The Fab fragments of monoclonal IgG to a merozoite surface antigen inhibit Plasmodium knowlesi invasion of erythrocytes

Thomas, Alan W.; Deans, Judith A.; Mitchell, G. H.; Alderson, Thomas; Cohen, S.

doi:10.1016/0166-6851(84)90112-9

Cited by 135 publications

(97 citation statements)

References 18 publications

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“…Similar results were reported by Dutta et al who examined the invasion-inhibitory activity of impure preparations of 4G2 Fab (43). This result is intriguingly reminiscent of similar findings reported in an early study of invasion-inhibition by a mAb specific for AMA1 of the simian malaria parasite P. knowlesi; in that work, the authors speculated that the enhanced potency of the Fab fragments might be a result of charge effects, removal of the Fc perhaps reducing electrostatic repulsion between the antibody and the negatively charged merozoite surface (61,62). It has been suggested that some invasion-inhibitory antibodies exert their activity by cross-linking AMA1 at the apical tip of the parasite, interfering with its redistribution across the parasite surface and possibly as a result blocking the secretion of other essential invasion-related proteins from the micronemes and/or rhoptries (44).…”

Section: Discussionsupporting

confidence: 54%

Fine Mapping of an Epitope Recognized by an Invasion-inhibitory Monoclonal Antibody on the Malaria Vaccine Candidate Apical Membrane Antigen 1

Collins¹,

Withers‐Martinez²,

Bentley

et al. 2007

Journal of Biological Chemistry

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102

115

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Antibodies that inhibit red blood cell invasion by the Plasmodium merozoite block the erythrocytic cycle responsible for clinical malaria. The invasion-inhibitory monoclonal antibody (mAb) 4G2 recognizes a conserved epitope in the ectodomain of the essential Plasmodium falciparum microneme protein and vaccine candidate, apical membrane antigen 1 (PfAMA1). Here we demonstrate that purified Fab fragments of 4G2 inhibit invasion markedly more efficiently than the intact mAb, suggesting that the invasion-inhibitory activity of this mAb is not due solely to steric effects and that the epitope lies within a functionally critical region of the molecule. We have taken advantage of a synthetic gene encoding a modified form of PfAMA1, and existing x-ray crystal structure data, to fully characterize this epitope. We first validate the gene by demonstrating that it fully complements the function of the authentic gene in P. falciparum. We then use it to identify a group of residues within the previously described domain II loop of PfAMA1 that are critical for recognition by mAb 4G2 and demonstrate that the epitope lies exclusively within this loop with no contributions from residues in other domains of the molecule. This is the first complete characterization of a conserved invasion-inhibitory epitope on PfAMA1. Our results will aid in the design of subunit vaccines designed to generate a broadly effective, focused anti-PfAMA1 protective immune response and may help elucidate the function of PfAMA1.Over half of the world's population is exposed to malaria (1). Until recently, the disease was controlled in or eradicated from a number of areas. However, the emergence of insecticide-resistant mosquito vectors and multidrug-resistant forms of the causative agent has contributed to resurgences of the disease, which in some cases have resulted in near catastrophic epidemics. As a result malaria remains a global problem, affecting many of the poorest nations and representing a serious threat to travelers. The development and implementation of effective new drugs and a vaccine is of paramount importance.Clinical malaria results from replication of protozoan parasites of the genus Plasmodium in circulating erythrocytes. Like most apicomplexan pathogens, the malaria merozoite invades its host cell in a multistep process initiated by reversible binding to receptors on the erythrocyte surface, followed by high affinity attachment via the apical end of the merozoite, and finally entry into a parasitophorous vacuole. Invasion is facilitated by the discharge of apical secretory organelles called micronemes and rhoptries. The type I integral membrane microneme protein apical membrane antigen 1 (AMA1) 2 is widely regarded as a leading candidate for inclusion in a malaria vaccine (2-5). Identified initially in the simian malaria species Plasmodium knowlesi as a target of a monoclonal antibody (mAb) that prevented erythrocyte invasion (6), homologues of AMA1 have been identified in all species of Plasmodium (6 -13) and in all other apicomplexa...

