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Development of an in vitro Plasmodium parasite killing assay for the evaluation of cell-mediated immune responses following vaccination with pre-erythrocytic malaria vaccine candidates


Vaccination against liver stage malaria antigens can induce T cell-mediated immunity to the disease [1]. A viral vector vaccination regime undergoing Phase 2b clinical testing uses chimpanzee adenovirus 63 (ChAd63) and Modified Vaccinia virus Ankara (MVA) encoding liver stage antigen Thrombospondin-Related Adhesion Protein (TRAP) fused to a malaria multi-epitope string (ME). This regime induces high frequencies of antigen-specific T cells, providing 21% sterile protection and a delay to patent parasitaemia in a further 36% of vaccinees, following controlled human Plasmodium falciparum malaria infection (CHMI). Monofunctional IFNg-producing CD8+ T cells correlate with vaccine-induced protection but the associated protective mechanisms remain unidentified [2]. Developing standardized immunological and functional assays is a research-specific aim of the WHO’s Malaria Vaccine Technology Roadmap, with emphasis on novel immunoassays for investigation of cellular products reflecting cell-mediated malaria immunity [3]. Development of an in vitro parasite killing assay is underway, which quantifies cell-mediated killing of Plasmodium-infected human hepatocytes and investigates the underlying functional mechanisms. Additionally, the assay aims to compliment in vivo CHMI studies.

Materials and methods

Human hepatoma cell lines were infected with transgenic P. berghei sporozoites expressing TRAP from P. falciparum. Freshly separated peripheral blood mononuclear cells from partial-HLA class I matched ChAd63.MVA ME-TRAP human vaccinees were enriched for CD8+ T cell populations. Following hepatocyte infection, enriched CD8+ T cells were added and incubated overnight. Level of infectivity was measured by flow cytometry through expression of GFP under a P. berghei promotor. TRAP-specific killing was calculated by subtraction of non-specific killing in wild type P. berghei-infected hepatocytes.


Transgenic P. berghei sporozoites with full replacement of wild type TRAP infect human hepatoma cells at >1% frequency. Preliminary results measure a 9.5-22% TRAP-specific reduction of infected hepatocytes after addition of CD8+ T cells from ChAd63.MVA ME-TRAP vaccinees. Equivalently, TRAP-specific parasite killing was not detected following addition of CD8+ T cells from control volunteers. Conclusions Successful use of transgenic parasites and the preliminary results obtained provide proof of assay concept for this in vitro system. Further optimization will permit the investigation of cell-mediated parasite killing of CD8+ T cells from a larger number of vaccinees and extend to exploration of vaccine-induced mechanisms of protection.


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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

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Bliss, C.M., Salman, A.M., Longley, R.J. et al. Development of an in vitro Plasmodium parasite killing assay for the evaluation of cell-mediated immune responses following vaccination with pre-erythrocytic malaria vaccine candidates. Malar J 13 (Suppl 1), P14 (2014).

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