Recombinant Human Antibodies Specific for the Pfs48/45 Protein of the Malaria Parasite Plasmodium falciparum

Roeffen, Will; Raats, Jos; Teelen, Karina; Hoet, Rene; Eling, W.M.C.; Venrooij, Walther J. van; Sauerwein, Robert W.

doi:10.1074/jbc.m100562200

Cited by 30 publications

(37 citation statements)

References 16 publications

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“…Seven days after membrane feed, the number of infected mosquitoes and oocyst density were determined. Sera, mouse serum from M-Pfs10C-immunized group; Control, mean of five feeders with serum from control immunization; AM, arrhythmic mean oocyst number in 20 dissected mosquitoes; TN, total number of oocysts in 20 dissected mosquitoes; TBA, percentage of TB activity (19). cated (10C) residues 159 -428 were cloned into pMAL-p2x (New England Biolabs), resulting in N-terminal MBP fusions.…”

Section: Methodsmentioning

confidence: 99%

“…To detect epitopes of Pfs48/45, various mAbs of mouse origin, 32F5 (epitope I) and 32F1 (epitope IIb) (6,20), or rat origin, 85RF45.1 (epitope I), 85RF45.2b (epitope IIb), 85RF45.3 (epitope III), and 85RF45.5 (epitope V) (7,19), were used. HRP-conjugated anti-mouse IgG was purchased from Dako (P0161).…”

Section: Methodsmentioning

confidence: 99%

“…Mature P. falciparum gametocytes (NF54 strain) were produced in an automated static culture system as described (7,18), isolated (19), and stored at Ϫ70°C until used. NF54 gametocytes were extracted in 25 mM Tris⅐HCl (pH 8.0) supplemented with 150 mM NaCl, 1.0% sodium desoxycholate, and 1 mM phenylmethylsulphonyl fluoride.…”

Section: Methodsmentioning

confidence: 99%

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Correctly folded Pfs48/45 protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in mice

Outchkourov¹,

Roeffen

Kaan³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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136

117

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Malaria kills >1 million people each year, in particular in subSaharan Africa. Although asexual forms are directly responsible for disease and death, sexual stages account for the transmission of Plasmodium parasites from human to the mosquito vector and therefore the spread of the parasite in the population. Development of a malaria vaccine is urgently needed to reduce morbidity and mortality. Vaccines against sexual stages of Plasmodium falciparum are meant to decrease the force of transmission and consequently reduce malaria burden. Pfs48/45 is specifically expressed in sexual stages and is a well established transmissionblocking (TB) vaccine candidate. However, production of correctly folded recombinant Pfs48/45 protein with display of its TB epitopes has been a major challenge. Here, we show the production of a properly folded Pfs48/45 C-terminal fragment by simultaneous coexpression with four periplasmic folding catalysts in Escherichia coli. This C-terminal fragment fused to maltose binding protein was produced at medium scale with >90% purity and a stability over at least a 9-month period. It induces uniform and high antibody titers in mice and elicits functional TB antibodies in standard membrane feeding assays in 90% of the immunized mice. Our data provide a clear perspective on the clinical development of a Pfs48/45-based TB malaria vaccine. M alaria parasites are spread in the population by Plasmodium-infected Anopheles mosquitoes. Successful transmission of malarial parasites from humans to mosquitoes depends on the presence and infectiousness of gametocytes in the peripheral blood and the number of Anopheles mosquitoes in the area. Transmission of Plasmodium falciparum can be blocked inside the mosquito by antibodies that have been ingested together with the gametocytes as part of a blood meal, interrupting the sporogonic cycle inside the mosquito (1).Pfs48/45 is a transmission-blocking (TB) target protein expressed by gametocytes (2-4) and present on the surface of the sporogonic (macrogametes) stages of the malaria parasites. Pfs48/45 plays a key role in parasite fertilization (5) and antibodies that exclusively target conformational epitopes of Pfs48/45 protein prevent fertilization (6, 7). Furthermore, antiPfs48/45 antibodies are present in human sera from endemic areas (8) and correlate with TB activity (8-10). The induction of antibodies after natural infection as observed in the field creates the highly beneficial potential of vaccine boosting in the endemic setting. TB vaccines might be applied alone or more likely as part of a combination vaccine or package of control measures depending on the intensity of malaria transmission (11).A strategy for vaccine development requires the production of correctly folded recombinant Pfs48/45 protein. Proper folding of many cysteine-rich proteins, including Pfs48/45, depends on correct formation of disulphide bridges. In eukaryotes the oxidizing environment of the endoplasmic reticulum (ER) provides a milieu for disulphide bonds formation. Plasmodium p...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Correctly folded Pfs48/45 protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in mice

Outchkourov¹,

Roeffen

Kaan³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

136

117

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“…We constructed a phage display library from the peripheral blood of human patients, as reported previously. Roeffen et al (27) showed their biological activity and invasion inhibition using competitive ELISA with neutralizing antibodies. Lundquist et al (18) showed the neutralizing activity with an antibody-dependent cellular assay.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, we decided to make neutralizing antibody fragments against PvRII from an scFv library of naturally infected malaria patients. Some human recombinant antibodies against Plasmodium have been reported using the MSP protein of Plasmodium falciparum or the AMA protein of Plasmodium chabaudi as targets (13,18,27,30). We constructed a phage display library from the peripheral blood of human patients, as reported previously.…”

