330 research outputs found
Immune sera recognize on erythrocytes a Plasmodium falciparum antigen composed of repeated amino acid sequences
No full textAbstract not availableRoss L. Coppel, Alan F. Cowman, Robin F. Anders, Albert E. Bianco, Robert B. Saint, Klaus R. Lingelbach, David J. Kemp & Graham V. Brow
Biochemical and biophysical investigations into key malaria parasite proteins
No full textPlasmodium falciparum, the most pestilential of the malaria parasite species, is
responsible for ~450,000 direct deaths annually. Clinical disease is a consequence
of the blood stage of the parasite’s lifecycle involving a plethora of host-parasite
interactions. Key to these interactions are the P. falciparum reticulocyte binding-like
homologue (PfRh) proteins responsible for binding erythrocyte receptors and
gaining entry to host cells. For example, PfRh4 binds to human complement
receptor-1 (CR1) on erythrocytes for sialic-acid-independent invasion. Another
protein important for invasion is the PfRh5-interacting protein (PfRipr), an
essential member of the PfRh5-associated invasion complex (PAIN-complex)
along with CyRPA, the cysteine-rich protective antigen. Loss of function of PfRipr
in P. falciparum parasites prevents erythrocyte entry and ablates Ca2+-influx into the
erythrocyte; essential events during invasion.
This study aimed to biochemically and structurally investigate truncated
recombinant versions of PfRh4 and PfRipr. Homology modelling suggested that
PfRh4 is rich in alpha-helical secondary structure. The sequence of PfRipr
suggested the presence of ten epidermal growth factor-like (EGF) modules, two
towards the N-terminus and eight in the C-terminal domain.
In this project, monoclonal antibodies made against recombinant PfRh4 were
shown, via indirect immunofluorescent assays, to localize to the apical tip of
merozoites. Monoclonal antibody 5H12, raised against PfRh4, reduces parasite
invasion of erythrocytes by ~75% in growth-inhibition assays with neuraminidase
pre-treated erythrocytes. Attempts to produce a stable truncated recombinant
PfRh4 protein for structural studies were unsuccessful. An ELISA-based assay
using ten alanine-scan mutants suggested the CR1-binding site lies outside of
amino acids 283 – 341 of PfRh4. PfRipr truncations, defined by the boundaries of EGF-like repeats predicted based
on sequence homology, were produced recombinantly in Escherichia coli and Pichia
pastoris. These proteins had a circular dichroism signature suggestive of β-strand-containing
proteins with disordered regions. EGF-containing PfRipr truncations
did not bind recombinant PfRh5 according to ELISA and size-exclusion
chromatography assays. EGFs 1-2, 5-7 and 7-10 of PfRipr did not bind CyRPA via
size-exclusion chromatography or NMR. Crystallisation trials performed on EGF
modules failed to yield crystals suitable for data collection. A 15N isotopically-labelled
sample of EGF5-7 gave good quality HSQC NMR spectra. A suite of
three-dimensional NMR spectra collected on a 13C,15N-EGF5-7 sample, at three
different temperatures, allowed for >86% of backbone assignments. T1/T2
relaxation analysis and heteronuclear NOE data were suggestive of an elongated,
rigid protein undergoing intermolecular self-association. Further evidence for
EGF5-7 being an elongated protein was provided via SAXS analysis. Chemical
shifts facilitated prediction of secondary structure in EGF 5-7 consistent with an
EGF-like fold. Melting studies performed on EGF5-7 showed no evidence of
denaturation over the temperature range 20 °C - 95 °C indicating a thermally-stable
protein. The addition of Ca2+ to the 15N-EGF5-7 sample caused chemical
shift perturbations consistent with high-affinity binding. The discovery of
inhibitory monoclonal antibodies recognising a conformational epitope on EGF7
provided evidence of the functional importance of this region within PfRipr.
