Subcutaneous immunization with a novel immunogenic candidate (urease) confers protection against Brucella abortus and Brucella melitensis infections

Brucellosis is a world prevalent endemic illness that is transmitted from domestic animals to humans. Brucella spp. exploits urease for survival in the harsh conditions of stomach during the gastrointestinal infection. In this study, we examined the immune response and the protection elicited by using recombinant Brucella urease (rUrease) vaccination in BALB/c mice. The urease gene was cloned in pET28a and the resulting recombinant protein was employed as subunit vaccine. Recombinant protein was administered subcutaneously and intraperitoneally. Dosage reduction was observed with subcutaneous (SC) vaccination when compared with intraperitoneal (IP) vaccination. rUrease induced mixed Th1–Th2 immune responses with high titers of specific IgG1 and IgG2a. In lymphocyte proliferation assay, splenocytes from IP and SC‐vaccinated mice displayed a strong recall proliferative response with high amounts of IL‐4, IL‐12 and IFN‐γ production. Vaccinated mice were challenged with virulent Brucella melitensis, B. abortus and B. suis. The SC vaccination route exhibited a higher degree of protection than IP vaccination (p value ≤ 0.05). Altogether, our results indicated that rUrease could be a useful antigen candidate for the development of subunit vaccines against brucellosis.     

Immunization with a single dose of a microencapsulated Brucella melitensis mutant enhances protection against wild-type challenge

The development of safe and efficacious immunization systems to prevent brucellosis is needed to overcome the disadvantages of the currently licensed vaccine strains that restrict their use in humans. Alginate microspheres coated with a protein of the parasite Fasciola hepatica (vitelline protein B [VpB]) and containing live Brucella melitensis attenuated mutant vjbR::Tn5 (BMEII1116) were evaluated for vaccine efficacy and immunogenicity in mice. A single immunization dose in BALB/c mice with the encapsulated vjbR mutant improved protection against wild-type B. melitensis 16M challenge compared to the nonencapsulated vaccine strain (P < 0.05). The encapsulated mutant was also shown to induce a sustained elevation of Immunoglobulin G levels. Cytokine secretion from spleen cells of mice vaccinated with the encapsulated vjbR::Tn5 revealed elevated secretion of gamma interferon and interleukin-12, but no interleukin-4, suggesting an induction of a T helper 1 response reflecting the enhanced immunity associated with microencapsulation. Together, these results suggest that microencapsulation of live attenuated organisms offers the ability to increase the efficacy of vaccine candidates.     

Aerosol infection of BALB/c mice with Brucella melitensis and Brucella abortus and protective efficacy against aerosol challenge

Brucellosis is a zoonotic disease with a worldwide distribution that can be transmitted via intentional or accidental aerosol exposure. In order to engineer superior vaccine strains against Brucella species for use in animals as well as in humans, the possibility of challenge infection via aerosol needs to be considered to properly evaluate vaccine efficacy. In this study, we assessed the use of an aerosol chamber to infect deep lung tissue of mice to elicit systemic infections with either Brucella abortus or B. melitensis at various doses. The results reveal that B. abortus causes a chronic infection of lung tissue in BALB/c mice and peripheral organs at low doses. In contrast, B. melitensis infection diminishes more rapidly, and higher infectious doses are required to obtain infection rates in animals similar to those of B. abortus. Whether this difference translates to severity of human infection remains to be elucidated. Despite these differences, unmarked deletion mutants BAΔasp24 and BMΔasp24 consistently confer superior protection to mice against homologous and heterologous aerosol challenge infection and should be considered viable candidates as vaccine strains against brucellosis.     

Immune responses and protection against experimental Brucella suis biovar 1 challenge in nonvaccinated or B. abortus strain RB51-vaccinated cattle

