title: Recombinant Brucella abortus proteins that induce proliferation and gamma-interferon secretion by CD4+ T cells from Brucella-vaccinated mice and delayed-type hypersensitivity in sensitized guinea pigs.
abstract: Optimal protective immunity to Brucella abortus infection is dependent on a coordinate interaction between different T-cell subsets which leads to an antigen-specific T-lymphocyte-mediated activation of macrophages, the main cellular reservoir for the bacterium. As an initial step in the identification of bacterial proteins that mediate cellular immunity, we have subcloned the B. Abortus ssb, uvrA, GroES, and GroEL genes into the prokaryotic expression vector pMAL-c2 using PCR. Escherichia coli DH5 alpha was transformed with the pMAL-ssb, pMAL-uvrA, pMAL-GroES, and pMAL-GroEL constructs separately, and gene expression was induced by isopropyl-beta-D-thiogalactopyranoside. The resulting fusion proteins were purified by affinity chromatography and confirmed by Western blot analysis using an anti-maltose-binding protein antibody. Furthermore, we have examined the pattern of T helper (Th) cell response from vaccinated BALB/c mice after in vitro stimulation with the recombinant (r) fusion proteins. In addition to T-cell proliferative responses, CD4+ T cells were tested for interleukin-2 (IL-2), IL-4, and gamma interferon (IFN-gamma) secretion. Primed CD4+ T cells proliferated to the rUvrA, rGroES, and rGroEL, but not to rSsb. The cytokine profile of the proliferating cells was characteristic of a Th1 type, as we detected IL-2 and IFN-gamma but not IL-4 in the T-cell culture supernatants. The recombinant B. abortus proteins were also screened in vivo to their ability to elicit DTH reaction in Brucella-sensitized guinea pigs. Moreover, the results of this study suggest that B. abortus rUvrA, rGroES, and rGroEL might be important sources of potentially protective molecules.
authors.affiliation: Department of Animal Health, University of Wisconsin-Madison, 53706-1581, USA.
authors.forename: S C
authors.initials: SC
authors.lastname: Oliveira
authors.forename: J S
authors.initials: JS
authors.lastname: Harms
authors.forename: M
authors.initials: M
authors.lastname: Banai
authors.forename: G A
authors.initials: GA
authors.lastname: Splitter
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D002004:Brucella Vaccine / Q000276:immunology*; D002003:Brucella abortus / Q000235:genetics / Q000276:immunology*; D017347:Brucella melitensis / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006168:Guinea Pigs; D006968:Hypersensitivity, Delayed / Q000209:etiology*; D007371:Interferon-gamma / Q000096:biosynthesis / Q000378:metabolism*; D008213:Lymphocyte Activation / Q000187:drug effects*; D051379:Mice; D008807:Mice, Inbred BALB C
chemical_list: D001426:Bacterial Proteins; D002004:Brucella Vaccine; D007371:Interferon-gamma