Novel ELISA method as exploratory tool to assess immunity induced by radiated attenuated sporozoites to decipher protective immunity

Abstract

Background: Whole parasite vaccines provide a unique opportunity for dissecting immune mechanisms and identify antigens that are targeted by immune responses which have the potential to mediate sterile protection against malaria infections. The radiation attenuated sporozoite (PfSPZ) vaccine has been considered the gold standard for malaria vaccines because of its unparalleled efficacy. The immunogenicity of this and other vaccines continues to be evaluated by using recombinant proteins or peptides of known sporozoite antigens. This approach, however, has significant limitations by relying solely on a limited number of known pathogen-associated immune epitopes. Using the full range of antigens expressed by the sporozoite will enable the comprehensive immune-profiling of humoral immune responses induced by whole parasite vaccines. To address this challenge, a novel ELISA based on sporozoites was developed.

Results: The SPZ-ELISA method described in this report can be performed with either freshly dissected sporozoites or with cryopreserved sporozoite lysates. The use of a fixative for reproducible coating is not required. The SPZ-ELISA was first validated using monoclonal antibodies specific for CSP and TRAP and then used for the characterization of immune sera from radiation attenuated sporozoite vaccinees.

Conclusion: Applying this simple and highly reproducible approach to assess immune responses induced by malaria vaccines, both recombinant and whole parasite vaccines, (1) will help in the evaluation of immune responses induced by antigenically complex malaria vaccines such as the irradiated SPZ-vaccine, (2) will facilitate and accelerate the identification of immune correlates of protection, and (3) can also be a valuable assessment tool for antigen discovery as well as down-selection of vaccine formulations and, thereby, guide vaccine design.

Keywords: Antibodies; Assay development; Correlates of protection; Immunity; Malaria; Plasmodium; Sporozoite.

Fig. 1

Comparison of coating conditions. Plasmodium falciparum sporozoites were coated at 50,000 (black bar) or 25,000 (white bar) sporozoites per well and then either air-dried or fixed using the fixatives indicated on the x-axis. Plates were then probed with a CSP-specific monoclonal antibody (clone 2A10) or control mAb 1D9. Data are expressed as mean OD (± SD) of quadruplicates of a representative experiment. The dashed line indicates the background activity [maximal ELISA signal obtained with the control mAb (OD < 0.05 for all conditions)]

Fig. 2

Differential reactivity of antibodies with air-dried vs. methanol fixed sporozoites. Various concentrations of intact sporozoites (a, b) or sporozoite lysates (c, d) were plated and air-dried or methanol fixed. Plates were then probed with either a CSP-specific mAb (2A10) (a, c) or pooled sera from RAS immune subjects (b, d). The dashed lines indicate the background activity [ELISA signal obtained with either the control mouse mAb 1D9 (OD < 0.05) or pre-immune serum pool (OD < 0.12)]. Asterisks indicate statistical significance (p < 0.05, paired T test)

Fig. 3

Air-dried sporozoite react significantly better with polyclonal RAS immune antibodies. Lysates were either dried onto ELISA plates or fixed with methanol onto the plate’s surface. Plates were then probed with two serum pools from protected (P) or non-protected (NP) individuals at a 1:2000 dilution. Asterisk indicates statistical significance (paired T test). Data are expressed as mean OD (SD). The background OD with the pre-immune serum pool was 0.13 ± 0.04 (30,000 spz) and 0.12 ± 0.05 (15,000 spz)

Fig. 4

CSP specific antibodies show distinct reaction patterns when testing air dried vs. methanol-fixed sporozoites. a CSP repeat-specific mAb 2A10, b CSP specific mAb TH3, c CSP-C-term specific TH1, d TRAP-specific mAb 5E2. The dashed lines indicate the background activity [ELISA signal obtained with the control mouse mAb 1D9 (OD < 0.05)].