Human Recombinant Antibodies againstPlasmodium falciparumMerozoite 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-3 194-257 ) 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.Clinical immunity to Plasmodium falciparum malaria is gradually acquired over a dozen years of intense exposure to the parasite (12). Acquired immunity to malaria has been termed premunition and is characterized as being nonsterile and incomplete (43). The exact mechanism responsible for premunition is not known with certainty. However, a number of clinical studies carried out in the early sixties (9, 16, 23)-and subsequently confirmed and extended in the nineties (2, 33)-showed an unambiguous antiparasitic effect of antibodies transferred from adults with immunity to malaria to malariainfected infants. Clinical effects observed in one of these studies correlated with the effect measured in the in vitro assay termed antibody-dependent cellular inhibition (ADCI) (2, 4). In the ADCI assay, immune antibody cooperates with monocytes in an in vitro malaria culture, and the antiparasitic effect is demonstrated by parasite growth inhibition. It has been shown that the antibody-merozoite complex by a contact-dependent mechanism stimulates the monocyte to secrete substances toxic to the asexual blood stages. The specific substances responsible for the subsequent, non-contact-dependent parasite growth inhibition include tumor necrosis factor alpha together with other molecules that are yet to be identified (4). The ADCI assay has been used for identification and characterization of the merozoite surface protein 3 (MSP-3) (27).An invariable structural feature of all reported MSP-3 sequences is the presence of three regions each of which contains three, four, or five conserved heptad repeat units. Previously published structural analyses suggest that the heptad repeat regions have an amphipathic alpha-helical secondary structure. A coiled-coil bundle conformation including these regions is a theoretical possibility supported by experimental data (24). The C-terminal part of MSP-3 contains a leucine zipper-like domain possibly implicated in dimerization and the formation...

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

“…The human recombinant anti-MSP-3 mAb RAM-1, specific for the MSP-3b peptide, was constructed with either IgG1 and IgG3 H chains as described previously (19). Recombinant anti-rhesus D (anti-RhD) mAb were constructed in IgG1, IgG2, IgG3, IgG4, IgA 1 , and IgA 2 versions (20,21).…”

Isolation and expression of UB05, a Plasmodium falciparum antigen recognised by antibodies from semi-immune adults in a high transmission endemic area of the Cameroonian rainforest

“…One such approach is differential immuno-screening using sera from semiimmune and susceptible subjects (9,10). Antibody transfer experiments suggest that immunoglobulin G (IgG) antibodies are critical effectors of protective immunity to malaria (11,12). Indeed, many of the malaria vaccines under investigation are designed to elicit protective antibodies (7,13).…”

Maternofetal transplacental transport of recombinant IgG antibodies lacking effector functions

“…23 The human IgG3DHinge antibodies have all 4 hinge region exons deleted, and lack a disulfide bridge between their light and heavy chains due to a cysteine to serine mutation at amino acid position 131 of the C H 1 domains (C131S). This allows the 2 light chains to form an interchain disulfide bridge through their C-terminal cysteines.…”

“…19 The anti-D antibodies, a-D (IgG1, IgG3DHinge, and IgG3DHinge:R435H), were termed IgG11GAN, HM51GAN, and HM5R435H1GAN, respectively, 19,22 and the anti-malaria antibodies, a-MSP-3 (IgG1 and IgG3), were termed IgG11RAM1 and IgG31RAM1. 23 All antibodies were produced and purified essentially as previously described. 19,22,23 Receptor-binding studies Microtiter plates were coated with equal amounts of antibodies (supplemental Figure 2, available on the Blood website) and incubated with glutathione Stransferase (GST)-fused recombinant soluble forms of human FcgRs (hFcgRI, hFcgRIIa, hFcgRIIb, hFcgRIIIa, and hFcgRIIIb).…”

“…23 All antibodies were produced and purified essentially as previously described. 19,22,23 Receptor-binding studies Microtiter plates were coated with equal amounts of antibodies (supplemental Figure 2, available on the Blood website) and incubated with glutathione Stransferase (GST)-fused recombinant soluble forms of human FcgRs (hFcgRI, hFcgRIIa, hFcgRIIb, hFcgRIIIa, and hFcgRIIIb). 24,25 All receptors were diluted in 13 phosphate-buffered saline (PBS)/Tween 20 pH 7.4 and added to the wells at a concentration of 1 mg/mL except for shFcgRI, which was added at a concentration of 0.25 mg/mL.…”