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Section: Discussionsupporting

confidence: 54%

Fine Mapping of an Epitope Recognized by an Invasion-inhibitory Monoclonal Antibody on the Malaria Vaccine Candidate Apical Membrane Antigen 1

Collins¹,

Withers‐Martinez²,

Bentley

et al. 2007

Journal of Biological Chemistry

Self Cite

102

115

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“…Domains I and II belong to the PAN module superfamily, suggesting that they may function in adhesion This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05-04 -0281) on July 6, 2005. to protein or carbohydrate receptors (Pizarro et al, 2005). Antibodies against AMA1 inhibit invasion ( Thomas et al, 1984;Deans et al, 1988;Hehl et al, 2000;Kocken et al, 2000;Mitchell et al, 2004), as do phage-displayed peptides derived from (Urquiza et al, 2000) or with affinity for Plasmodium AMA1 (Li et al, 2002;Keizer et al, 2003). Together with trans-species complementation experiments (Triglia et al, 2000) and heterologous expression experiments (Fraser et al, 2001;Kato et al, 2005), these data suggest that AMA1 plays a role in host cell invasion, perhaps as an adhesin.…”

Section: Introductionmentioning

confidence: 69%

Conditional Expression ofToxoplasma gondiiApical Membrane Antigen-1 (TgAMA1) Demonstrates That TgAMA1 Plays a Critical Role in Host Cell Invasion

Mital

Meissner

Soldati

et al. 2005

MBoC

229

250

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Toxoplasma gondii is an obligate intracellular parasite and an important human pathogen. Relatively little is known about the proteins that orchestrate host cell invasion by T. gondii or related apicomplexan parasites (including Plasmodium spp., which cause malaria), due to the difficulty of studying essential genes in these organisms. We have used a recently developed regulatable promoter to create a conditional knockout of T. gondii apical membrane antigen-1 (TgAMA1). TgAMA1 is a transmembrane protein that localizes to the parasite's micronemes, secretory organelles that discharge during invasion. AMA1 proteins are conserved among apicomplexan parasites and are of intense interest as malaria vaccine candidates. We show here that T. gondii tachyzoites depleted of TgAMA1 are severely compromised in their ability to invade host cells, providing direct genetic evidence that AMA1 functions during invasion. The TgAMA1 deficiency has no effect on microneme secretion or initial attachment of the parasite to the host cell, but it does inhibit secretion of the rhoptries, organelles whose discharge is coupled to active host cell penetration. The data suggest a model in which attachment of the parasite to the host cell occurs in two distinct stages, the second of which requires TgAMA1 and is involved in regulating rhoptry secretion. INTRODUCTIONToxoplasma gondii is one of the one most common protozoan parasites of humans, infecting approximately one-third of the world's population. The disease caused by an acute T. gondii infection, toxoplasmosis, can be life threatening in the congenitally infected fetus and immunocompromised hosts. Like all members of the Phylum Apicomplexa, including Plasmodium spp. (the causative agents of malaria) and Cryptosporidium parvum (a significant worldwide cause of waterborne illness), T. gondii is an obligate intracellular parasite. Host cell invasion is a critical step in the pathogenesis of the diseases caused by apicomplexan parasites, including toxoplasmosis. T. gondii represents an excellent model system for studying the relatively conserved process of apicomplexan invasion, because of the ease with which this parasite can be cultured and genetically manipulated (Roos et al., 1994;Black and Boothroyd, 2000;Kim and Weiss, 2004).Host cell invasion by apicomplexan parasites is a complex, multistep process (reviewed in Black and Boothroyd, 2000;Chitnis and Blackman, 2000). In T. gondii, the asexual stage tachyzoites move over solid surfaces, including cells, by an unusual form of substrate-dependent gliding motility (Sibley et al., 1998;Hakansson et al., 1999). After the apical end of a parasite comes in contact with the host cell membrane, apical secretory organelles (micronemes and rhoptries) sequentially discharge their contents (Dubremetz et al., 1993;Carruthers and Sibley, 1997) and a zone of tight interaction forms between the two cells (Nichols and O'Connor, 1981;Dubremetz et al., 1985;Grimwood and Smith, 1995). An invagination in the host cell plasma membrane develops at the point ...

