Mice immune responses to Brucella abortus heat shock proteins. Use of baculovirus recombinant-expressing whole insect cells,purified Brucella abortus recombinant proteins,and a vaccinia virus recombinant as immunogens

Brucella abortus resists the microbicidal mechanisms of macrophages, and the expression of its heat shock proteins (HSPs) such as GroEL, GroES and HtrA may play a role in this resistance. Bacterial HSPs can be very immunogenic, inducing protective immunity in various types of bacterial infections. However, the significance of immune responses directed against B. abortus HSPs in the protection against brucellosis is currently unresolved. To elucidate the role of these proteins in protection against Brucella challenge, individual, divalent or trivalent baculovirus (BV) recombinants of B. abortus GroEL, GroES and/or HtrA were injected into BALB/c mice either as protein-expressing whole cells or as purified proteins. The preparations were given to mice in combination with Freund's or Ribi adjuvant, respectively. In addition, some mice were primed with a vaccinia virus-GroEL recombinant, followed by inoculation with purified GroEL-Ribi adjuvant combination. Antibodies were observed against B. abortus GroEL and HtrA, but not against GroES. Cellular immune response was demonstrated by observing significant IFN-gamma release by lymphocytes of mice immunized with the purified HtrA-Ribi adjuvant combination. However, none of the mice inoculated with individual, divalent or trivalent HSP-expressing cells combined with complete Freund's adjuvant or inoculated with purified B. abortus HSPs combined with Ribi adjuvant, were protected against challenge with B. abortus virulent strain 2308. Priming with vaccinia virus-GroEL recombinant and boosting with GroEL-Ribi combination did not induce protective immunity. Based on the results obtained, we suggest that although humoral and cell-mediated immune responses are induced, but protective immune response is not induced by B. abortus HSPs.     

Immune responses of mice to vaccinia virus recombinants expressing either Listeria monocytogenes partial listeriolysin or Brucella abortus ribosomal L7/L12 protein

The Brucella abortus L7/L12 gene encoding ribosomal protein L7/L12 and the Listeria monocytogenes partial hly gene encoding the protective region of the hemolysin (partial listeriolysin, pLLO) were cloned into vaccinia virus by homologous recombination to produce recombinants WRL7/L12 and WRpLLO, respectively. The ability of these recombinants to induce humoral, cell mediated and protective immune response in mice was assessed. Although mice inoculated with WRL7/L12 recombinant produced antibodies specific to vaccinia virus and L7/L12 antigens, they were not protected against a virulent challenge with B. abortus 2308 strain. In contrast, mice inoculated with WRpLLO were protected against a challenge with virulent L. monocytogenes. Stimulation with purified fusion listeriolysin protein (MBP-LLO), but not with unrelated control protein (MBP), induced splenocytes from WRpLLO-inoculated mice to secrete significantly higher amounts of IFN-gamma than saline inoculated mice. Mice inoculated with either WRpLLO or WRL7/L12 recombinants produced predominantly IgG2a isotype antibody responses, indicative of a Th1 type of immune response. The protective potential of the WRpLLO recombinant correlated with the level of IFN-gamma produced in these mice.     

Biological properties of RB51; a stable rough strain of Brucella abortus

A rifampin-resistant mutant of Brucella abortus, designated RB51, was derived by repeated passage of strain 2308 on Trypticase soy supplemented with 1.5% agar and varying concentrations rifampin or penicillin. The RB51 colonies absorbed crystal violet and RB51 cell suspensions autoagglutinated, indicating a rough type colonial morphology for this strain. No O-chain component was detected in lipopolysaccharide (LPS) extracted from RB51 on SDS-PAGE gels stained with silver. Western blot analysis with the monoclonal antibody BRU 38, which is specific for the perosamine homopolymer O-chain of smooth Brucella LPS, indicated that the LPS of RB51 is highly deficient in O-chain when compared with the parenteral smooth strain 2308 or rough strain 45/20. Biochemically, RB51 resembles parental strain 2308 in its ability to utilize erythritol. Intraperitoneal inoculation of RB51 into mice results in a splenic colonization which is cleared within four weeks post infection. RB51 does not revert to smooth colony morphology upon passage in vivo (mice) or in vitro. Mice infected with RB51 produce antibodies against B. abortus antigens including class 2 and 3 outer membrane proteins but not against the O-chain. Furthermore, rabbits, goats and cattle hyperimmunized with sonicates of RB51 develop antibodies to B. abortus cellular antigens but do not develop antibodies specific for the O-chain. Immunization of mice with 1 x 10(8) viable RB51 organisms confers significant protection against challenge with virulent B. abortus strain 2308.     

