Leif Kofoed Nielsen

A Novel Antibody-Dependent Cellular Cytotoxicity Mechanism Involved in Defense against Malaria Requires Costimulation of Monocytes FcγRII and FcγRIII

Clinical experiments have shown that the Ab-dependent cell-mediated inhibition of Plasmodium falciparum is a major mechanism controlling malaria parasitemia and thereby symptoms. In this study, we demonstrate that a single merozoite per monocyte (MN) is sufficient to trigger optimal antiparasitic activity. Using particulate Ag as pseudomerozoites, we show that only Ags, and no other parasite-derived factor, are required to trigger MN activation and that a single Ag is as potent as the complex combination of Ags constituting the merozoite surface. Moreover, we found that soluble Ags binding at least two Abs are as effective as the parasite at stimulating MN and that nonmalarial Ags are as efficient provided they are targeted by cytophilic Abs. Indeed, only cytophilic IgGs are potent and, in agreement with immunoepidemiological findings, IgG3 is superior to IgG1. Very low Ab concentrations (>700 pM), i.e., in the range of molecules having a hormonal effect, are effective, in contrast to Abs having a direct, neutralizing effect. Finally, Ab-dependent cell-mediated inhibition proved to require the synergistic activation of both FcγRIIa and FcγRIIIa which both distinguish it from other Ab-dependent cellular cytotoxicity and implies that all MN are not equally effective. These findings have both fundamental and practical implications, particularly for vaccine discovery.

Quality assessment of a placental perfusion protocol

Validation of in vitro test systems using the modular approach with steps addressing reliability and relevance is an important aim when developing in vitro tests in e.g. reproductive toxicology. The ex vivo human placental perfusion system may be used for such validation, here presenting the placental perfusion model in Copenhagen including control substances. The positive control substance antipyrine shows no difference in transport regardless of perfusion media used or of terms of delivery (n=59, p<0.05). Negative control studies with FITC marked dextran correspond with leakage criteria (<3 ml h(-1) from the fetal reservoir) when adding 2 (n=7) and 20mg (n=9) FITC-dextran/100 ml fetal perfusion media. Success rate of the Copenhagen placental perfusions is provided in this study, including considerations and quality control parameters. Three checkpoints suggested to determine success rate revealed that 15% of the cannulated placentae received in one year (n=202) were successfully perfused.

Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D☆

A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection of specific Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively.

Phage-display libraries of murine and human antibody Fab fragments.

We provide efficient and detailed procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab fragments displayed on the surface of filamentous phage. In addition, protocols for producing and using ultra-electrocompetent cells, for producing Fab phages from libraries, and for selecting antigen binders by panning are presented. The latter protocol includes a procedure for trypsin elution of bound phage.

Human Recombinant Antibodies against Plasmodium falciparum Merozoite Surface Protein 3 Cloned from Peripheral Blood Leukocytes of Individuals with Immunity to Malaria Demonstrate Antiparasitic Properties

ABSTRACT Immunoglobulins from individuals with immunity to malaria have a strong antiparasitic effect when transferred to Plasmodium falciparum malaria infected patients. One prominent target of antiparasitic antibodies is the merozoite surface antigen 3 (MSP-3). We have investigated the antibody response against MSP-3 residues 194 to 257 (MSP-3194-257) on the molecular level. mRNA from peripheral blood leukocytes from clinically immune individuals was used as a source of Fab (fragment antibody) genes. A Fab-phage display library was made, and three distinct antibodies designated RAM1, RAM2, and RAM3 were isolated by panning. Immunoglobulin G1 (IgG1) and IgG3 full-length antibodies have been produced in CHO cells. Reactivity with the native parasite protein was demonstrated by immunofluorescence microscopy, flow cytometry, and immunoblotting. Furthermore, the antiparasitic effect of RAM1 has been tested in vitro in an antibody-dependent cellular inhibition (ADCI) assay. Both the IgG1 and the IgG3 versions of the antibody show an inhibitory effect on parasite growth.