Twenty Hereford heifers approximately 9 months of age were vaccinated with saline (control) or 2 × 10(10) CFU of the Brucella abortus strain RB51 (RB51) vaccine. Immunologic responses after inoculation demonstrated significantly greater (P < 0.05) antibody and proliferative responses to RB51 antigens in cattle vaccinated with RB51 than in the controls. Pregnant cattle received a conjunctival challenge at approximately 6 months of gestation with 10(7) CFU of B. suis bv. 1 strains isolated from naturally infected cattle. The fluorescence polarization assay and the buffered acid plate agglutination test had the highest sensitivities in detecting B. suis-infected cattle between 2 and 12 weeks after experimental infection. Serologic responses and lymphocyte proliferative responses to B. suis antigens did not differ between control and RB51 vaccinees after experimental infection. No abortions occurred in cattle in either treatment group after challenge, although there appeared to be an increased incidence of retained placenta after parturition in both the control and the RB51 vaccination treatment groups. Our data suggest that the mammary gland is a preferred site for B. suis localization in cattle. Vaccination with RB51 did not reduce B. suis infection rates in maternal or fetal tissues. In conclusion, although B. suis is unlikely to cause abortions and fetal losses in cattle, our data suggest that RB51 vaccination will not protect cattle against B. suis infection after exposure.     

Intact purine biosynthesis pathways are required for wild-type virulence of Brucella abortus 2308 in the BALB/c mouse model

Brucella abortus 2308 derivatives with mini-Tn5 insertions in purE, purL, and purD display significant attenuation in the BALB/c mouse model, while isogenic mutants with mini-Tn5 insertions in pheA, trpB, and dagA display little or no attenuation in cultured murine macrophages or mice. These experimental findings confirm the importance of the purine biosynthesis pathways for the survival and replication of the brucellae in host macrophages. In contrast to previous reports, however, these results indicate that exogenous tryptophan and phenylalanine are available for use by the brucellae in the phagosomal compartment.     

Deletion of wboA enhances activation of the lectin pathway of complement in Brucella abortus and Brucella melitensis

Brucella spp. are gram-negative intracellular pathogens that survive and multiply within phagocytic cells of their hosts. Smooth organisms present O polysaccharides (OPS) on their surface. These OPS help the bacteria avoid the bactericidal action of serum. The wboA gene, coding for the enzyme glycosyltransferase, is essential for the synthesis of O chain in Brucella. In this study, the sensitivity to serum of smooth, virulent Brucella melitensis 16M and B. abortus 2308, rough wboA mutants VTRM1, RA1, and WRR51 derived from these two Brucella species, and the B. abortus vaccine strain RB51 was assayed using normal nonimmune human serum (NHS). The deposition of complement components and mannose-binding lectin (MBL) on the bacterial surface was detected by flow cytometry. Rough B. abortus mutants were more sensitive to the bactericidal action of NHS than were rough B. melitensis mutants. Complement components were deposited on smooth strains at a slower rate compared to rough strains. Deposition of iC3b and C5b-9 and bacterial killing occurred when bacteria were treated with C1q-depleted, but not with C2-depleted serum or NHS in the presence of Mg-EGTA. These results indicate that (i) OPS-deficient strains derived from B. melitensis 16M are more resistant to the bactericidal action of NHS than OPS-deficient strains derived from B. abortus 2308, (ii) both the classical and the MBL-mediated pathways are involved in complement deposition and complement-mediated killing of Brucella, and (iii) the alternative pathway is not activated by smooth or rough brucellae.     

Deletion of the BCSP31 gene of Brucella abortus by replacement.

The 31-kDa salt-extractable immunogenic protein, BCSP31, was deleted from several Brucella abortus strains by replacement with a marker gene encoding resistance to the antibiotics kanamycin and neomycin. The BCSP31 gene replacement plasmids, constructed with ColE1-derived vectors, were introduced by electroporation into B. abortus strain 19 (S19), into a rough variant of B. abortus S19, and into B. abortus S2308, and antibiotic-resistant transformants were isolated. B. abortus S19 is an attenuated strain used as a vaccine for prevention of bovine brucellosis in the United States, and B. abortus S2308 is a commonly used challenge strain. The antibiotic-resistant isolates were all obtained by recombination; none were spontaneous mutants. Loss of the gene encoding BCSP31 and presence of the marker gene were confirmed by Southern analysis. Vector sequences were either absent or linked to the genome, indicating that ColE1-derived plasmids are not maintained in B. abortus. Survival of B. abortus mutant strains in the macrophagelike cell line J774 and in HeLa cells was examined and shown to be indistinguishable from that of the parental strain.     

