Screening of potential vaccine candidates against pathogenic Brucella spp. using compositive reverse vaccinology.

Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in humans and various animals. The threat of brucellosis has increased, yet currently available live attenuated vaccines still have drawbacks. Therefore, subunit vaccines, produced using protein antigens and having the advantage of being safe, cost-effective and efficacious, are urgently needed. In this study, we used core proteome analysis and a compositive RV methodology to screen potential broad-spectrum antigens against 213 pathogenic strains of Brucella spp. with worldwide geographic distribution. Candidate proteins were scored according to six biological features: subcellular localization, antigen similarity, antigenicity, mature epitope density, virulence, and adhesion probability. In the RV analysis, a total 32 candidate antigens were picked out. Of these, three proteins were selected for assessment of immunogenicity and preliminary protection in a mouse model: outer membrane protein Omp19 (used as a positive control), type IV secretion system (T4SS) protein VirB8, and type I secretion system (T1SS) protein HlyD. These three antigens with a high degree of conservation could induce specific humoral and cellular immune responses. Omp19, VirB8 and HlyD could substantially reduce the organ bacterial load of B. abortus S19 in mice and provide varying degrees of protection. In this study, we demonstrated the effectiveness of this unique strategy for the screening of potential broad-spectrum antigens against Brucella. Further evaluation is needed to identify the levels of protection conferred by the vaccine antigens against wild-type pathogenic Brucella species challenge.

Mannosylated chitosan nanoparticles loaded with FliC antigen as a novel vaccine candidate against Brucella melitensis and Brucella abortus infection.

Brucellosis is a worldwide bacterial zoonosis disease. Live attenuated Brucella vaccines have several drawbacks. Thus development of a safe and effective vaccine for brucellosis is a concern of many scientists. FliC protein contributes in virulence of Brucella; hence, it is a promising target for brucellosis vaccine. In this study, Mannosylated Chitosan Nanoparticles (MCN) loaded with FliC protein were synthesized as a targeted vaccine delivery system. The immunogenicity and protective efficacy of FliC and FliC-MCN against Brucella infection were evaluated in BALB/c mice. After cloning, expression and purification, FliC protein was loaded on MCN. The particle size, loading efficiency and in vitro release of the NPs were determined. Our investigation revealed that FliC and FliC-MCN could significantly increase specific IgG response (higher IgG2a titers). Besides, spleen cells from immunized mice produced high level of IFN-γ and IL-2 and low level IL-10 cytokines. Immunization with FliC and FliC-MCN conferred significant degree of protection against B. melitensis 16 M and B. abortus 544 infections. Overall these results indicate that FliC protein would be a novel potential antigen candidate for the development of a subunit vaccine against B. melitensis and B. abortus. Moreover, MCN could be used as an adjuvant and targeted vaccine delivery system.

Rev1 wbdR tagged vaccines against Brucella ovis.

Sheep brucellosis is a worldwide extended disease caused by B. melitensis and B. ovis, two species respectively carrying smooth or rough lipopolysaccharide. Vaccine B. melitensis Rev1 is used against B. melitensis and B. ovis but induces an anti-smooth-lipopolysaccharide response interfering with B. melitensis serodiagnosis, which precludes its use against B. ovis where B. melitensis is absent. In mice, Rev1 deleted in wbkC (Brucella lipopolysaccharide formyl-transferase) and carrying wbdR (E. coli acetyl-transferase) triggered antibodies that could be differentiated from those evoked by wild-type strains, was comparatively attenuated and protected against B. ovis, suggesting its potential as a B. ovis vaccine.

Effects of Thermoextracts of Brucella S and L Forms on Lipid Peroxidation and Antioxidant Defense in Organs of Laboratory Animals.

The dynamics of LPO marker malondialdehyde formation and peroxidase-destroying activity was studied in homogenized organs of guinea pigs, immunized with thermoextracts from S and L forms Brucella abortus I-206. The L form brucella thermoextract exhibited a lower reactogenicity and adequately activated the antioxidant system, due to which the destructive effects of ROS could be partially neutralized during the vaccinal process.

