Novel sporozoite-based ELISpot assay to assess frequency of parasite-specific B cells after vaccination with irradiated sporozoites

Abstract

Background: Whole parasite vaccination is an efficacious strategy to induce sterile immunity and to prevent malaria transmission. Understanding the mechanism and response of immune cells to vaccines plays a critical role in deciphering correlates of protection against infection and disease. Immunoassays, such as ELISpot, are commonly used to assess the immunogenicity of vaccines towards T cells and B cells. To date, these assays only analyse responses to specific antigens since they are based on recombinant parasite-derived proteins or peptides. There is the need for an agnostic approach that allows the evaluation of all sporozoite-associated antigens.

Methods: ELISpot plates coated with a defined amount of lysed Plasmodium falciparum sporozoites were used to assess the frequency of sporozoite-specific B cells in peripheral blood mononuclear cells from donors immunized with either a recombinant malaria vaccine or irradiated sporozoites.

Results: This report describes the assay conditions for a specific and sensitive sporozoite-based B cell ELISpot assay. The assay development considers the quality of sporozoite preparation as well as the detection threshold of the frequency of antigen-specific B cells. The assay enables the detection of sporozoite-specific IgM and IgG-producing B cells. Moreover, the assay can detect sporozoite-reactive B cells from subjects that were either vaccinated with the radiation attenuated sporozoite vaccine or a recombinant pre-erythrocytic vaccine.

Conclusion: The newly developed sporozoite-based B cell ELISpot enables the monitoring of changes in the frequency of sporozoite-specific B cells. Applying this assay to assess the potency of vaccination regimens or seasonal changes in B cell populations from subjects residing in malaria-endemic areas will provide an opportunity to gain insight into immune mechanisms involved in protection and/or disease.

Keywords: Antibodies; B cells; ELISpot; Immunity; Malaria; Sporozoite; Whole parasite vaccine.

Fig. 1

Impact of sporozoite preparation on signal strength. ELISpot plates were coated with lysates of sporozoite (30,000/well) either dissected using the Ozaki method (white bars) or by preparing salivary glands (black bars). 5 × 10⁵ PBMC/well from four different CSP-immune donors were plated. Data are expressed as the average number of sporozoite-reactive B cells (error bar represents standard deviation of triplicate wells)

Fig. 2

CSP-immune PBMC react with sporozoite lysates in an ELISpot format. Wells were either coated with sporozoite lysate (30,000/well, salivary gland preparation) or recombinant CSP protein (positive control) or BSA (negative control) and titrations of CSP-immune PBMC (indicated on x-axis) plated to determine the optimal cell concentration. Data (representative experiment) are expressed as the average number of reactive B cells (error represents SD of triplicate wells)

Fig. 3

Enrichment of B cells prior to plating onto the sporozoite-ELISpot improves the assay sensitivity. Three CSP-immune donors (a, b, and c representing the donors) were tested to measure the parasite response for pre-immune, post 3 (i.e., pre challenge), and post challenge (4 weeks after controlled human malaria infection) time points. 5 × 10⁵ PBMC/well (black symbol) or 10⁵ purified B cells/well (white symbol) were plated. Data expressed as mean (SD) of triplicate wells

Fig. 4

Detection of parasite-specific, IgM- and IgG-producing B cells. Parasite-specific IgM response (a). Parasite-specific IgG response (b). Data are represented as the mean number of sporozoite-specific spots per 10⁵ B cells (triplicate wells). Time points tested: pre-immune, post 3 (i.e., pre-challenge), post challenge (4 weeks after controlled malaria infection). BSA-coated (BSA) wells demonstrate non-specific binding of antibodies to the plate. Fill of bars indicates donors (n = 2)

Fig. 5

Sporozoite-ELISpot can discern between protected and non-protected RAS vaccinated subjects. Box plots represent the frequency of CSP-specific (a) or SPZ-specific (b) IgG-spots per 10⁵ plated B cells from protected (n = 8) and non-protected (n = 4) subjects per time point. Time points: pre-immune and pre-challenge (i.e., 2 weeks after last immunization, day of challenge)