Pathogenic brucella bacteria have developed strategies to persist for prolonged periods of time in host cells, avoiding innate immune responses. Here we show that the cyclic beta-1,2-glucans (CbetaG) synthesized by brucella is important for circumventing host cell defenses. CbetaG acted in lipid rafts found on host cell membranes. CbetaG-deficient mutants failed to prevent phagosome-lysosome fusion and could not replicate. However, when treated with purified CbetaG or synthetic methyl-beta-cyclodextrin, the mutants were able to control vacuole maturation by avoiding lysosome fusion, and this allowed intracellular brucella to survive and reach the endoplasmic reticulum. Fusion between the endoplasmic reticulum and the brucella-containing vacuole depended on the brucella virulence type IV secretion system but not on CbetaG. Brucella CbetaG is thus a virulence factor that interacts with lipid rafts and contributes to pathogen survival.

Bacterial vectors may offer many advantages over other antigen delivery systems for cancer vaccines. We engineered a Salmonella typhimurium vaccine strain to deliver the NY-ESO-1 tumor antigen (S. typhimurium-NY-ESO-1) through a type III protein secretion system. The S. typhimurium-NY-ESO-1 construct elicited NY-ESO-1-specific CD8+ and CD4+ T cells from peripheral blood lymphocytes of cancer patients in vitro. Oral administration of S. typhimurium-NY-ESO-1 to mice resulted in the regression of established NY-ESO-1-expressing tumors. Intratumoral inoculation of S. typhimurium-NY-ESO-1 to NY-ESO-1-negative tumors resulted in delivery of antigen in vivo and led to tumor regression in the presence of preexisting NY-ESO-1-specific CD8+ T cells. Specific T cell responses against at least 2 unrelated tumor antigens not contained in the vaccine were observed, demonstrating epitope spreading. We propose that antigen delivery through the S. typhimurium type III secretion system is a promising novel strategy for cancer vaccine development.

Null cyclic ␤-1,2-glucan synthetase mutants (cgs mutants) were obtained from Brucella abortus virulent strain 2308 and from B. abortus attenuated vaccinal strain S19. Both mutants show greater sensitivity to surfactants like deoxycholic acid, sodium dodecyl sulfate, and Zwittergent than the parental strains, suggesting cell surface alterations. Although not to the same extent, both mutants display reduced virulence in mice and defective intracellular multiplication in HeLa cells. The B. abortus S19 cgs mutant was completely cleared from the spleens of mice after 4 weeks, while the 2308 mutant showed a 1.5-log reduction of the number of brucellae isolated from the spleens after 12 weeks. These results suggest that cyclic ␤-1,2-glucan plays an important role in the residual virulence of the attenuated B. abortus S19 strain. Although the cgs mutant was cleared from the spleens earlier than the wild-type parental strain (B. abortus S19) and produced less inflammatory response, its ability to confer protection against the virulent strain B. abortus 2308 was fully retained. Equivalent levels of induction of spleen gamma interferon mRNA and anti-lipopolysaccharide (LPS) of immunoglobulin G2a (IgG2a) subtype antibodies were observed in mice injected with B. abortus S19 or the cgs mutant. However, the titer of anti-LPS antibodies of the IgG1 subtype induced by the cgs mutant was lower than that observed with the parental S19 strain, thus suggesting that the cgs mutant induces a relatively exclusive Th1 response.Brucella abortus is an intracellular pathogen that causes abortion in bovines and can infect humans. Abortion in cattle is the consequence of the tropism that the bacterium has for the placenta of pregnant animals, in which it multiplies intracellularly (10). Brucellosis in humans is primarily a disease of the reticuloendothelial system, in which the bacteria multiply inside the phagocytic cell; the intermittent release of bacteria from the cells into the bloodstream causes undulant fever (17, 29). Brucellosis does not spread among humans; consequently, eradication of the disease from the natural reservoirs, cattle, pigs, sheep, goats, and other susceptible animals, will lead to elimination of human infection. In regions with high prevalence of the disease, the only way of controlling and eventually eradicating this zoonosis is by vaccination of all susceptible hosts and elimination of infected animals.Vaccination represents an important tool for the control of bovine brucellosis. One of the most used vaccines is the attenuated strain B. abortus S19 obtained spontaneously from the virulent strain B. abortus 2308 (24, 25, 26, 29). Live attenuated B. abortus S19 has served for many years as an effective vaccine to prevent brucellosis in cattle (8,18). The genetic defect that leads to attenuation of this strain has not yet been defined. B. abortus S19 has lost some essential unknown mechanism of virulence. Despite this fact, the vaccinal strain conserves some degree of virulence, being pathogenic for humans (37), and...

