Identification of pre-erythrocytic malaria antigens that target hepatocytes for killing in vivo and contribute to protection elicited by whole-parasite vaccination.

Lin Chen,Marissa Vignali,Xiaohong Peng,Gladys J Keitany,Isaac Mohar,Claire Gibson,Fusheng Huang,Ruobing Wang,Ian N Crispe,Anne Charlotte Gruner

doi:10.1371/journal.pone.0102225

Abstract

Pre-erythrocytic malaria vaccines, including those based on whole-parasite approaches, have shown protective efficacy in animal and human studies. However few pre-erythocytic antigens other than the immunodominant circumsporozoite protein (CSP) have been studied in depth with the goal of developing potent subunit malaria vaccines that are suited for use in endemic areas. Here we describe a novel technique to identify pre-erythrocytic malaria antigens that contribute to protection elicited by whole-parasite vaccination in the mouse model. Our approach combines immunization with genetically attenuated parasites and challenge with DNA plasmids encoding for potential protective pre-erythrocytic malaria antigens as luciferase fusions by hydrodynamic tail vein injection. After optimizing the technique, we first showed that immunization with Pyfabb/f−, a P. yoelii genetically attenuated parasite, induces killing of CSP-presenting hepatocytes. Depletion of CD8+ but not CD4+ T cells diminished the killing of CSP-expressing hepatocytes, indicating that killing is CD8+ T cell-dependent. Finally we showed that the use of heterologous prime/boost immunization strategies that use genetically attenuated parasites and DNA vaccines enabled the characterization of a novel pre-erythrocytic antigen, Tmp21, as a contributor to Pyfabb/f− induced protection. This technique will be valuable for identification of potentially protective liver stage antigens and has the potential to contribute to the understanding of immunity elicited by whole parasite vaccination, as well as the development of effective subunit malaria vaccines.

Highlights

Malaria remains a daunting public health challenge in spite of global elimination efforts that have significantly reduced incidence and death caused by this parasitic disease
We detected high levels of luciferase activity in the liver of all mice in both groups as early as 4 h post Hydrodynamic Tail Vein Injection (HTVI), with expression peaking 4 to 8 h post HTVI and remaining high 7 d post HTVI (Figure 1A and B). This result shows that the PyCSP-luciferase fusion protein is expressed in a stable and persistent manner in the liver of mice injected with plasmid DNA through HTVI, and that by measuring whole-body bioluminescence in vivo, in vivo imaging system (IVIS) allows the real-time monitoring of the abundance of luciferase-fused tagged proteins
The use of immunization strategies that result in late liver stage (LS) or early blood stages (BS) arrest results in the development of immunity against a broad spectrum of antigens expressed by LS-infected hepatocytes and as consequence, in enhanced protection against sporozoite challenge [19,44]

Summary

Introduction

Malaria remains a daunting public health challenge in spite of global elimination efforts that have significantly reduced incidence and death caused by this parasitic disease. Since all clinical symptoms derive from the blood stages (BS), malaria vaccines that block parasite development during preerythrocytic (PE) stages prevent all human disease symptoms [2]. With up to 100% efficacy in human trials, live attenuated wholeparasite vaccines have been most effective to date, and include sporozoites that have been radiation-, drug-, or geneticallyattenuated (reviewed in [3]). All of these can invade hepatocytes but subsequently arrest at different points during the liver stage (LS) or early in the BS of the life cycle of the parasite, while simultaneously inducing immune responses that protect against subsequent challenge with wild type sporozoites (wt spz). The PfSPZ vaccine was recently reported to protect 80% of volunteers who received 4–5 doses of intravenously administered irr-spz [10], in line with the vaccine efficacy required for eradication as per recent WHO guidelines [11]

Methods

Results

Conclusion