Inmunologia

G Model VETIMM-8506;

No. of Pages 8

ARTICLE IN PRESS
Veterinary Immunology and Immunopathology xxx (2011) xxx–xxx

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Veterinary Immunology and Immunopathology
journal homepage: www.elsevier.com/locate/vetimm

Evaluation of a nanobody phage display library constructed from a Brucella-immunised camel
A.Q. Abbady a,∗ , A. Al-Mariri a , M.Zarkawi b , A. Al-Assad c , S. Muyldermans d,e
a b c d e

Department of Molecular Biology and Biotechnology, AECS, P.O. Box 6091, Damascus, Syria Department of Agriculture, AECS, P.O. Box 6091, Damascus, Syria General Commission for Scientific Agricultural Research (GCSAR), Deir El-Hagar Station for Camel Researches, Damascus, Syria Laboratorium voor Cellulaire en Moleculaire Immunologie, VrijeUniversiteit Brussel, Pleinlaan 2, B-1050 Brussel, Belgium Department of Cellular and Molecular Interactions, VIB, Brussels, Belgium

a r t i c l e

i n f o

a b s t r a c t
Brucella are invasive gram-negative bacteria that multiply and survive within eukaryotic cells causing brucellosis. Syrian (and Middle East) health and economy sectors are still affected by this disease causing a seriousnational problem that needs to be solved. Here, a strategy was developed to introduce a new generation of binders, known as Nanobodies (Nbs) in our combat against Brucella. These Nbs, recombinant single-domain variable fragments derived from camelid heavy-chain antibodies are very stable and highly soluble, making them a useful tool in numerous biotechnological and medical applications. In this workand without having access to purified antigens (Ags), a camel was immunised successfully with heat-killed Brucella melitensis strain Riv1 as demonstrated by the high titer of Ag-specific heavy-chain antibodies in the serum. Lymphocytes of the immunised camel were isolated and their Nb genes were cloned in a relatively large library of 108 individual transformants, of which 81% contained an insertwith the proper size of a Nb gene. Phage display expression of the Nbs from this library and pannings on the Brucella lysate resulted in a clear enrichment of three distinct Nb-displaying phages (phage-Nbs), referred to as NbBruc01, 02 and 03, with specificity for Brucella. Producing these binders in a pure, soluble form, as well as identifying their specific targets, which are likely to beimmunodominant Ags in Brucella, is expected to open wide perspectives for following the vaccination, diagnosis and treatment of brucellosis. © 2011 Published by Elsevier B.V.

Article history: Received 2 November 2010 Received in revised form 4 March 2011 Accepted 6 April 2011 Keywords: Camel Nanobody Brucella Brucellosis Phage display

1. Introduction Brucella is a small invasive, non-motile,gram-negative coccobacillus that multiplies and survives within a broad range of eukaryotic cells. Brucellosis, also known as undulant fever or Mediterranean fever, is caused by this genus of bacteria and is still one of the major, highly transmissible, zoonotic diseases affecting people and animals (Pappas et al., 2005). While it has been successfully eradicated from

∗ Corresponding author. Tel.: +963966292797. E-mail address: aabady@aec.org.sy (A.Q. Abbady). 0165-2427/$ – see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.vetimm.2011.04.004

most developed countries, the situation of this disease in particular countries, e.g. Syria, is rapidly worsening. In the affected areas, it still has a major impact on the health of both man and domestic animals, particularly cattle,sheep, goats and camels with concomitant economic consequences (Darwish and Benkirane, 2001; Pappas et al., 2006). While classical serological techniques are widely used for diagnosing human and animal brucellosis, these techniques depend mainly on the detection of antibodies (Abs) to lipopolysaccharides (LPS) that represent the immunodominant surface antigen (Ag) (Forestier et al., 1999). However,...