Encapsulated Brucella ovis Lacking a Putative ATP-Binding Cassette Transporter (ΔabcBA) Protects against Wild Type Brucella ovis in Rams.

This study aimed to evaluate protection induced by the vaccine candidate B. ovis ΔabcBA against experimental challenge with wild type B. ovis in rams. Rams were subcutaneously immunized with B. ovis ΔabcBA encapsulated with sterile alginate or with the non encapsulated vaccine strain. Serum, urine, and semen samples were collected during two months after immunization. The rams were then challenged with wild type B. ovis (ATCC25840), and the results were compared to non immunized and experimentally challenged rams. Immunization, particularly with encapsulated B. ovis ΔabcBA, prevented infection, secretion of wild type B. ovis in the semen and urine, shedding of neutrophils in the semen, and the development of clinical changes, gross and microscopic lesions induced by the wild type B. ovis reference strain. Collectively, our data indicates that the B. ovis ΔabcBA strain is an exceptionally good vaccine strain for preventing brucellosis caused by B. ovis infection in rams.

Vaccination with recombinant L7/L12-truncated Omp31 protein induces protection against Brucella infection in BALB/c mice.

Brucellosis is the most common bacterial zoonotic disease worldwide and no vaccine is available for the prevention of human brucellosis. In humans, brucellosis is mostly caused by Brucella melitensis and Brucella abortus. The Outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved in human Brucella pathogens. In the present study, we evaluated the humoral and cellular immune responses induced by a fusion protein designed based on the Truncated form of Omp31 (TOmp31) and L7-L12 antigens. Vaccination of BALB/c mice with the recombinant fusion protein (rL7/L12-TOmp31) provided the significant protection level against B. melitensis and B. abortus challenge. Moreover, rL7/L12-TOmp31 elicited a strong specific IgG response (higher IgG2a titers) and significant IFN-γ/IL2 production and T-cell proliferation was also observed. The T helper1 (Th1) oriented response persisted for 12 weeks after the first immunization. The rL7/L12-TOmp31 could be a new potential antigen candidate for the development of a subunit vaccine against B. melitensis and B. abortus.

Protection to respiratory challenge of Brucella abortus strain 2308 in the lung.

Brucella is amongst the top 5 causes of zoonotic disease worldwide. Infection is through ingestion, inhalation or contact exposure. Brucella is characterized as a class B pathogen by Centers of Disease Control and Prevention (CDC). Currently, there are no efficacious vaccines available in people. Currently available USDA approved vaccines for animals include B. abortus strain RB51 and B. melitensis Rev1. Protection is mediated by a strong innate and CD4 Th1, CD8 Tc1 immune response. If protective vaccines can be developed, disease in people and animals can be controlled. While strain RB51 protects in cattle, and against intraperitoneal challenge in mice, it does not protect against respiratory challenge. Therefore, we assessed the efficacy of strain RB51 combined with different TLR agonists, and O-side chain from LPS, to enhance protection against respiratory challenge with strain 2308. We hypothesized that TLR agonists and O-side chain would enhance protection. Strains RB51 with TLR2 agonist, RB51 with TLR4 agonist and strain 19 provided significant protection in the lung. Protection using strain RB51 with TLR agonists was associated with increased IgG2a and IgG1 in the (bronchoalveolar lavage) BAL and serum, and increased IgA (serum). Splenocytes from strain RB51 with TLR2 vaccinated mice up-regulated antigen specific interferon-gamma and TNF-alpha production. Vaccination and challenge resulted in significant increases in activated dendritic cells (DCs), and increased CD4 and CD8 cells in the BAL. Overall, this study demonstrates the ability of TLR agonists 2 and 4 to up-regulate strain RB51 mediated protection in the lung to respiratory challenge against strain 2308.

Escheriosome-mediated delivery of recombinant ribosomal L7/L12 protein confers protection against murine brucellosis.