We report here that a bacterial protease inhibitor from Brucella spp. called U-Omp19 behaves as an ideal constituent for a vaccine formulation against infectious diseases. When co-administered orally with an antigen (Ag), U-Omp19: i) can bypass the harsh environment of the gastrointestinal tract by inhibiting stomach and intestine proteases and consequently increases the half-life of the co-administered Ag at immune inductive sites: Peyer's patches and mesenteric lymph nodes while ii) it induces the recruitment and activation of antigen presenting cells (APCs) and increases the amount of intracellular Ag inside APCs. Therefore, mucosal as well as systemic Ag-specific immune responses, antibodies, Th1, Th17 and CD8(+) T cells are enhanced when U-Omp19 is co-administered with the Ag orally. Finally, this bacterial protease inhibitor in an oral vaccine formulation confers mucosal protection and reduces parasite loads after oral challenge with virulent Toxoplasma gondii.

bBrucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis. Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant for Brucella intracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratorygrown Brucella. To define the potential role of cyclophilins in Brucella virulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. The Brucella abortus ⌬cypAB mutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, the B. abortus ⌬cypAB mutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. The B. abortus ⌬cypAB mutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important for Brucella virulence and survival in the host cells. C yclophilins (Cyps) are folding helper enzymes that belong to the enzyme class of peptidyl prolyl cis/trans isomerases (PPIases; EC 5.2.1.8). In addition to cyclophilins, PPIases also includes FK506-binding proteins (FKBPs) and parvulins. These three families of proteins that have no sequence or structural homology can be distinguished by being inhibited by the immunosuppressive compounds cyclosporine, FK506, and rapamycin, respectively (1, 2). PPIases catalyze the cis/trans isomerization of peptidyl prolyl bonds. This reaction requires free energy and as a consequence is a slow process at lower temperatures, being the rate-limiting step in protein folding (3). PPIases are thought to be important for the correct folding of nascent proteins as well as their refolding (4-6). It is postulated that conformational isomerization by PPIases controls the activity of target proteins, regulating the interaction with other partner proteins to form complexes (3, 7).Cyclophilins are evolutionary conserved and have been found in all organisms analyzed to date, with the exception of Mycoplasma genitalium and some members of the Archaea (8). They are ubiquitously distributed proteins and like the other PPIases are critical for cell adaptation under stress conditions (9). Cyclophilins have been reported to be involved in several processes, such as adaptation to environmental stress, cell cycle control, signal transduction, and transcriptional regulation (8, 10-12). In addition, they have been implicated in the virulence of fungal and parasitic pathogens (13-17). Recent reports have shown the involvement of PPIases in stress tolerance and pathogenesis of bacteria, su...

