An Opsonic Phagocytosis Assay for Plasmodium falciparum Sporozoites

Abstract

Plasmodium falciparum malaria remains the deadliest parasitic disease worldwide. Vaccines targeting the preerythrocytic sporozoite and liver stages have the potential to entirely prevent blood-stage infection and disease, as well as onward transmission. Sporozoite surface and secreted proteins are leading candidates for inclusion in a preerythrocytic stage-specific, antibody-based vaccine. Preclinical functional assays to identify humoral correlates of protection in vitro and to validate novel sporozoite protein targets for inclusion in multisubunit vaccines currently do not consider the interaction of sporozoite-targeting antibodies with other components of the immune system. Here, we describe the development of a simple flow cytometric assay to quantitatively assess the ability of antibodies directed against P. falciparum sporozoites to facilitate their phagocytosis. We demonstrate that this sporozoite opsonic phagocytosis assay (SOPA) is compatible with both monoclonal antibodies and human immune serum and can be performed using cryopreserved P. falciparum sporozoites. This simple, accessible assay will aid with the assessment of antibody responses to vaccination with Plasmodium antigens and their interaction with phagocytic cells of the immune system. KEYWORDS Plasmodium falciparum, antibodies, assay development, malaria, opsonization, phagocytosis, sporozoite, vaccines T he parasite Plasmodium falciparum causes hundreds of millions of clinical malaria infections and half a million deaths annually (1). The parasite is transmitted by deposition of sporozoite stages into the skin through the bite of infected Anopheles mosquitoes. Motile sporozoites then move in the dermis to locate a capillary, gaining access to the circulation, through which they sequester in the liver. Here sporozoites select and infect hepatocytes, and the parasite then develops as a liver stage, producing tens of thousands of red blood cell-infectious merozoites that initiate blood-stage infection. Stages prior to merozoite emergence from hepatocytes are collectively known as preerythrocytic (PE) stages. The PE stages of infection are asymptomatic and are caused by a relatively small number of parasites. Elimination of PE stages prevents progression to blood stages of infection, responsible for the clinical symptoms of malaria and onward transmission of the parasite (2). These features render PE stages attractive vaccine targets.Sporozoite surface and secreted proteins are rational antibody targets for vaccines that aim to block sporozoite infection. The most advanced PE stage-specific vaccine candidate consists of the circumsporozoite protein (CSP), which is abundantly expressed on the sporozoite surface. Immunization with a CSP-based vaccine, RTS,S/AS01, a virus-like particle expressing CSP adjuvanted with AS01, achieved modest protection in phase 3 pediatric clinical trials in areas where malaria is endemic that was in part mediated by antibodies (3, 4). An alternative approach, vaccination with live-attenuated sporozoites that...

In vitro and in vivo inhibition of malaria parasite infection by monoclonal antibodies against Plasmodium falciparum circumsporozoite protein (CSP)

“…The WRAIR intravenous challenge model bypasses skin immunity 60 and protection may rely on a multitude of anti-parasitic effects of CSP antibodies including inhibition of motility and traversal 61 , Fc mediated opsonization 62 , complement mediated killing 63 , blocking of proteolytic processing of CSP 64 , or blocking of a putative receptor-ligand interaction 65 . This model measures protection in the blood which requires inactivation of 100% of the parasites in the inoculum and is sensitive to the biological and technical variability between challenge experiments for example the variation in protection outcomes between 24 and 48 h challenge with mAbs MGG4 and 311.…”

Protective effects of combining monoclonal antibodies and vaccines against the Plasmodium falciparum circumsporozoite protein

“…Antibodies can prevent malaria by inhibiting sporozoites (SPZ; the infectious form of Plasmodium parasites transmitted by mosquitos) before they invade hepatocytes in the liver. Antibody-mediated SPZ inhibition can be achieved by direct neutralization (e.g., blocking parasite motility [2] or invasion of hepatocytes [3,4]) or by engaging Fc-mediated effector functions such as opsonophagocytosis [5] or complement fixation [6]. Most anti-SPZ antibodies induced following natural infection or immunization with attenuated SPZ target the circumsporozoite protein (CSP), the most abundant surface protein on SPZ [7].…”

Role of Opsonophagocytosis in Immune Protection against Malaria

“…A major shortcoming of many protocols is the requirement for components that are not easily obtainable. For example, phagocytosis assays described for analyzing malaria-immune sera call for fresh/cryopreserved malaria parasites [ 87 ] or primary human phagocytes [ 67 ]. The use of fresh sporozoites is not feasible for many laboratories or are rather costly (in case of cryopreserved sporozoites).…”