Brucellosis is an important zoonotic disease that causes abortion in cattle and undulant fever, arthritis, endocarditis and meningitis in human. In spite of the fact that immunization could be an efficient measure to control brucellosis, not a single ideal vaccine against this important disease has been developed so far. In order to develop an effective vaccine against Brucella abortus (B. abortus), various protective immunodominant gene/protein products of the pathogen have been studied in combination with different adjuvants. For example, recombinant ribosomal protein L7/L12 (rL7/L12) although an interesting T-cell antigen, normally failed to evoke protective immune response when used in free form. In the present study we have demonstrated that Escherischia coli (E. coli) lipid liposome (escheriosome)-mediated cytosolic delivery of recombinant rL7/L12 protein can elicit strong immunological responses in the Balb/c mice. In contrast, egg PC/Chol liposome entrapped rL7/L12, in a manner similar to its free form, was found to impart relatively poor immune response. Furthermore, escheriosome entrapped rL7/L12 protein elicited high IgG2a isotype response suggestive of its relevance in imparting protection against brucellosis in mice. Altogether the present study is a clear indicative of the possible use of escheriosome-based delivery of rL7/L12 protein to induce protective immune responses against experimental murine brucellosis.

A combined DNA vaccine encoding BCSP31, SOD, and L7/L12 confers high protection against Brucella abortus 2308 by inducing specific CTL responses.

We constructed a combined DNA vaccine comprising genes encoding the antigens BCSP31, superoxide dismutase (SOD), and L7/L12 and evaluated its immunogenicity and protective efficacy. Immunization of mice with the combined DNA vaccine offered high protection against Brucella abortus (B. abortus) infection. The vaccine induced a vigorous specific immunoglobulin G (IgG) response, with higher IgG2a than IgG1 titers. Cytokine profiling performed at the same time showed a biased Th1-type immune response with significantly increased interferon-gamma and tumor necrosis factor-alpha stimulation. CD8(+), but not CD4(+), T cells accumulated at significantly higher levels after administration of the vaccine. Granzyme B-producing CD8(+) T cells were significantly higher in number in samples prepared from combined DNA-vaccinated mice compared with S19-vaccinated mice, demonstrating that the cytotoxicity lysis pathway is involved in the response to Brucella infection. The success of our combined DNA vaccine in a mouse model suggests its potential efficacy against brucellosis infection in large animals.

Improved immunogenicity of a vaccination regimen combining a DNA vaccine encoding Brucella melitensis outer membrane protein 31 (Omp31) and recombinant Omp31 boosting.

In the present study, we report an attempt to improve the immunogenicity of the Omp31 antigen by a DNA prime-protein boost immunization regimen. We immunized BALB/c mice with an Omp31 DNA vaccine (pCIOmp31) followed by boosting with recombinant Omp31 (rOmp31) in incomplete Freund's adjuvant and characterized the resulting immune responses and the protective efficacy against Brucella ovis and B. melitensis infection. Immunoglobulin G1 (IgG1) and IgG2a titers were higher in sera from pCIOmp31/rOmp31-immunized mice than in sera from mice immunized with pCIOmp31 or rOmp31 alone. Splenocytes from pCIOmp31/rOmp31-immunized mice produced significantly higher levels of gamma interferon than did those from mice given rOmp31 alone. In contrast, interleukin 2 (IL-2) production levels were comparable between the two groups of immunized mice. Cells from all immunized mice produced undetectable levels of IL-4. Notably, rOmp31 stimulated IL-10 production in the pCIOmp31/rOmp31-immunized group but not in the pCIOmp31- or rOmp31-immunized group. Although the prime-boost regimen induced specific cytotoxic responses, these responses could not reach the levels achieved by the pCIOmp31 immunization. In conclusion, pCIOmp31 priming followed by rOmp31 boosting led to moderately improved protection against a challenge with B. ovis or B. melitensis.

Brucella abortus vaccine strain RB51 produces low levels of M-like O-antigen.