Role in virulence of a Brucella abortus protein exhibiting lectin-like activity

Brucella abortus is a facultative, intracellular zoonotic pathogen which can cause undulant fever in humans and abortions in cattle. A 14-kDa protein of B. abortus was previously identified to be immunogenic in animals infected with Brucella spp. In this study, we discovered that the 14-kDa protein possessed immunoglobulin binding and hemagglutination properties that appeared to be based on the protein's lectin-like properties. Hemagglutination inhibition experiments suggested that the 14-kDa protein has affinity towards mannose. Disruption of the gene encoding the 14-kDa protein in virulent B. abortus strain 2308 induced a rough-like phenotype with an altered smooth lipopolysaccharide (LPS) immunoblot profile and a significant reduction in the bacterium's ability to replicate in mouse spleens. However, the mutant strain was stably maintained in mouse spleens at 2.0 to 2.6 log(10) CFU/spleen from day 1 to week 6 after intraperitoneal inoculation with 4.65 log(10) CFU. In contrast to the case for the smooth virulent strain 2308, in the rough attenuated strain RB51 disruption of the 14-kDa protein's gene had no effect on the mouse clearance pattern. These findings indicate that the 14-kDa protein of B. abortus possesses lectin-like properties and is essential for the virulence of the species, probably because of its direct or indirect role in the synthesis of smooth LPS.     

Deletion of purE attenuates Brucella melitensis infection in mice.

We previously showed that a purE mutant (delta purE201) of Brucella melitensis 16M is attenuated for growth in cultured human monocytes (E. S. Drazek, H. H. Houng, R. M. Crawford, T. L. Hadfield, D. L. Hoover, and R. L. Warren, Infect. Immun. 63:3297-3301, 1995). To determine if this strain is attenuated in animals, we compared the growth of the delta purE201 mutant with that of strain 16M in BALB/c mice. The number of bacteria in the spleen and spleen weight peaked for both strains between 1 and 2 weeks postinfection (p.i.), though the number of delta purE201 cells was significantly less than the number of 16M cells recovered from the spleens of infected mice. During the next 6 weeks, delta purE201 was essentially eliminated from infected mice (three of five mice sterile; < 100 CFU in two of live mice at 8 weeks p.i.), whereas bacteria persisted at a high level in the spleens of 16M-infected mice (about 106 CFU per spleen). The number of bacteria in the livers and lungs of mice infected with either strain paralleled those in the spleen. Mice infected with 16M had a strong inflammatory response, developing dramatic and prolonged splenomegaly (five to eight times normal spleen weight) and producing serum interleukin-6. In contrast, mice infected with delta purE201 developed only mild, transient splenomegaly at 1 week p.i. and produced no interleukin-6 in their serum. We further characterized the host response to infection by measuring changes in immune spleen cell populations by flow cytometry. CD4- and CD8-positive lymphocytes declined by I week in both experimental groups, while MAC-1-positive cells increased. T-cell subpopulations remained low or declined further, and MAC-1 cells increased to three times normal levels during 8 weeks of infection with 16M but returned to normal by 4 weeks after infection with delta purE201. These results document infectivity and attenuation of delta purE201 and suggest that it should be further evaluated as a potential vaccine.     

Vaccination with Brucella abortus rough mutant RB51 protects BALB/c mice against virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis.

Vaccination of BALB/c mice with live Brucella abortus RB51, a stable rough mutant, produced protection against challenge with virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis. Passive-transfer experiments indicated that vaccinated mice were protected against B. abortus 2308 through cell-mediated immunity, against B. ovis PA through humoral immunity, and against B. melitensis 16M through both forms of immunity. Live bacteria were required for the induction of protective cell-mediated immunity; vaccination with whole killed cells of strain RB51 failed to protect mice against B. abortus 2308 despite development of good delayed-type hypersensitivity reactions. Protective antibodies against the heterologous species were generated in vaccinated mice primarily through anamnestic responses following challenge infections. Growth of the antigenically unrelated bacterium Listeria monocytogenes in the spleens of vaccinated mice indicated that nonspecific killing by residual activated macrophages contributed minimally to protection. These results encourage the continued investigation of strain RB51 as an alternative vaccine against heterologous Brucella species. However, its usefulness against B. ovis would be limited if, as suggested here, epitopes critical for protective cell-mediated immunity are not shared between B. abortus and B. ovis.     

