Rebecca A. O'Donnell

Functional conservation of the malaria vaccine antigen MSP-1 19 acrossdistantly related Plasmodium species

The C-terminal region of Plasmodium falciparum merozoite surface protein 1 (MSP-119) is at present a leading malaria vaccine candidate. Antibodies against the epidermal growth factor-like domains of MSP-119are associated with immunity to P. falciparum and active immunization with recombinant forms of the molecule protect against malaria challenge in various experimental systems. These findings, with the knowledge that epidermal growth factor-like domains in other molecules have essential binding functions, indicate the importance of this protein in merozoite invasion of red blood cells. Despite extensive molecular epidemiological investigations, only limited sequence polymorphism has been identified in P. falciparum MSP-119 (refs. 9–11). This indicates its sequence is functionally constrained, and is used in support of the use of MSP-119 as a vaccine. Here, we have successfully complemented the function of most of P. falciparum MSP-119 with the corresponding but highly divergent sequence from the rodent parasite P. chabaudi. The results indicate that the role of MSP-119 in red blood cell invasion is conserved across distantly related Plasmodium species and show that the sequence of P. falciparum MSP-119 is not constrained by function.

Evidence That Invasion-Inhibitory Antibodies Specific for the 19-kDa Fragment of Merozoite Surface Protein-1 (MSP-119) Can Play a Protective Role against Blood-Stage Plasmodium falciparum Infection in Individuals in a Malaria Endemic Area of Africa

The C-terminal 19-kDa fragment of Plasmodium falciparum merozoite surface protein-1 (MSP-119) is a target of protective Abs against blood-stage infection and a leading candidate for inclusion in a human malaria vaccine. However, the precise role, relative importance, and mechanism of action of Abs that target this protein remain unclear. To examine the potential protective role of Abs to MSP-119 in individuals naturally exposed to malaria, we conducted a treatment time to infection study over a 10-wk period in 76 residents of a highland area of western Kenya during a malaria epidemic. These semi-immune individuals were not all equally susceptible to reinfection with P. falciparum following drug cure. Using a new neutralization assay based on transgenic P. falciparum expressing the P. chabaudi MSP-119 orthologue, individuals with high-level MSP-119-specific invasion-inhibitory Abs (>75th percentile) had a 66% reduction in the risk of blood-stage infection relative to others in the population (95% confidence interval, 3–88%). In contrast, high levels of MSP-119 IgG or IgG subclass Abs measured by enzyme immunoassay with six different recombinant MSP-119 Ags did not correlate with protection from infection. IgG Abs measured by serology and functional invasion-inhibitory activity did not correlate with each other. These findings implicate an important protective role for MSP-119-specific invasion inhibitory Abs in immunity to blood-stage P. falciparum infection, and suggest that the measurement of MSP-119 specific inhibitory Abs may serve as an accurate correlate of protection in clinical trials of MSP-1-based vaccines.

A genetic screen for improved plasmid segregation reveals a role for Rep20 in the interaction of Plasmodium falciparum chromosomes

Bacterial plasmids introduced into the human malaria parasite Plasmodium falciparum replicate well but are poorly segregated during mitosis. In this paper, we screened a random P.falciparum genomic library in order to identify sequences that overcome this segregation defect. Using this approach, we selected for parasites that harbor a unique 21 bp repeat sequence known as Rep20. Rep20 is one of six different repeats found in the subtelomeric regions of all P.falciparum chromosomes but which is not found in other eukaryotes or in other plasmodia. Using a number of approaches, we demonstrate that Rep20 sequences lead to dramatically improved episomal maintenance by promoting plasmid segregation between daughter merozoites. We show that Rep20+, but not Rep20−, plasmids co‐localize with terminal chromosomal clusters, indicating that Rep20 mediates plasmid tethering to chromosomes, a mechanism that explains the improved segregation phenotype. This study implicates a direct role for Rep20 in the physical association of chromosome ends, which is a process that facilitates the generation of diversity in the terminally located P.falciparum virulence genes.

