Candida Infection Biology – fungal armoury, battlefields ... - FINSysB

scFv Phage Display Library against Complex
Immunomodulatory Glycans
Abhishek Saxena 1 , Soumya Palliyil 1,2 , Keith A. Charlton 3 , Alistair J.P.
Brown 1 & Andrew J.R. Porter 1,2,3
1 Department of Molecular and Cell Biology, Institute of Medical Sciences, Aberdeen Medical
School, University of Aberdeen, Aberdeen AB25 2ZD, UK; 2 Scottish Biologics Facility,
University of Aberdeen, Aberdeen AB25 2ZD, UK; 3 ImmunoSolv Ltd, Liberty Research centre,
University of Aberdeen, Aberdeen AB25 2ZP, UK
The concept of Antibody therapy against infectious diseases is still in its infancy.
Numerous studies in last two decades have focused on anti-glycan antibodies that
recognize fungal cell surface. The presence of these antibodies in blood has been
used to diagnose fungal infection. Candida albicans cell wall -glucan and -mannan
are very well established diagnostic and therapeutic targets because they represent
the most abundant cell surface epitopes responsible for generation of unique
antibodies against fungus. We have constructed a scFv M13 phage display library
against complex glycans of Candida albicans. To generate an antibody response
and construct a library, a Welch breed/Suffolk sheep was hyper-immunized (IgG
titres > 60,000) against a Candida albicans hyphal cell wall preparation. Binding
analysis of crude/purified polyclonal serum IgG indicates that cell wall glycans are
indeed primary targets because they dominate the immune response of the sheep.
We further demonstrated that polyclonal IgG has in vitro hyphae neutralization
potential in growth inhibition assay. A scFv library has been constructed by cloning
the immunoglobulin variable gene repertoire of peripheral blood lymphocytes
isolated from this hyper-immune animal. Variable heavy and variable light gene
segments of immunoglobulins were PCR amplified and linked through a cellulase
linker. Linked scFv segments were cloned into the phagemid vector pHEN2a. The
size of the VH-V and VH-V library in E.coli TG1 cells was 3.8 x 10 10 and 1.1 x 10 8
respectively. Repertoire diversity was confirmed by sequencing random clones
(approximately 45 clones) and CDRs of VH and V /V genes were found to be 100%
different. We have a panel of early monoclonal hits against both CA -glucan and
-mannan antigens. We are currently interested in pulling monoclonal phages from
library against variety of immunomodulatory glycans.
We are grateful to the European Commission for funding the FINSysB Marie Curie Initial Training Network
(PITN-GA-2008-214004).
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