The work described in this thesis provides methods for the industrially-scalable
production and biophysical investigations of P. pastoris or E. coli-produced
disulfide-rich P. falciparum antigens of interest to vaccinologists
Expression of Plasmodium falciparum blood-stage antigens in Escherichia coli: detection with antibodies from immune humans
No full textMany proteins produced by blood stages of the malaria parasite Plasmodium falciparum are natural immunogens in man. As an approach to determining which of these are relevant to protective immunity we have constructed an expression library of P. falciparum cDNA sequences, cloned in Escherichia coli. The cDNA sequences were inserted into the beta-galactosidase gene of an ampicillin-resistant derivative of the temperature-sensitive lysogenic bacteriophage lambda gt11. About 5% of the resulting clones expressed P. falciparum sequences as polypeptides fused to beta-galactosidase. We have identified many clones that express P. falciparum antigens by immunological screening in situ with antibodies from immune human sera that inhibit P. falciparum growth in vitro. The antigen-positive clones contain P. falciparum cDNA sequences, as determined by hybridization. Some express polypeptides that are larger than beta-galactosidase and react both with antibodies to beta-galactosidase and with antibodies from humans immune to P. falciparum. The cloned P. falciparum antigens should facilitate new approaches to the identification of potential vaccine molecules.David J. Kemp, Ross L. Coppel, Alan F. Cowman, Robert B. Saint, Graham V. Brown, and Robin F. Ander
Efficient measurement of opsonising antibodies to plasmodium falciparum merozoites
No full textBACKGROUND Antibodies targeting merozoites are important in protection from malaria. Therefore, merozoite surface proteins are attractive vaccine candidates. There is a need for robust functional assays to investigate mechanisms of acquired immunity and vaccine efficacy. To date, the study of merozoite phagocytosis has been confounded by the complexity and variability of in vitro assays. METHODOLOGY/PRINCIPAL FINDINGS We have developed a new flow cytometry-based merozoite phagocytosis assay. An optimized merozoite preparation technique produced high yields of merozoites separated from haemozoin. Phagocytosis by the undifferentiated THP-1 monocytic cell line was mediated only by Fc Receptors, and was therefore ideal for studying opsonising antibody responses. The assay showed robust phagocytosis with highly diluted immune sera and strong inter-assay correlation. The assay effectively measured differences in opsonisation-dependent phagocytosis among individuals. CONCLUSIONS/SIGNIFICANCE This highly reproducible assay has potential applications in assessing the role of opsonic phagocytosis in naturally acquired immunity and vaccine trials.Danika L. Hill, Emily M. Eriksson, Amandine B. Carmagnac, Danny W. Wilson, Alan F. Cowman, Diana S. Hansen, Louis Schofiel
Targeted Disruption of <i>PfRh3</i> Shows It Is Not Essential for the 3D7ΔRh2b Chymotrypsin-Resistant Pathway
No full text<div><p>(A) Disruption of the <i>PfRh3</i> gene in 3D7. The pCC4-<i>Rh3</i> plasmid contains the blasticidin-S deaminase selectable marker, a negative selectable marker (A. G. Maier and A. F. Cowman, unpublished data), and 5′ and 3′ <i>Rh3</i> regions. <i>PfRh3</i> is shown with homologous target sequences (shaded regions). The double-crossover integration events are shown for 3D7, resulting in the deletion of a 5′ region of the gene. Restriction enzymes are C (ClaI) and X (XbaI), with fragment sizes shown for a C/X digestion. RT-PCR amplification target is shown as a black bar.</p><p>(B) Southern blot of genomic DNA from parasites shown digested with ClaI and XbaI and probed with the 5′ <i>Rh3</i> flank from the pCC4-<i>Rh3</i> vector.</p><p>(C) RT-PCR of <i>PfRh2a</i> and <i>PfRh3</i> from 3D7 and three knockout lines.</p><p>(D) Invasion into enzyme-treated erythrocytes expressed as a percentage of that into untreated erythrocytes for 3D7 and three knockout lines. Values above each column indicate the mean % invasion, with error bars representing the 95% CI from three independent assays.</p></div
Defining the antigenic diversity of Plasmodium falciparum Apical Membrane Antigen 1 and the requirements for a multi-allele vaccine against malaria
No full textApical Membrane Antigen 1 (AMA1) is a leading malaria vaccine candidate and a target of naturally-acquired human immunity. Plasmodium falciparum AMA1 is polymorphic and in vaccine trials it induces strain-specific protection. This antigenic diversity is a major roadblock to development of AMA1 as a malaria vaccine and understanding how to overcome it is essential. To assess how AMA1 antigenic diversity limits cross-strain growth inhibition, we assembled a panel of 18 different P. falciparum isolates which are broadly representative of global AMA1 sequence diversity. Antibodies raised against four well studied AMA1 alleles (W2Mef, 3D7, HB3 and FVO) were tested for growth inhibition of the 18 different P. falciparum isolates in growth inhibition assays (GIA). All antibodies demonstrated substantial cross-inhibitory activity against different isolates and a mixture of the four different AMA1 antibodies inhibited all 18 isolates tested, suggesting significant antigenic overlap between AMA1 alleles and limited antigenic diversity of AMA1. Cross-strain inhibition by antibodies was only moderately and inconsistently correlated with the level of sequence diversity between AMA1 alleles, suggesting that sequence differences are not a strong predictor of antigenic differences or the cross-inhibitory activity of anti-allele antibodies. The importance of the highly polymorphic C1-L region for inhibitory antibodies and potential vaccine escape was assessed by generating novel transgenic P. falciparum lines for testing in GIA. While the polymorphic C1-L epitope was identified as a significant target of some growth-inhibitory antibodies, these antibodies only constituted a minor proportion of the total inhibitory antibody repertoire, suggesting that the antigenic diversity of inhibitory epitopes is limited. Our findings support the concept that a multi-allele AMA1 vaccine would give broad coverage against the diversity of AMA1 alleles and establish new tools to define polymorphisms important for vaccine escape.Damien R. Drew, Anthony N. Hodder, Danny W. Wilson, Michael Foley, Ivo Mueller, Peter M. Siba, Arlene E. Dent, Alan F. Cowman, James G. Beeso
Revealing a Parasite's Invasive Trick
No full textA study of an important parasite reveals the core structure of the apicomplexan moving junction.</jats:p
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