Protection of mice against Brucella abortus infection by inoculation with monoclonal antibodies recognizing Brucella O-antigen

Monoclonal antibodies recognizing the O-polysaccharide portion of Brucella abortus strain 2308 provided BALB/c mice with passive protection against challenge exposure with the homologous strain. Numbers of colony-forming organisms in the spleen were reduced by IgM and IgG monoclonal antibodies. Active immunization of mice, using B abortus 2308S lipopolysaccharide, resulted in production of IgM antibody at 14 days. Clearance of organisms in the actively immunized mice after challenge exposure at 14 days was nearly identical to that in passively immunized mice. Mice either passively or actively immunized were effectively protected from 0 to 28 days. Bacterial colonization of the spleen was observed to increase in both groups of mice at 56 days and indicated that humoral responses were effective in eliminating the organism in the early stages of infection, but other immune mechanisms were necessary for protection of mice in the later stage of infection with virulent strains of B abortus.     

Monoclonal antibodies directed against Brucella abortus cell surface antigens

A panel of monoclonal antibodies (MAb) has been raised against Brucella abortus cell surface antigens from mice immunized with either heat/phenol treated or UV killed bacterial suspensions of B. abortus. The hybridomas were screened by either a microagglutination procedure or by an indirect enzyme immunoassay (EIA) on sonicated bacterial preparations. From a large number of MAb generated by various procedures, two distinct types of MAb emerged. The most numerous type was capable of agglutinating B. abortus and reacting with a soluble preparation of lipopolysaccharide (LPS). A second type was not capable of agglutinating the bacterial suspensions or of binding to the soluble LPS preparation but reacted with an antigen present in bacterial sonicates. Two MAb of this type react differentially with sonicates prepared from virulent and avirulent strains of B. abortus. There appeared to be sufficient evidence from our analysis of the relative degree of cross reaction with antigens present on a range of B. abortus strains and Brucella and xenogenic bacterial species to conclude that each of the seven MAb was recognising a separate antigenic site on the B. abortus cell surface.     

Detection of Brucella abortus in mammalian tissue, using biotinylated, whole genomic DNA as a molecular probe

A method has been developed for the detection of Brucella abortus in complex tissue homogenates. The technique uses tissue homogenization in the presence of sucrose and Triton X-100 and subsequent filtration through a 5-microns pore size filter to remove mammalian nuclei and cellular debris. The DNA from the bacteria is then extracted, dot blotted onto nitrocellulose, and hybridized with a biotinylated probe of B abortus strain 19 DNA. In the present study, BALB/C mice were inoculated intraperitoneally with either 10(9) or 10(11) B abortus strain 2308S organisms. After 6 days, the mice were euthanatized by cervical dislocation and the livers were removed, weighed, and the appearance of each was noted. The tissues were homogenized, and a viable cell count was performed to determine the number of bacteria in each organ. The DNA was extracted, blotted onto nitrocellulose, and hybridized with the Brucella probe. The biotin label was detected by use of a commercially available streptavidin/alkaline phosphatase system. In control experiments, the technique detected 10(5) organisms in a mixture of bacteria and 1 g of rat liver. The technique also detected 10(7) B abortus organisms/g of tissue from experimentally inoculated mice. The probe was specific for Brucella and had no affinity for contaminating bovine or bacterial DNA.     

Brucella abortus INTA2, a novel strain 19 (Delta)bp26::luc (Delta)bmp18 double mutant lacking drug resistance markers

Brucella abortus INTA2, a novel mutant strain, was constructed by inactivation of two B. abortus S19 genes: bp26 and bmp18, with the objective of obtaining a mutant strain that could be compatible with a diagnostic test and have less residual virulence than strain 19. The double mutant was constructed by replacing a large section of the bp26 coding region with the luciferase (luc) coding gene, resulting in mutant strain B. abortus M1luc, followed by partial deletion of bmp18 coding sequence. Both genes were inactivated by allelic replacement assisted by sacB counter-selection. Luciferase expression was evaluated and confirmed that it is a valid marker in the construction of mutant strains. When B. abortus INTA2 was inoculated in BALB/c mice, significantly fewer colony forming units (CFUs) were recovered from mice spleens during initial phase of infection. No splenomegaly was observed in strain INTA2-immunized mice at any time suggesting that strain INTA2 has lost some residual virulence of the parental strain. Nevertheless, similar protection levels against virulent challenge were observed in mice immunized with strains INTA2 or S19. Although strain INTA2 would still induce O-side antibodies, it does not express BP26. This would allow differentiation of INTA2-vaccinated animals from animals infected with field strains by measuring anti-BP26 antibodies, either by an agglutination test or ELISA using BP26 as antigen. Altogether these results indicate that B. abortus INTA2 might be a promising vaccine strain against brucellosis.     

