Abstract
The development of an efficacious vaccine against the Plasmodium parasite remains a top priority. Previous research has demonstrated the ability of a prime-boost virally vectored sub-unit vaccination regimen, delivering the liver-stage expressed malaria antigen TRAP, to produce high levels of antigen-specific T cells. The liver-stage of malaria is the main target of T cell-mediated immunity, yet a major challenge in assessing new T cell inducing vaccines has been the lack of a suitable pre-clinical assay. We have developed a flow-cytometry based in vitro T cell killing assay using a mouse hepatoma cell line, Hepa1-6, and Plasmodium berghei GFP expressing sporozoites. Using this assay, P. berghei TRAP-specific CD8+ T cell enriched splenocytes were shown to inhibit liver-stage parasites in an effector-to-target ratio dependent manner. Further development of this assay using human hepatocytes and P. falciparum would provide a new method to pre-clinically screen vaccine candidates and to elucidate mechanisms of protection in vitro.
Highlights
Malaria, caused by the parasite Plasmodium, remains a serious public health concern in developing countries where it is a significant cause of morbidity and mortality [1], and promotes the cycle of poverty
We defined our gating strategy based on selection of viable hepatocytes, with positive green fluorescent protein (GFP) expression representing P. berghei infected cells (Fig. 1); this method of measuring infectivity has previously been shown to correlate with the number of infected cells as per traditional microscopy or PCR [32]
We have described the development of an improved murine in vitro assay and used it to demonstrate the ability of P. berghei ortholog of PfTRAP (PbTRAP)-specific CD8+ T cell enriched splenocytes to inhibit liver-stage parasites
Summary
Malaria, caused by the parasite Plasmodium, remains a serious public health concern in developing countries where it is a significant cause of morbidity and mortality [1], and promotes the cycle of poverty. Vaccines are considered one of the most cost-effective public health tools, yet a highly efficacious vaccine against malaria has yet to be achieved. One promising avenue has been the use of viral vectored vaccines to deliver the pre-erythrocytic antigen thrombospondin-related adhesion protein (TRAP) [2], known as SSP2. This vaccination regimen provided 21% sterile efficacy and a higher rate of partial efficacy manifest as delay to patency in a controlled human malaria infection trial; protection was associated with interferon-gamma (IFN-γ) producing CD8+ T cells [3], suggesting TRAP might be the target of cell-.
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