Salmonella typhimurium engineered to deliver cancer/testis antigen NY-ESO-1 through type III secretion (S typhimurium-NY-ESO-1) was shown to be an efficient cancer vaccine construct in mice and to stimulate NY-ESO-1-specific CD8 ؉ /CD4 ؉ T cells in vitro in patients with cancer with NY-ESO-1 spontaneous immunity. We also showed that individuals without spontaneous immunity to NY-ESO-1 had specific CD4 ؉ T-cell precursors with high avidity to NY-ESO-1 under tight control by CD4 ؉ CD25 ؉ regulatory T (Treg) cells. We IntroductionNaturally occurring CD4 ϩ CD25 ϩ regulatory T (Treg) cells play an essential role in maintaining immunologic balance and preventing the development of autoimmunity. [1][2][3][4] Accumulating evidence shows that Treg cell populations are also crucial for controlling antitumor immune responses. In mice, depletion of Treg cell populations enhances spontaneous and vaccine-induced antitumor T-cell responses 5-8 and stimulation of CD4 ϩ CD25 ϩ Treg cells by immunization with self-antigens enhances the development of chemically induced primary tumors 9 and of pulmonary metastases following injection of transplantable tumor cells. 10 In humans, the presence of high numbers of CD4 ϩ CD25 ϩ Treg cells or a high ratio of CD4 ϩ CD25 ϩ Treg cells to CD8 ϩ T cells at the local tumor site is correlated with unfavorable prognosis. 11,12 From these results, it is becoming an important priority to find strategies for controlling Treg cells in the cancer vaccine field.NY-ESO-1, a germ-cell protein, was originally found by SEREX (serological identification of antigens by recombinant expression cloning) using the serum of an esophageal cancer patient. [13][14][15] Its expression pattern and the frequent finding of humoral and cellular immune responses against this antigen in patients with cancer with NY-ESO-1-expressing tumors make NY-ESO-1 one of the most intriguing cancer vaccine targets. [14][15][16][17] In monitoring a large series of patients with cancer, humoral responses to NY-ESO-1 were found to be correlated with the presence of peripheral CD8 ϩ T cells against NY-ESO-1, suggesting the involvement of CD4 ϩ helper T (Th) cells in coordinating these responses. [15][16][17][18] It was indeed confirmed that effector CD4 ϩ Th cell responses to NY-ESO-1 were only observed in patients with cancer who had antibodies against NY-ESO-1. 15,18 However, it has recently been shown that NY-ESO-1-specific CD4 ϩ T-cell precursors are also present in patients with NY-ESO-1-expressing tumors but without NY-ESO-1-specific antibody as well as in healthy individuals, and that CD4 ϩ CD25 ϩ Treg cells play a critical role in keeping these NY-ESO-1-specific precursors under control. 15,[19][20][21] The preexisting NY-ESO-1-specific CD4 ϩ T-cell precursors of seronegative and healthy individuals are exclusively from a naive (CD4 ϩ CD25 Ϫ CD45RA ϩ ) repertoire with high avidity to antigen and are highly sensitive to Treg cells, while spontaneously induced NY-ESO-1-specific CD4 ϩ T cells of seropositive patients are derived fro...

Type III protein secretion systems, which are organelles with the capacity to deliver bacterial proteins into host cells, have been adapted to deliver heterologous antigens for vaccine development. A limitation of these antigen delivery systems is that some proteins are not amenable to secretion through this pathway. We show here that proteins from the simian and human immunodeficiency viruses that are not permissive for secretion through a Salmonella enterica serovar Typhimurium type III secretion system can be modified to travel this secretion pathway by introduction of discrete mutations. Proteins optimized for secretion were presented more efficiently via the major histocompatibility complex class I pathway and were able to induce a better immune response.

Biosynthesis of periplasmic cyclic 1,2-p-glucans in Brucella ovis strain RE01 98 and B. abortus strain Sl9 was found to be carried out by membrane-bound enzymes that use UDP-glucose (UDP-Glc) as donor substrate. Contrary to what happens in species of the genera Agrobacterium and Rhizobium, the accumulation of the reaction products in Brucella appeared not to be osmotically regulated. Incubation of permeabilized cells with UDP-[14C]GI~ led to the formation of soluble neutral cyclic 1,2-p-glucans and [14C]glucoselabelled glucoproteins. PAGE of pulse-chase experiments carried out with permeabilized cells showed that the molecular mass of the labelled protein was indistinguishable from Agrobacterium tumefaciens A348 and Rhizobium fredii USDA191 glucoproteins known to be intermediates in the synthesis of cyclic glucans. Brucella total membrane preparations were less efficient than permeabilized cells in the formation of cyclic glucan; this was attributed to defective cyclization. Accumulation of protein intermediates having oligosaccharides of high molecular mass that were not released from the protein was observed after chase with 2 mM UDP-Glc. This defect was not observed when permeabilized cells were used as enzyme preparation, thus suggesting that in Brucella a factor(s) that was lost or inactivated upon the preparation of membranes was required for the effective regulation between elongation and cyclization reactions.