Brucella abortus RB51 is a rough (R) stable vaccine strain used in cattle and is believed to be devoid of O-side chain. We analyzed by use of a panel of monoclonal antibodies (MAbs) directed against seven previously defined O-polysaccharide (O-PS) epitopes the O-chain expression in strain RB51. Two MAbs specific for the C/Y (A=M) and C (M>A) epitopes showed low bindings in ELISA to strain RB51. O-chain expression was further confirmed by Western blot after SDS-PAGE of strain RB51. In particular, the MAb of C (M>A) specificity, showing preferential binding to M-dominant smooth (S) Brucella strains, revealed in strain RB51 a typical smooth-lipopolysaccharide (S-LPS) pattern which resembled that of M-dominant S-LPS. Thus, the results clearly show that strain RB51 produces low levels of M-like O-antigen.

Safety and immunogenicity of Brucella abortus strain RB51 vaccine in pregnant cattle.

OBJECTIVE: To determine the safety and immunogenicity of Brucella abortus strain RB51 as a vaccine in pregnant cattle. ANIMALS: 12 Polled Hereford heifers obtained from a brucellosis-free herd and bred on site at 16 months of age to a brucellosis-free bull. PROCEDURE: Pregnant heifers were vaccinated at 6 months' gestation with 10(9) colony-forming units of B abortus strain RB51 (n = 5), 3 x 10(8) colony-forming units of B abortus strain 19 (n = 5), or sterile pyrogenfree saline solution (n = 2). Samples were periodically collected for serologic testing and lymphocyte blastogenesis assays. At full gestation, heifers were euthanatized and specimens were collected for bacteriologic culture, histologic analysis, and lymphocyte blastogenesis assay, using various antigenic stimuli. RESULTS: None of the strain RB51- or strain 19-vaccinates aborted or had gross or microscopic lesions at necropsy that were consistent with brucellosis. Maternal blood mononuclear cells from strain RB51- and strain 19-vaccinates had proliferative responses to gamma-irradiated strain RB51 and strain 19 that were greater than responses by cells from nonvaccinated controls. In contrast, maternal superficial cervical lymph node cells from strain 19-vaccinates had proliferative responses to gamma-irradiated strain RB51 or strain 19 bacteria greater than those of cells from RB51-vaccinates and nonvaccinated controls. None of the heifers vaccinated with strain RB51 developed antibodies detected by use of the standard tube agglutination test, but all developed antibodies to strain RB51 that reacted in a dot ELISA, using irradiated strain RB51 as antigen. CONCLUSIONS: Pregnant cattle can be safely vaccinated with strain RB51 without subsequent abortion or placentitis. Furthermore, strain RB51 is immunogenic in pregnant cattle, resulting in humoral and cell-mediated immune responses, but does not interfere with serologic diagnosis of field infections.

Evaluation of serological tests for diagnosis of Brucella melitensis infection of goats.

Five serological assays were evaluated for the diagnosis of brucellosis in goats: the rose bengal test (RBT), complement fixation test (CFT), radial immunodiffusion (RID) with Brucella and Yersinia enterocolitica O:9 polysaccharides, counterimmunoelectrophoresis (CIEP) with cytosol, and enzyme-linked immunosorbent assay (ELISA) with polyclonal and protein G conjugates and smooth lipopolysaccharide (S-LPS), native hapten polysaccharide (NH), or cytosol antigens. For optimal sensitivity, RBT had to be used with sera-antigen at a 3:1 dilution. In the RID test, Brucella melitensis biotype 1 NH could not be replaced by Brucella abortus biotype 1 or Y. enterocolitica 0:9 polysaccharides. In the ELISA, S-LPS and NH gave similar results and the protein G conjugate increased the specificity. With the sera from 55 B. melitensis culture-positive goats, the sensitivity was 100% for RBT, CFT (titer > or = 4), and ELISA with S-LPS or NH; 94% for RID; and 93% for CIEP. All tests were negative (100% specific) when testing the sera from 127 brucella-free goats. Larger discrepancies among the results of the serological tests were obtained with sera from goats of areas where brucellosis is endemic. When the sera of 20 young goats vaccinated subcutaneously (10(9) CFU of B. melitensis Rev 1) and bled 6 months later were examined, the specificities were as follows: NH ELISA, 60%; CFT and S-LPS ELISA, 75%; RBT, 80%; CIEP, 90%; and RID, 94%. With the sera from 10 young goats vaccinated conjunctivally (10(9) CFU of B. melitensis Rev 1) all tests were 100% specific 4 months after vaccination. The proportion of goats giving a positive reaction after vaccination decreased faster in RID than in other tests.

