An Improved Theileria parva Sporozoite Seroneutralization Assay for the Identification of East Coast Fever Immune Correlates

Abstract

Background: Immune correlates of protection are ideal tools to predict treatment or vaccine efficacy. However, the accuracy of the immune correlate and the capability to robustly predict the outcome of a vaccine candidate are determined by the performance of the in vitro immunoassay used. Several Theileria parva sporozoite seroneutralization assays have previously been used to assess antibody functional activities; however, a common limitation has been the need for fresh material, target cells and sporozoites, and operator-to-operator bias. An improved assay represents a positive step toward overcoming challenges associated with variability and it might provide a more reliable means of establishing an immune correlate with protection after sub-unit vaccine administration.

Methods: Herein, we describe key improvements, among them, (1) the use of frozen parasites and target cells to avoid batch-to-batch variations and (2) the development of a new assay read-out based on the detection of infected cells through flow cytometry, instead of the use of Giemsa staining and microscopic evaluation, in order to improve the reproducibility of the results.

Results: The improved seroneutralization assay is not only able to detect the individual neutralizing capacity of antibodies; it also detects the additive effect of antibody combinations.

Conclusions: This effect is described for the first time in Theileria parva and is of great interest for new antigen discovery and/or the epitope discovery of already known antigens like p67, opening a new avenue for the identification of ECF immune correlates of protection and the in vitro down-selection of new Theileria parva vaccine candidates, thereby contributing to reducing the use of animals in challenge experiments.

Keywords: East Coast fever; Theileria parva; antibodies; antigen; seroneutralization assay; sporozoites.

Figure 1

The percentage of infected cells was monitored using intracellular staining from day 8 to 12. The mean (column) and standard deviation (bars) are represented.

Figure 2

The neutralizing capacity of antibodies was assessed individually and in combination. The median and lower and upper quartile are represented by the box and the minimum and maximum values are represented by a vertical line.