A New Rodent Model to Assess Blood Stage Immunity to the Plasmodium falciparum Antigen Merozoite Surface Protein 119 Reveals a Protective Role for Invasion Inhibitory Antibodies

Antibodies capable of inhibiting the invasion of Plasmodium merozoites into erythrocytes are present in individuals that are clinically immune to the malaria parasite. Those targeting the 19-kD COOH-terminal domain of the major merozoite surface protein (MSP)-119 are a major component of this inhibitory activity. However, it has been difficult to assess the overall relevance of such antibodies to antiparasite immunity. Here we use an allelic replacement approach to generate a rodent malaria parasite (Plasmodium berghei) that expresses a human malaria (Plasmodium falciparum) form of MSP-119. We show that mice made semi-immune to this parasite line generate high levels of merozoite inhibitory antibodies that are specific for P. falciparum MSP-119. Importantly, protection from homologous blood stage challenge in these mice correlated with levels of P. falciparum MSP-119–specific inhibitory antibodies, but not with titres of total MSP-119–specific immunoglobulins. We conclude that merozoite inhibitory antibodies generated in response to infection can play a significant role in suppressing parasitemia in vivo. This study provides a strong impetus for the development of blood stage vaccines designed to generate invasion inhibitory antibodies and offers a new animal model to trial P. falciparum MSP-119 vaccines.

Functional analysis of proteins involved in Plasmodium falciparum merozoite invasion of red blood cells

Plasmodium falciparum causes the most lethal form of malaria in humans and is responsible for over two million deaths per year. The development of a vaccine against this parasite is an urgent priority and potential protein targets include those on the surface of the asexual merozoite stage, the form that invades the host erythrocyte. The development of methods to transfect P. falciparum has enabled the construction of gain‐of‐function and loss‐of‐function mutants and provided new strategies to analyse the role of parasite proteins. In this review, we describe the use of this technology to examine the role of merozoite antigens in erythrocyte invasion and to address their potential as vaccine candidates.

A Set of Glycosylphosphatidyl Inositol-Anchored Membrane Proteins of Plasmodium falciparum Is Refractory to Genetic Deletion

ABSTRACT Targeted gene disruption has proved to be a powerful approach for studying the function of important ligands involved in erythrocyte invasion by the extracellular merozoite form of the human malaria parasite, Plasmodium falciparum. Merozoite invasion proceeds via a number of seemingly independent alternate pathways, such that entry can proceed with parasites lacking particular ligand-receptor interactions. To date, most focus in this regard has been on single-pass (type 1) membrane proteins that reside in the secretory organelles. Another class of merozoite proteins likely to include ligands for erythrocyte receptors are the glycosylphosphatidyl inositol (GPI)-anchored membrane proteins that coat the parasite surface and/or reside in the apical organelles. Several of these are prominent vaccine candidates, although their functions remain unknown. Here, we systematically attempted to disrupt the genes encoding seven of the known GPI-anchored merozoite proteins of P. falciparum by using a double-crossover gene-targeting approach. Surprisingly, and in apparent contrast to other merozoite antigen classes, most of the genes (six of seven) encoding GPI-anchored merozoite proteins are refractory to genetic deletion, with the exception being the gene encoding merozoite surface protein 5 (MSP-5). No distinguishable growth rate or invasion pathway phenotype was detected for the msp-5 knockout line, although its presence as a surface-localized protein was confirmed.

A novel family of apicomplexan glideosome-associated proteins with an inner membrane-anchoring role.

The phylum Apicomplexa are a group of obligate intracellular parasites responsible for a wide range of important diseases. Central to the lifecycle of these unicellular parasites is their ability to migrate through animal tissue and invade target host cells. Apicomplexan movement is generated by a unique system of gliding motility in which substrate adhesins and invasion-related proteins are pulled across the plasma membrane by an underlying actin-myosin motor. The myosins of this motor are inserted into a dual membrane layer called the inner membrane complex (IMC) that is sandwiched between the plasma membrane and an underlying cytoskeletal basket. Central to our understanding of gliding motility is the characterization of proteins residing within the IMC, but to date only a few proteins are known. We report here a novel family of six-pass transmembrane proteins, termed the GAPM family, which are highly conserved and specific to Apicomplexa. In Plasmodium falciparum and Toxoplasma gondii the GAPMs localize to the IMC where they form highly SDS-resistant oligomeric complexes. The GAPMs co-purify with the cytoskeletal alveolin proteins and also to some degree with the actin-myosin motor itself. Hence, these proteins are strong candidates for an IMC-anchoring role, either directly or indirectly tethering the motor to the cytoskeleton.