Brucella abortus HtrA functions as an authentic stress response protease but is not required for wild-type virulence in BALB/c mice

A second mutation has recently been identified in the previously described Brucella abortus htrA mutant PHE1. As a result of this finding, a new B. abortus htrA mutant, designated RWP11, was constructed to evaluate the biological function of the Brucella HtrA protease. RWP11 is more sensitive to oxidative killing in vitro and less resistant to killing by cultured murine neutrophils and macrophages than the virulent parental strain 2308 but is not attenuated in BALB/c mice through 4 weeks postinfection. The in vitro phenotype of B. abortus RWP11 is consistent with the proposed function of bacterial HtrA proteases as components of a secondary line of defense against oxidative damage. The in vivo phenotype of this mutant, however, indicates that, unlike the corresponding Salmonella and Yersinia proteins, Brucella HtrA does not play a critical role in virulence in the mouse model.     

Mechanism of resistance to virus challenge in mice infected with Brucella abortus

Summary Mice infected intraperitoneally or intravenously with liveB. abortus showed increased resistance to intraperitoneal infection with Mengo virus. The resistant state lasted for at least 3 weeks.When the bacteria were given by the intraperitoneal route, passage of virus from the peritoneal cavity to the circulation was inhibited. By analogy to findings with polycarboxylate-treated mice, it was concluded that this trapping of virus in the peritoneal cavity contributed to the development of resistance to virus infection.In mice which were protected by intravenous injection of brucellae, passage of the challenge virus from the peritoneal cavity to the blood was not inhibited. Replication of virus in the spleens of these mice was decreased. However, no interferon was detectable in the sera or spleens, and no increased interf eron response to Mengo virus was observed, suggesting that interferon was not responsible for the observed inhibition of virus replication. It is proposed thatB. abortus infection potentiated phagocytic and virucidal activity of reticuloendothelial cells and thereby depressed apparent virus production in the spleen. This view was supported by the observation that uptake and subsequent clearance of a non-replicating picornavirus (poliovirus) were enhanced in the spleens ofB. abortus-infected mice.Assistant of the National University of Zaire, Kinshasa, Republic of Zaire, and Fellow of the Belgian O.C.D.Fellow of the Belgian N.F.W.O.     

Decay-accelerating factor confers protection against complement-mediated podocyte injury in acute nephrotoxic nephritis

SUMMARY: Decay-accelerating factor (DAF or CD55) is one of a set of regulators that function to protect self cells from deposition of autologous C3b on their surfaces. Its relative importance in vivo, however, is incompletely understood. As one approach to address this issue, we induced nephrotoxic serum (NTS) nephritis in wild-type mice and Daf1 gene-floxed mice devoid of renal DAF expression. For these experiments NTS IgG was administered at a dose (0.5 mg iv) that requires complement for glomerular injury. After 18 hours, renal injury was assessed by proteinuria and by histologic, immunohistochemical, and electron microscopic analyses of kidneys. Fifteen normal and 15 DAF-deficient mice were studied. Baseline albuminuria in the Daf1(-/-) mice was 115.9 +/- 41.4 microg/mg creatinine as compared with 85.7 +/- 32.3 microg/mg creatinine in their Daf1(+/+) littermates (p = 0.075). After administration of NTS IgG, albuminuria increased to 2001.7 +/- 688.7 microg/mg creatinine as compared with 799.7 +/- 340.5 microg/mg creatinine in the controls (p = 0.0003). Glomerular histology was similar in Daf1(-/-) and Daf1(+/+) mice, with essentially no infiltrating leukocytes. In contrast, electron microscopy revealed severe podocyte fusion in the Daf1(-/-) mice but only mild focal changes in the controls. Immunohistochemical staining showed equivalent deposition of the administered (sheep) NTS IgG in the Daf1(-/-) and Daf1(+/+) animals. This contrasted with marked deposition of autologous murine C3 in the former and minimal deposition in the latter. The results show that DAF is essential physiologically for protecting glomeruli against autologous complement attack initiated by the classical pathway.     

