Cloning, expression and antigenic analysis of an iron regulated surface expose protein of B. pertussis

J. ALVAREZ HAYES, E. ERBEN, Y. LAMBERTI, J. GORGOJO, H. VALDEZ, G. PRINCIPI, F. MASCHI, M. AYALA, CECILIA CARBONE , AND M. E. RODRIGUEZ

Baltimore, MD, USA

Simposio; Ninth International Bordetella Symposium; 2010

Successful microbial pathogens have developed mechanisms to overcome host iron restriction, including production and utilization of iron chelators, and direct removal of iron from host proteins via specific bacterial cell surface receptors. Because these are key factors for bacterial survival, they gain importance as potential targets for the development of vaccines and therapeutic agents. By mean of comparative proteomic and immune proteomics we identified antigens that although present in the infective phenotype are absent in current vaccines. Among them, IRP1-3, a putative outer membrane-associated protein, showed a particularly strong reaction with human IgG purified from pooled sera of pertussis-infected individuals. IRP1-3 was detected in outer membrane subproteome highly over-expressed under iron restriction. Antibodies against this protein might be protective in different ways. Computer analysis showed IRP1-3 as a dimeric outer membrane protein potentially involved in iron uptake with significant homology with lactoferrin receptors of other Gram-negative bacteria. Experimental data confirmed the surface-exposure of this protein and showed a 50-fold increased under iron starvation irrespective of the bacterial virulence state. Nucleotide sequence analysis of the IRP1-3 upstream region confirmed the absence of a putative promoter, a BvgA binding site. IRP1-3 was then cloned and expressed in E. coli for further studies. Whole cell ELISA studies showed that antibodies raised against rIRP1-3 recognized the native protein on bacterial surface. These antibodies proved effective in the promoting bacterial phagocytosis by human PMN, a key protecting activity against this pathogen. Accordingly, not only active but also passive immunization proved protective against infection even in monovalent vaccine formulation. Splenocyte re-stimulations assays demonstrated that mice immunization with rIRP1-3 elicited a Th1/Th2 mixed response.These results together with immunoblots analysis that showed that IRP1-3 is conserved in clinical isolates of B. pertussis suggest that this protein might be an interesting novel vaccine candidate.