The Fine Specificity, but Not the Invasion Inhibitory Activity, of 19-Kilodalton Merozoite Surface Protein 1-Specific Antibodies Is Associated with Resistance to Malarial Parasitemia in a Cross-Sectional Survey in The Gambia

ABSTRACT In a cross-sectional survey of 187 Gambian children and adults, we have analyzed prevalence, fine specificity, and 19-kilodalton merozoite surface protein 1 (MSP-119)-specific erythrocyte invasion inhibitory activity of antibodies to MSP-119 but find no significant association between any of these parameters and prevalence or density of malarial parasitemia, except that, after correcting for total anti-MSP-119 antibody levels, individuals with anti-MSP-119 antibodies that compete with an invasion inhibitory monoclonal antibody (12.10) were significantly less likely to have malaria infections with densities of ≥1,000 parasites/μl than were individuals without such antibodies. This association persisted after correction for age and ethnic origin.

Deletion of the Plasmodium falciparum Merozoite Surface Protein 7 Gene Impairs Parasite Invasion of Erythrocytes

ABSTRACT Merozoite surface proteins have been implicated in the initial attachment to the host red blood cell membrane that begins the process of invasion, an important step in the life cycle of the malaria parasite. In Plasmodium falciparum, merozoite surface proteins include several glycosylphosphatidyl inositol-anchored proteins and peripheral proteins attached to the membrane through protein-protein interactions. The most abundant of these proteins is the merozoite surface protein 1 (MSP1) complex, encoded by at least three genes: msp1, msp6, and msp7. The msp7 gene is part of a six-member multigene family in Plasmodium falciparum. We have disrupted msp7 in the Plasmodium falciparum D10 parasite, as confirmed by Southern hybridization. Immunoblot and indirect immunofluorescence analyses confirmed the MSP7 null phenotype of D10ΔMSP7 parasites. The synthesis, distribution, and processing of MSP1 were not affected in this parasite line. The level of expression and cellular distribution of the proteins MSP1, MSP3, MSP6, MSP9, and SERA5 remained comparable to those for the parental line. Furthermore, no significant change in the expression of MSP7-related proteins, except for that of MSRP5, was detected at the transcriptional level. The lack of MSP7 was not lethal at the asexual blood stage, but it did impair invasion of erythrocytes by merozoites to a significant degree. Despite this reduction in efficiency, D10ΔMSP7 parasites did not show any obvious preference for alternate pathways of invasion.

MSP119 miniproteins can serve as targets for invasion inhibitory antibodies in Plasmodium falciparum provided they contain the correct domains for cell surface trafficking

Antibodies from malaria‐exposed individuals can agglutinate merozoites released from Plasmodium schizonts, thereby preventing them from invading new erythrocytes. Merozoite coat proteins attached to the plasma membrane are major targets for host antibodies and are therefore considered important malaria vaccine candidates. Prominent among these is the abundant glycosylphosphatidylinositol (GPI)‐anchored merozoite surface protein 1 (MSP1) and particularly its C‐terminal fragment (MSP119) comprised of two epidermal growth factor (EGF)‐like modules. In this paper, we revisit the role of agglutination and immunity using transgenic fluorescent marker proteins. We describe expression of heterologous MSP119‘miniproteins’ on the surface of Plasmodium falciparum merozoites. To correctly express these proteins, we determined that GPI‐anchoring and the presence of a signal sequence do not allow default export of proteins from the endoplasmic reticulum to merozoite surface and that extra sequence elements are required. The EGFs are insufficient for correct trafficking unless they are fused to additional residues that normally reside upstream of this fragment. Antibodies specifically targeting the surface‐expressed miniprotein can inhibit erythrocyte invasion in vitro despite the presence of endogenous MSP1. Using a line expressing a green fluorescent protein–MSP1 fusion protein, we demonstrate that one mode of inhibition by antibodies targeting the MSP119 domain is the rapid agglutinating of merozoites prior to erythrocyte attachment.