Detecting fetomaternal hemorrhage by flow cytometry.

Purpose of reviewThe aim of this review is to summarize the most recent developments in the area of detection of fetomaternal hemorrhage by flow cytometry. Recent findingsMaternal red blood cell chimerism is readily detectable by flow cytometry. Fetal and maternal red blood cells differ in their content of fetal hemoglobin (α2γ2). Fetal red blood cells contain fetal hemoglobin, and normal maternal red blood cells contain some percentage of fetal hemoglobin in a background of normal adult hemoglobin. All blood group systems with allelic differences between mother and fetus are readily applicable for detection of fetomaternal hemorrhage by fetal hemoglobin. SummaryFetal hemoglobin for detection of fetomaternal hemorrhage is an accurate clinical diagnostic procedure for investigation of anemia in fetus and newborn.

Next-generation sequencing: proof of concept for antenatal prediction of the fetal Kell blood group phenotype from cell-free fetal DNA in maternal plasma.

Maternal immunization against KEL1 of the Kell blood group system can have serious adverse consequences for the fetus as well as the newborn baby. Therefore, it is important to determine the phenotype of the fetus to predict whether it is at risk. We present data that show the feasibility of predicting the fetal KEL1 phenotype using next‐generation sequencing (NGS) technology.

Human Anti-Rhesus D IgG1 Antibody Produced in Transgenic Plants

Transgenic plants represent an alternative to cell culture systems for producing cheap and safe antibodies for diagnostic and therapeutic use. To evaluate the functional properties of a ‘plantibody’, we generated transgenic Arabidopsis plants expressing full-length human IgG1 against the Rhesus D antigen, which is responsible for alloimmunization of RhD– mothers carrying an RhD+ fetus. Anti-RhD extracted from plants specifically reacted with RhD+ cells in antiglobulin technique, and elicited a respiratory burst in human peripheral blood mononuclear cells. Plant-derived antibody had equivalent properties to CHO cell-produced anti-RhD antibody, indicating its potential usefulness in diagnostic and therapeutic programs.

Is current serologic RhD typing of blood donors sufficient for avoiding immunization of recipients

BACKGROUND: Avoiding immunization with clinically important antibodies is a primary objective in transfusion medicine. Therefore, it is central to identify the extent of D antigens that escape routine RhD typing of blood donors and to improve methodology if necessary.

Maternofetal transplacental transport of recombinant IgG antibodies lacking effector functions

The neonatal Fc receptor (FcRn) directs the transfer of maternal immunoglobulin G (IgG) antibodies across the placenta and thus provides the fetus and newborn with passive protective humoral immunity. Pathogenic maternal IgG antibodies will also be delivered via the placenta and can cause alloimmunity, which may be lethal. A novel strategy to control pathogenic antibodies would be administration of a nondestructive IgG antibody blocking antigen binding while retaining binding to FcRn. We report on 2 human IgG3 antibodies with a hinge deletion and a C131S point mutation (IgG3ΔHinge) that eliminate complement activation and binding to all classical Fcγ receptors (FcγRs) and to C1q while binding to FcRn is retained. Additionally, 1 of the antibodies has a single point mutation in the Fc (R435H) at the binding site for FcRn (IgG3ΔHinge:R435H). We compared transplacental transport with wild-type IgG1 and IgG3, and found transport across trophoblast-derived BeWo cells and ex vivo placenta perfusions with hierarchies as follows: IgG3ΔHinge:R435H>wild-type IgG1≥IgG3ΔHinge and IgG3ΔHinge:R435H=wild-type IgG1=wild-type IgG3>>>IgG3ΔHinge, respectively. Collectively, IgG3ΔHinge:R435H was transported efficiently from the maternal to the fetal placental compartment. Thus, IgG3ΔHinge:R435H may be a good candidate for transplacental delivery of a nondestructive antibody to the fetus to combat pathogenic antibodies.