Efficacy of Brucella suis strain 2 vaccine against Brucella ovis in rams.

The protective efficacy against Brucella ovis of live vaccine Brucella suis strain 2 (S2) and Brucella melitensis strain Rev 1 has been evaluated in rams. Fourteen 4-month-old Brucella-free Aragonesa rams were vaccinated conjunctivally with 2 x 10(9) c.f.u. S2. Sixteen rams of the same breed, condition and age were conjunctivally vaccinated the same day with 1.6 x 10(9) Rev 1. Thirteen rams were unvaccinated controls. Eight months after vaccination all rams were challenged with 6 x 10(9) c.f.u. B. ovis and slaughtered 2 months thereafter for bacteriological and pathological studies. The percentage of infection in the group vaccinated with Rev 1 (43.7%) was significantly lower (p < 0.05) than that of the S2-vaccinated animals (78.6%) and unvaccinated controls (84.6%). No significant differences were found when comparing the percentages of infection corresponding to S2-vaccinated and control groups. The degree of infection (percentage of necropsy samples infected) was significantly lower in Rev 1-vaccinated (13%) than in S2-vaccinated (36.9%) or control groups (47.4%) (p < 0.001). However, no significant differences were found when comparing S2-vaccinated and control groups.

Vacunas en la brucelosis humana / Vaccines in human brucellosis

Hace casi 100 años la brucelosis humana fue considerada por Zammit y Horrocks como una zoonosis, sólo 18 años después que Bruce identificó el agente etiológico de la fiebre de Malta. A lo largo de los años, el avance científico se ha visto encaminado primordialmente al control de la brucelosis animal en los aspectos de diagnóstico y vacunación. En cuanto a la enfermedad en el humano, el progreso más importante ha sido la quimioterapia, aunque en la última década el diagnóstico correcto ha sido un asunto de preocupación general. Las estrategias de erradicación de la brucelosis se divide en tres categorías: 1) Erradicación de la brucelosis animal; 2) Mejoramiento de las medidas de higiene individual y saneamiento y 3) Inmunización. Las vacunas contra la brucelosis han sido desarrolladas a lo largo de tres líneas: 1) vacunas vivas preparadas con cepas atenuadas como B. abortus cepa 19 y B. melitensis cepa Rev.1; 2) células completas inactivadas, como B. abortus cepa 45/20 y B. melitensis H-38 que se administran con adyuvante oleoso y 3) vacunas preparadas con fracciones celulares. Todas ellas han sido usadas ampliamente en el control de la brucelosis en animales y sólo en algunos casos muy particulares en humanos

Preferential suppression of delayed-type hypersensitivity by L-156,602, a C5a receptor antagonist.

In the course of our screening for in vivo immunomodulating substances in which sheep red blood cells (SRBC) and heat-killed Brucella abortus cells (thymus dependent and independent antigens, respectively) for antibody production assays, and trinitrobenzene sulfonic acid (TNBS) for delayed-type hypersensitivity (DTH) assay were adopted as antigens, we detected a DTH-specific suppressive activity. The producing organism was isolated from a soil sample collected in Ushiku City, Ibaraki, Japan and identified with Streptomyces sp. A1502 (FERM P-12448). The active component was identified with L-156,602, a C5a receptor antagonist. L-156,602 suppressed both TNBS-induced and TNP-SRBC-induced DTH while it enhanced antibody production against SRBC, Brucella abortus, and TNP-SRBC. L-156,602 significantly suppressed DTH induced by direct injection of type 1 helper T cells and its relevant antigen into hind-footpads, indicating that the efferent phase of DTH was affected by L-156,602. The results demonstrated that L-156,602 preferentially suppressed the DTH response.

Responses of ewes to B. melitensis Rev1 vaccine administered by subcutaneous or conjunctival routes at different stages of pregnancy.

