Initial host defense to bacterial infection is executed by innate immunity, and therefore the main goal of this study was to examine the contribution of Toll-like receptors (TLRs) during Brucella abortus infection. CHO reporter cell lines transfected with CD14 and TLRs showed that B. abortus triggers both TLR2 and TLR4. In contrast, lipopolysaccharide (LPS) and lipid A derived from Brucella rough (R) and smooth (S) strains activate CHO cells only through TLR4. Consistently, macrophages from C3H/HePas mice exposed to R and S strains and their LPS produced higher levels of tumor necrosis factor alpha (TNF-␣) and interleukin-12 compared to C3H/HeJ, a TLR4 mutant mouse. The essential role of TLR4 for induction of proinflammatory cytokines was confirmed with diphosphoryl lipid A from Rhodobacter sphaeroides. Furthermore, to determine the contribution of TLR2 and TLR4 in bacterial clearance, numbers of Brucella were monitored in the spleen of C3H/HeJ, C3H/HePas, TLR2 knockout, and wild-type mice at 1, 3, and 6 weeks following B. abortus infection. Interestingly, murine brucellosis was markedly exacerbated at weeks 3 and 6 after infection in animals that lacked functional TLR4 (C3H/HeJ) compared to C3H/HePas that paralleled the reduced gamma interferon production by this mouse strain. Finally, by mass spectrometry analysis we found dramatic differences on the lipid A profiles of R and S strains. In fact, S lipid A was shown to be more active to trigger TLR4 than R lipid A in CHO cells and more effective in inducing dendritic cell maturation. In conclusion, these results indicate that TLR4 plays a role in resistance to B. abortus infection and that S lipid A has potent adjuvant activity.

The immunogenicity and protective ef®cacy of a DNA vaccine encoding the GroEL heatshock gene from Brucella abortus was tested in BALB/c mice immunised by intramuscular (i.m.) needle injection or epidermally by gene gun. The Brucella GroEL gene was ampli®ed by PCR and cloned into two different mammalian expression vectors pCMV-link and pCMV-tPA. The D17 cell line was transfected with both constructs and GroEL transcripts were detected by Northern blot. To determine the level of protein synthesised, transfected cell lysates were then submitted to Western blot. The nonsecreted form of the recombinant GroEL produced by the pCMV-link construct was detected in much greater amount than the secreted form of the protein produced by the pCMV-tPA construct. After immunisation, a strong anti-GroEL IgG response was detected in mice vaccinated by i.m. injection or gene gun only when the pCMV-link/ GroEL plasmid was used. Regarding the pattern of immune response induced, i.m. needle injection raised a predominantly Th1 response with mostly IgG2a-speci®c antiGroEL and high levels of IFN-ã produced by splenic T cells. Gene gun immunisation induced a Th0 type of immune response in mice characterised by a high IgG1/IgG2a ratio, and IL-4 and interferon (IFN)-ã production. Even though a distinct pattern of immune response was generated depending upon the immunisation route used, neither method engendered a signi®cant level of protection with the GroEL DNA vaccine.

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.

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