equivalence assay will inform future vaccine design and possibly even adjuvant selection. This methodology can be adapted to other antigens and disease models, when a panel of relevant mAbs exists, and could offer a unique tool for comparing and down-selecting vaccine formulations.

Keywords: antigen-specificity; circumsporozoite protein; equivalency; malaria; protection; serology; vaccine.

Figure 1

Overview of the CBASQE assay. (A) The 10-spot MSD plate accommodates up to ten different antigens per well (left column). Wells were coated with a mixture containing seven U-plex coupled peptides (spots 4-6 were not used). Serially diluted test serum was mixed with the Sulfo-tag labeled mAb reporter mix and added to the U-plex plates. Loss/reduction in luminescence is seen when serum antibodies successfully competed off the Sulfo-tag labeled reporter mix. (B) Schematic of the CSP protein visualizing the regions represented by the peptides used in this study and where epitope-specific mAbs are binding. (C) Analysis workflow for conducting the serological equivalence assay.

Figure 2

Serological profile of RTS,S vaccinees (NCT00075049) as determined in the CBASQE assay. Data expressed as ng/ml epitope-specific antibody concentration in sera of protected (n=18, (A)) or non-protected (n=18, (B)) RTS,S vaccinees. Equivalency was determined using mAbs CIS43, 317, 236, and 369. Results for N-terminus omitted since no reactivity of the sera was measured. Asterisks indicate statistical significance between protected vs. non-protected RTS,S vaccinees (two-sample T-test, *p< 0.05, **< 0.005).

Figure 3

Antibody profile of protected vs. non-protected RTS,S immunized vaccinees to CSP regions. Correlation matrices indicate the relationship between the equivalencies of the antibody responses to the various CSP epitope specific mAbs. The color and size of the dots (scale below graphs) indicate the degree of correlation between the different CSP peptides (small to large indicating low to high correlation). Correlation matrices stratified by protective status ((A) = protected, (B) = non-protected individuals).

Figure 4

Agreement between ELISA- and CBASQE (Equivalence) results dependent on protective status. Log-transformed ELISA titers (y-axis) and Equivalence (x-axis) results for repeat-specific and C-terminal responses were stratified by protective status: responses of sera from protected (n=18, top panels) and non-protected individuals (n=18, bottom panels).