An experiment to determine the innocuousness and the humoral immune response of B. melitensis Rev1 vaccine was carried out in pregnant ewes. Thirty-three ewes were vaccinated on day 55 of pregnancy: 9 ewes received 2 X 10(9) Rev1 of French origin by the subcutaneous route, 9 ewes were conjunctivally vaccinated with 5 X 10(8) of the same strain, 7 ewes were subcutaneously vaccinated with 10(9) Rev1 of Spanish origin and 8 ewes received 1.8 X 10(9) Spanish strain Rev1 by conjunctival route. Another group (8 animals) were subcutaneously vaccinated with a reduced dose (10(6) of the French strain between days 90 and 120 of pregnancy. Finally, 20 ewes were vaccinated on day 120 of pregnancy with the French strain of which 11 received 10(9) Rev1 by the subcutaneous route and 9 were conjunctivally vaccinated with the same dose. Rev1 strain was isolated from the vaginal discharge of a significantly higher number of ewes vaccinated subcutaneously than in conjunctivally vaccinated ewes (24/35 versus 9/26 respectively; P less than 0.001). No difference between the two vaccine strains was recorded in the number of ewes excreting Rev1. Ewes subcutaneously vaccinated on day 55 of pregnancy aborted more frequently than conjunctivally vaccinated ewes (11/16 versus 2/17, respectively), hence a significantly reduced (P less than 0.001) length of pregnancy. Although 10 of the 11 animals subcutaneously vaccinated on day 120 of pregnancy excreted Rev1 strain, only one aborted. There was no significant effect of the time of vaccination on both abortions and length of pregnancy in ewes conjunctivally vaccinated with the French strain.(ABSTRACT TRUNCATED AT 250 WORDS)

An evaluation of the stability of Brucella abortus strain 19 reduced dosage lyophilized vaccines produced by different methods.

Replicate lots of multiple dose lyophilized Brucella abortus strain 19 vaccine were prepared having four different cell concentrations and four different fill volumes. In addition, two different size vials utilizing cells prepared by three different methods were studied. This vaccine was maintained at four different storage temperatures and the viability determined periodically. There was no appreciable loss in viability that could be attributed to the method of cell preparation; however, marked differences in viability were associated with cell concentration and/or prelyophilization fill volume and the two vial sized. The optimal prelyophilization cell suspension contained 25 to 35 X 10(9) viable Brucella 1 per ml. The better lyophilization vials had dimensions which minimized the surface to volume ratio of the postlyophilization pellet. Based on these studies it was concluded that production of large volumes of reduced dosage lyophilized vaccine is feasible and that the vaccine produced will meet specifications currently being implemented in the United States. These specification will require that each two ml dose of vaccine contain between three and ten X 10(9) viable organisms of B. abortus strain 19.

Comparative efficacy of an experimental S45/20 bacterin and a reduced dose of strain 19 vaccine against bovine brucellosis.

Yearling beef heifers were vaccinated with 2 doses of an experimental Brucella abortus S45/20 bacterin or a reduced dose of strain 19 (S19) vaccine, or were used as nonvaccinated controls. None of the vaccinated heifers was positive by the acidified plate, Rivanol, complement-fixation, or card tests 5 months after inoculation. Sixteen of 21 heifers given S45/20 bacterin with trehalose dimycolate adjuvant had delayed hypersensitivity when skin was tested 7 months after initial vaccination, whereas none of the S19 vaccinates or controls was reactive. After natural breeding, pregnant heifers were challenge exposed at 3.5 to 5 months of gestation with 2.5 X 10(7) viable B abortus S2308 organisms in the conjunctival sac. Abortions occurred as follows: 16/19 (84.2%) of heifers given S45/20 bacterin, 6/12 (50.0%) of heifers vaccinated with S19, and 17/18 (94.4%) of the nonvaccinated controls. Of the 10 heifers that calved normally, only 3 (1 in each group) were seropositive. A correlation was not seen between skin hypersensitivity and acquired resistance. Possible reasons for the poor protection with a reduced dose of S19 vaccine and the failure of S45/20 bacterin to enhance cellular resistance are discussed.