Chaperonin GroEL a Brucella immunodominant antigen identified using Nanobody and MALDI-TOF-MS technologies.

The deployment of today's antibodies that are able to distinguish Brucella from the closely similar pathogens, such as Yersinia, is still considered a great challenge since both pathogens share identical LPS (lipopolysaccharide) O-ring epitopes. In addition, because of the great impact of Brucella on health and economy in many countries including Syria, much effort is going to the development of next generation vaccines, mainly on the identification of new immunogenic proteins of this pathogen. In this context, Brucella-specific nanobodies (Nbs), camel genetic engineered heavy-chain antibody fragments, could be of great value. Previously, a large Nb library was constructed from a camel immunized with heat-killed Brucella. Phage display panning of this 'immune' library with Brucella total lysate resulted in a remarkable fast enrichment for a Nb referred to as NbBruc02. In the present work, we investigated the main characteristics of this Nb that can efficiently distinguish under well-defined conditions the Brucella from other bacteria including Yersinia. NbBruc02 showed a strong and specific interaction with its antigen within the crude lysate as tested by a surface plasmon resonance (SPR) biosensor and it was also able to pull down its cognate antigen from such lysate by immuno-capturing. Using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), NbBruc02 specific antigen was identified as chaperonin GroEL, also known as heat shock protein of 60 kDa (HSP-60), which represents a Brucella immunodominant antigen responsible of maintaining proteins folding during stress conditions. Interestingly, the antigen recognition by NbBruc02 was found to be affected by the state of GroEL folding. Thus, the Nb technology applied in the field of infectious diseases, e.g. brucellosis, yields two outcomes: (1) it generates specific binders that can be used for diagnosis, and perhaps treatment, and (2) it identifies the immunogenic candidate antigens for developing vaccines.

Evaluation of a nanobody phage display library constructed from a Brucella-immunised camel.

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 serious national 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 work and 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 10(8) individual transformants, of which 81% contained an insert with 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 be immunodominant Ags in Brucella, is expected to open wide perspectives for following the vaccination, diagnosis and treatment of brucellosis.

ICBP90 belongs to a new family of proteins with an expression that is deregulated in cancer cells.

ICBP90 (Inverted CCAAT box Binding Protein of 90 kDa) is a recently identified nuclear protein that binds to one of the inverted CCAAT boxes of the topoisomerase IIalpha (TopoIIalpha) gene promoter. Here, we show that ICBP90 shares structural homology with several other proteins, including Np95, the human and mouse NIRF, suggesting the emergence of a new family of nuclear proteins. Towards elucidating the functions of this family, we analysed the expression of ICBP90 in various cancer or noncancer cell lines and in normal or breast carcinoma tissues. We found that cancer cell lines express higher levels of ICBP90 and TopoIIalpha than noncancer cell lines. By using cell-cycle phase-blocking drugs, we show that in primary cultured human lung fibroblasts, ICBP90 expression peaks at late G1 and during G2/M phases. In contrast, cancer cell lines such as HeLa, Jurkat and A549 show constant ICBP90 expression throughout the entire cell cycle. The effect of overexpression of E2F-1 is more efficient on ICBP90 and TopoIIalpha expression in noncancer cells (IMR90, WI38) than in cancer cells (U2OS, SaOs). Together, these results show that ICBP90 expression is altered in cancer cell lines and is upregulated by E2F-1 overexpression with an efficiency depending on the cancer status of the cell line.