Ontogeny of Th1 memory responses against a Brucella abortus conjugate

Protective immune responses to intracellular pathogens such as Brucella abortus are characteristically Th1-like. Recently we demonstrated that heat-killed B. abortus (HKBa), a strong Th1 stimulus, conjugated to ovalbumin (HKBA-OVA), but not B. abortus alone, can alter the antigen-specific cytokine profile from Th2- to Th1-like. In this report we study the ability of a single injection of B. abortus to switch a Th2 to a Th1 response in immature mice. One-day- and 1-week-old mice were given a single injection of B. abortus in the absence or presence of OVA, and at maturity mice were challenged with an allergenic preparation, OVA with alum (OVA-A). B. abortus given without OVA did not diminish the subsequent Th2 response in either age group. In contrast, mice receiving a single injection of B. abortus-OVA at the age of 1 week, but not those injected at the age of 1 day, had reversal of the ratio of OVA-specific Th1 to Th2 cells and decreased immunoglobulin E levels after allergen challenge as adults. Within 6 h both 1-day- and 1-week-old mice expressed interleukin-12 p40 mRNA following either B. abortus or B. abortus-OVA administration. However, only the 1-week-old mice exhibited increased expression of gamma interferon (IFN-gamma) mRNA. The absence of the early IFN-gamma response in 1-day-old mice may explain their inability to generate a Th1 memory response. These results suggest that at early stages of immune development, responses to intracellular bacteria may be Th2- rather than Th1-like. Furthermore, they suggest that the first encounter with antigen evokes either a Th1- or a Th2-like response which becomes imprinted, so that subsequent memory responses conform to the original Th bias. This has implications for protection against infectious agents and development of allergic responses.     

Deletion in the gene BruAb2_0168 of Brucella abortus strains: diagnostic challenges

Three Brucella abortus strains were isolated from joint hygromas from cows in northern Togo. Two deletions in the 5′ side of the gene BruAb2_0168 were identified. As this gene is used for species identification, these deletions have consequences for diagnostic procedures. Multiple locus variable number of tandem repeat (VNTR) analysis was therefore performed for species identification. The strains showed unique VNTR profiles, providing some of the first genotypic data from West Africa. More molecular and epidemiological data are needed from the region, in order to better understand transmission patterns and develop suitable diagnostic assays.     

Identification and characterization of a Brucella abortus ATP-binding cassette transporter homolog to Rhizobium meliloti ExsA and its role in virulence and protection in mice

Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not fully understood yet. Furthermore, genes that allow Brucella to reach the intracellular niche and to interact with host cells need to be identified. Using the genomic survey sequence (GSS) approach, we identified the gene encoding an ATP-binding cassette (ABC) transporter of B. abortus strain S2308. The deduced amino acid sequence encoded by this gene exhibited 69 and 67% identity with the sequences of the ABC transporters encoded by the exsA genes of Rhizobium meliloti and Mesorhizobium loti, respectively. Additionally, B. abortus ExsA, like R. meliloti and M. loti ExsA, possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Furthermore, ortholog group analysis placed B. abortus ExsA in ortholog group 6 of ABC transporters more likely to be involved in bacterial pathogenesis. In R. meliloti, ExsA is an exopolysaccharide transporter essential for alfalfa root nodule invasion and establishment of infection. To test the role of ExsA in Brucella pathogenesis, an exsA deletion mutant was constructed. Replacement of the wild-type exsA by recombination was demonstrated by Southern blot analysis of Brucella genomic DNA. Decreased survival in mice of the Brucella DeltaexsA mutant compared to the survival of parental strain S2308 demonstrated that ExsA is critical for full bacterial virulence. Additionally, the B. abortus exsA deletion mutant was used as a live vaccine. Challenge experiments revealed that the exsA mutant strain induced superior protective immunity in BALB/c mice compared to the protective immunity induced by strain S19 or RB51.     

Regulation of Brucella abortus catalase

All aerobic organisms have mechanisms that protect against oxidative compounds. Catalase, peroxidase, superoxide dismutase, glutathione, and thioredoxin are widely distributed in many taxa and constitute elements of a nearly ubiquitous antioxidant metabolic strategy. Interestingly, the regulatory mechanisms that control these elements are rather different depending on the nature of the oxidative stress and the organism. Catalase is well documented to play an important role in protecting cells from oxidative stress. In particular, pathogenic bacteria seem to use this enzyme as a defensive tool against attack by the host. To investigate the significance of catalase in hostile environments, we made catalase deletion mutations in two different B. abortus strains and used two-dimensional gel analysis, survival tests, and adaptation experiments to explore the behavior and role of catalase under several oxidative stress conditions. These studies show that B. abortus strains that do not express catalase activity exhibit increased sensitivity to hydrogen peroxide. We also demonstrate that catalase expression is regulated in this species, and that preexposure to a sublethal concentration of hydrogen peroxide allows B. abortus to adapt so as to survive subsequent exposure to higher concentrations of hydrogen peroxide.