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“…However, the specific epitope recognized appears to be critical since Ab fragments with different specificities have been shown to reduce parasitemia. 35,36 The unique Abs described here will permit delineation of human immune effector mechanisms in vivo, in experiments using mice transgenic for human FcR and complement genes. Such in vivo experiments are not possible with human malaria parasites.…”

Section: Discussionmentioning

confidence: 99%

Novel antimalarial antibodies highlight the importance of the antibody Fc region in mediating protection

Pleass

Ogun

McGuinness

et al. 2003

Blood

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Parasite drug resistance and difficulties in developing effective vaccines have precipitated the search for alternative therapies for malaria. The success of passive immunization suggests that immunoglobulin (Ig)-based therapies are effective. To further explore the mechanism(s) by which antibody mediates its protective effect, we generated human chimeric IgG1 IntroductionMalaria continues to kill approximately 2 million people each year. 1 The success of passive immunization in humans and animals suggests that immunoglobulin (Ig)-based therapies could be effective. 2,3 Manipulating antibody (Ab) genes allows the design of Ig with defined class and specificity, targeting protective epitopes on the parasite surface. An appropriate target is the 19-kDa C-terminal region of merozoite surface protein 1 (MSP1 19 ). This polypeptide displays limited sequence polymorphism, 4 is expressed by all vertebrate life-cycle stages, 5 and acts as a major target of the erythrocyte invasion-inhibitory Ab response in individuals immune to Plasmodium falciparum malaria. 6 The mechanisms whereby Ig mediates protective immunity in malaria remain unclear. 7 A primary role of Ab appears to involve blocking of merozoite invasion of erythrocytes. 8 However, studies using human Ab suggest that the protective effects are mediated through interaction with specific receptors for the Ig Fc region (FcR) expressed on various effector cells. 9,10 Using Plasmodium yoelii in a murine model to address these possibilities, we generated human chimeric IgG1 and IgA1 Abs recognizing an epitope on P yoelii MSP1 19 . We have investigated the potential of these intact Abs, and a novel diabody with specificity for both MSP1 19 and a human receptor for IgG Fc, Fc␥RI, as novel malaria therapeutics. Materials and methods Construction of expression vectorsVariable heavy (VH) and light (VL) complementary DNAs (cDNAs) were cloned from mouse hybridomas B10 (anti-MSP1 19 ) and M22 (anti-human Fc␥RI) by reverse transcriptase-polymerase chain reaction (RT-PCR) using degenerate primers (5Ј-SARGTNMARCTGCAGSAGTC-3Ј and 5Ј-TGAGGAGACGGTGACCGTGGTYCCTTGGCCCC-3Ј for VH and 5Ј-GAYATTGAGCTCACMCARWCTMCA-3Ј and 5Ј-CCGTTTBAKCTC-GAGCTTKGTSCC-3Ј for VL). 11,12 An alternative primer incorporating a BssHII restriction enzyme site was used to reamplify B10 VH to facilitate subcloning. The IgA1 heavy-chain constant region was subcloned as a BamHI-SalI fragment from an ␣ chain gene-containing plasmid 13 into pAD-Gal4-2.1 (Stratagene, La Jolla, CA) and then as a BamHI-XbaI fragment into pVHExpress (a kind gift from Andrew Bradbury, Los Alamos National Laboratory, Los Alamos, NM), 14 replacing the ␥1 constant domains to yield pVHExpress ␣1H. The VH genes were inserted into pVHExpress and pVHExpress ␣1H, upstream of the ␥1 or ␣1 constant regions, respectively, and the VL genes into pVKExpress upstream of the human gene. 14 To generate the Fc␥RI x MSP1 19 diabody, an overlapping PCR-based strategy was used. First, the VH and VL regions of B10 and M22 were amplified. The 5Ј VH primers inco...

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The Fab fragments of monoclonal IgG to a merozoite surface antigen inhibit Plasmodium knowlesi invasion of erythrocytes

Cited by 135 publications

References 18 publications

Fine Mapping of an Epitope Recognized by an Invasion-inhibitory Monoclonal Antibody on the Malaria Vaccine Candidate Apical Membrane Antigen 1

Fine Mapping of an Epitope Recognized by an Invasion-inhibitory Monoclonal Antibody on the Malaria Vaccine Candidate Apical Membrane Antigen 1

Conditional Expression ofToxoplasma gondiiApical Membrane Antigen-1 (TgAMA1) Demonstrates That TgAMA1 Plays a Critical Role in Host Cell Invasion

Novel antimalarial antibodies highlight the importance of the antibody Fc region in mediating protection

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