Article
Efficient antibody rescue from antigen-specific human B lymphocytes following Epstein-Barr virus-transformation
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Published: | January 29, 2014 |
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Malaria tropica, caused by Plasmodium falciparum still poses a major challenge with regard to global health care as well as to the economy in endemic countries. There is no efficient vaccine at hand, and multiple drug-resistant parasite strains are emerging. As yet, passive vaccination strategies with recombinant full-size human monoclonal antibodies are not pursued despite the fact that it has been shown early that these may confer immediate immunity toward as well as effectively cure malaria infections by Plasmodium falciparum.
Several technologies exist that allow the isolation and production of human full-size antibodies or antibody fragments, such as the techniques of Fab- or scFv-phage display, the use of transgenic mice possessing human immunoglobulin loci, as well as the transformation of B cells by Epstein-Barr Virus (EBV). Here, we would like to present a methodology that efficiently joins EBV transformation of peripheral B lymphocytes, V gene rescue, and recombinant antibody production in plants.
In an attempt to harness the protective immunoglobulin repertoire of Ghanaian donors IgG+/CD22+ B lymphocytes from peripheral blood were selected by means of fluorescently labeled antigens and flow cytometry. Antigen-specific B cells were subsequently infected with Epstein-Barr virus. After 3–4 weeks ELISA and spectratyping served to assess secretion of desired IgG by and the clonality of the obtained lymphoblastoid cultures. Sequences of variable regions of promising candidates were amplified, cloned into plant-expression vectors, and expressed in the context of human full-size IgG1κ1 in N. benthamiana. Integrity, binding characteristics, and functionality were verified by Western blots, ELISA, surface plasmon resonance (SPR) measurements, confocal immunofluorescence microscopy of parasites, and growth inhibition assays (GIA) in vitro.
Several human monoclonal antibodies directed against Plasmodium falciparum were successfully isolated which possess different affinities toward their epitopes and different characteristics of growth inhibition.
The recombinant production of these antibodies in plants allows the large scale production at moderate cost, which may build the base for affordable therapeutic anti-malarial antibodies.