Section: Discussionmentioning

confidence: 99%

Single-Chain Antibody Fragment Specific forPlasmodium vivaxDuffy Binding Protein

Kim

Hwang

Lee

et al. 2007

Clin Vaccine Immunol

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Phage display of single-chain variable fragment (scFv) antibodies is a powerful tool for selecting important, useful, and specific human antibodies. We constructed a library from three patients infected with Plasmodium vivax. Panning on recombinant PvRII enriched a population of scFvs that recognized region II of the P. vivax Duffy binding protein (DBP). Three clones of scFvs that reacted with PvRII were selected, and their biological functions were analyzed. These scFvs inhibited erythrocyte binding to DBP. Clone SFDBII92 had the greatest affinity (dissociation constant ‫؍‬ 3.62 ؋ 10 ؊8 M) and the greatest inhibition activity (50% inhibitory concentration Ϸ 2.9 g/ml) to DBP. Thus, we demonstrated that human neutralizing antibody could be made from malaria patients using phage display and that these neutralizing scFvs should prove valuable for developing both passive and active immunization strategies based on DBP.Malaria caused by Plasmodium vivax is responsible for substantial morbidity in Asia and Central and South America (19). Merozoites of Plasmodium must attach to and invade red blood cells (RBCs) to begin asexual reproduction of the parasite, making this brief event a critical phase in the parasite life cycle. Invasion occurs quickly through a complex, multistep process that follows a distinct sequence of events involving numerous molecules expressed on the surface of the merozoite and in the apical organelles (1,4,6,7). This cascade of events represents potential targets for reducing or eliminating the blood stages of malarial parasites (21,25,31).The Duffy binding protein (DBP) of P. vivax interacts with Duffy antigen receptor for chemokines (DARC) on the RBC during junction formation between the merozoite and RBC (1, 2, 16, 34). The P. vivax DBP (PvDBP) is a 140-kDa protein that belongs to a family of erythrocyte-binding proteins characterized by a functionally conserved cysteine-rich region (1,6,12). This cysteine-rich region is in DBP region II (DBP II), which contains the binding motifs necessary for adhering to DARC on the erythrocyte surface (9, 10, 29). The critical binding motif has been mapped to a 170-amino-acid segment between cysteines 4 and 8 in the cysteine-rich region (26,28,29). Studies have shown that although the cysteine residues are conserved, other regions of DBP II are highly polymorphic (3, 32, 36). However, the hypervariable region of DBP II is located on the sites remote from the DARC-binding site and does not alter the capacity of the protein to bind DARC-positive erythrocytes (28, 33).Phage display antibodies offer a way to produce high-affinity single-chain variable fragment (scFv) derivatives of human antibodies of "natural host" origin (8). Our goal was to produce human monoclonal antibodies against the DARC-binding region of DBP II of P. vivax (PvRII). To do so, we constructed a combinatorial phage display library using peripheral blood mononuclear cells from three patients infected naturally with P. vivax. Subsequently, anti-PvRII human scFvs that had neutralizing activi...

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The presence of citrullinated proteins is not specific for rheumatoid synovial tissue

Vossenaar¹,

Smeets²,

Kraan³

et al. 2004

Arthritis & Rheumatism

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143

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Objective. Antibodies directed toward citrullinated proteins (e.g., anti-cyclic citrullinated peptide antibodies) are highly specific for rheumatoid arthritis (RA) and are produced locally at the site of inflammation. Although the presence of citrullinated proteins in rheumatoid synovium has been described in the literature, it is uncertain whether their presence is specific for RA. The present study was undertaken to investigate this.Methods. The local production of the anticitrullinated protein antibodies was investigated by comparing the concentration of the antibodies (corrected for the total amount of IgG present) in paired samples of serum and synovial fluid from RA patients. The presence of citrullinated proteins in the synovial tissue was investigated by immunohistochemical analysis of synovial tissue from RA patients and from patients with other arthropathies, using a variety of specific antibodies to citrullinated proteins.Results. In RA patients, anti-citrullinated protein antibodies constituted a 1.4-fold higher proportion of IgG in synovial fluid compared with serum, which is indicative of a local production of the antibodies. Immunohistochemical staining of citrullinated proteins was observed in the lining layer, the sublining layer, and in extravascular fibrin deposits in inflamed synovial tissue from RA as well as non-RA patients.Conclusion. The presence of citrullinated proteins in the inflamed synovium is not specific for RA, but rather, it may be an inflammation-associated phenomenon. The high specificity of the anti-citrullinated protein antibodies is, therefore, most likely the result of an abnormal humoral response to these proteins.

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Recombinant Human Antibodies Specific for the Pfs48/45 Protein of the Malaria Parasite Plasmodium falciparum

Cited by 30 publications

References 16 publications

Correctly folded Pfs48/45 protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in mice

Correctly folded Pfs48/45 protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in mice

Single-Chain Antibody Fragment Specific forPlasmodium vivaxDuffy Binding Protein

The presence of citrullinated proteins is not specific for rheumatoid synovial tissue

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