Molecular and immunological characterisation of recombinant Brucella abortus glyceraldehyde-3-phosphate-dehydrogenase, a T- and B-cell reactive protein that induces partial protection when co-administered with an interleukin-12-expressing plasmid in a DNA vaccine formulation

Abstract

To identify antigens of Brucella spp. that are potentially involved in stimulating a protective T-cell-mediated immune response, previous studies identified 10 clones from a Brucella abortus 2308 genomic library with primed lymphocytes as probes. One selected positive clone (182) contained an insert of 1.2 kb which was identified, sequenced and characterised. The deduced amino acid sequence of the open reading frame (ORF) revealed 82% and 81% identity to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymes from Agrobacterium tumefaciens and Xanthobacter flavus, respectively. Southern blot analysis demonstrated that the gap gene is present in only one copy in the Brucella genome. B. abortus GAPDH was then expressed in Escherichia coli as a fusion protein with the maltose-binding protein (MBP). To demonstrate the functional activity of Brucella GAPDH, E. coli gap mutants were transformed with a Brucella pMAL-gap construct. Genetic complementation was achieved and as a result E. coli mutants were able to grow on glucose or other carbon source medium. The humoral and cellular immune responses to the recombinant (r) GAPDH were characterised. In Western blots, sera from naturally infected cattle and sheep showed antibody reactivity against rGAPDH. In response to in-vitro stimulation by rGAPDH, splenocytes from mice vaccinated with rGAPDH or B. abortus S19 were able to produce ª-interferon and tumour necrosis factor-AE but not interleukin (IL)-4. Furthermore, gap associated with murine IL-12 gene in a DNA vaccine formulation partially protected mice against experimental infection.

TheBrucella abortusPhosphoglycerate Kinase Mutant Is Highly Attenuated and Induces Protection Superior to That of Vaccine Strain 19 in Immunocompromised and Immunocompetent Mice

“…The pgk gene was isolated through screening of the B. abortus S2308 genomic library, using the gap (glyceraldehyde-3-phosphate-dehydrogenase) gene fragment as a DNA probe (36). In this screening process a clone of approximately 22 kb was obtained and partially sequenced using primer walking to obtain the open reading frame (ORF) of the B. abortus pgk gene.…”

“…Previous research in our laboratory has identified Brucella genes coding for metabolic enzymes (27,36). Among them is pgk, which encodes the phosphoglycerate kinase (PGK).…”

Protective Immunity Elicited by a Divalent DNA Vaccine Encoding Both the L7/L12 and Omp16 Genes ofBrucella abortusin BALB/c Mice

“…As B. abortus is an intracellular pathogen, DNA vaccination should be a good countermeasure to protect the host from its infection. Actually, extensive research on a B. abortus DNA vaccine has been performed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (32), bacterioferritin (1), P39 (1), GroEL heat shock gene (17), ribosome recycling factor-homologous protein (CP24) (4), superoxide dismutase (22,27), and others. These vehicles illustrate that DNA vaccination should provide a good countermeasure to protect the host from B. abortus infection.…”

Inhibition of Glyceraldehyde-3-Phosphate Dehydrogenase Activity by Antibodies Present in the Cerebrospinal Fluid of Patients with Multiple Sclerosis

“…This observation has led to speculation that the autoimmune response to GAPDH might be primarily initiated by foreign GAPDH, present on the surface of bacteria, viruses, and parasites, and subsequent cross-reaction with human GAPDH (55)(56)(57). People with an increased predisposition to autoimmune diseases might have a higher risk of developing MS (1,58).…”