Unresponsiveness of vaccinated BALB/c mice to a second inoculation of lipopolysaccharide from Brucella abortus strain 2308

A study was conducted to determine whether the protection induced in mice by a primary inoculation of lipopolysaccharide from Brucella abortus would be enhanced by a second inoculation given at different time intervals. Protection was challenged by exposure of the mice to a virulent culture of B. abortus strain 2308. Reduced mean viable count and/or splenic weights were the criteria of protection. There was no significant difference (P greater than 0.05) in the protective responses among mice given a single inoculation. Vaccinated mice were significantly (P less than 0.05) better protected than were nonvaccinated mice. Mice given vaccinal inoculations simultaneous with challenge exposure were less protected (P less than 0.001) than were mice vaccinated prior to challenge, but were better protected (P less than 0.010) than were nonvaccinated mice.     

Identification of a virulence factor for Brucella abortus infection in BALB/c mice

Immunogenic or pathogenic factors of recombinant proteins (rBCSP20, rBCSP-31, and rBCSP45 of Brucella abortus strain 19) for mice were compared with factors of a proteinase K-treated lipopolysaccharide extracted from B abortus strain 2308. Mice were vaccinated with 4 products, using different inoculation schedules and were challenge exposed with a virulent culture of B abortus strain 2308. Blood samples were collected 2 weeks after vaccination and at necropsy and sera were obtained. Spleens were cultured for B abortus at necropsy (3 to 4 weeks after challenge exposure). Mice given proteinase K-treated lipopolysaccharide alone or in conjunction with rBCSP20 or rBCSP45 proteins were protected, but mice given rBCSP31 on the same day as challenge exposure were not. Vaccination with recombinant proteins alone neither provide protection nor significantly (P greater than 0.05) increase the pathogenic effect of the challenge-exposure culture. Seemingly, rBCSP31 might be a virulence factor of B abortus.     

中国牛种布鲁菌弱毒疫苗株A19 SNP位点的研究

为鉴别我国牛种布鲁菌弱毒疫苗株A19与野生菌株,运用生物信息学方法结合基因测序,对疫苗株A19基因组SNP位点分析筛选,选取其中部分SNP位点,通过与布鲁菌常见种、生物型标准参考菌株和弱毒疫苗株基因组SNP位置核苷酸测序比较,验证SNP位点的A19特异性。结果表明,共筛选获得A19基因组29个SNP位点,验证ClpX G825-C825、LysR A605-C605、Omp2b G503-A503这3个SNP位点为A19（或S19）特异,揭示了A19基因组SNP位点分布情况,为疫苗株 A19与野生菌株鉴别提供了分子依据。     

Specific lymphocyte stimulation in cattle naturally infected with strains of Brucella abortus and cattle vaccinated with Brucella abortus strain 19.

Cell-mediated immune responses in cattle naturally infected with strains of Brucella abortus and in cattle vaccinated with B abortus strain 19 during calfhood were studied by an in vitro lymphocyte-stimulation procedure. Lymphocytes were prepared from peripheral bovine blood by the Ficoll-diatrizoate technique, suspended in RPMI-1640 medium (1.5 X 10(6) lymphocytes/ml), cultured with B abortus-soluble antigen or phytohemagglutinin, and incubated for 6 days. Sixteen hours prior to termination of incubation, cultures were labeled with 1 muCi of [3H]thymidine (3HdT) and, after harvesting, assayed for 3HdT incorporation into DNA by liquid scintillation spectrometry. Lymphocytes from cattle with bacteriologically confirmed isolation of B abortus underwent a significantly higher lymphocyte stimulation with B abortus-soluble antigen than did cattle vaccinated with B abortus strain 19 during calfhood (P less than 0.005). Standard seroagglutination tests were conducted simultaneously with lymphocyte-stimulation tests, but there was no apparent correlation between levels of humoral antibodies and the cell-mediated immune responses as measured by in vitro specific lymphocyte stimulation.     

Analysis and in vivo assay of B. abortus agglutinating and non-agglutinating antibodies

Studies were made of physicochemical and immunochemical characteristics of Brucella abortus agglutinating and non-agglutinating antibodies in the sera of cattle repeatedly injected with living B. abortus (Strain 1119). Both agglutinating and non-agglutinating antibody were shown to be IgG1, and by immunodiffusion against rabbit anti-cattle gamma-globulin, agglutinating antibody gave a precipitation line of identity with that given by non-agglutinating antibody. Whilst agglutinating antibody increased clearance of antigen from the blood of passively protected mice, non-agglutinating antibody did not enhance clearance. Determination of the spleen infection index in mice pre-treated with agglutinating and non-agglutinating antibody showed that in animals passively immunized with non-agglutinating antibody the number of living (infecting) bacteria was approximately 4 times higher than in the case of agglutinating antibody. The possible potentiation of chronic B. abortus infection by non-agglutinating antibody is discussed.     

Cloning and kinetics of expression of Brucella abortus heat shock proteins by baculovirus recombinants

In an effort to develop genetically engineered Brucella abortus (BA) vaccines, the genes encoding heat shock proteins (HSPs) GroEL, GroES, and HtrA were cloned and expressed in the BAC-TO-BAC Baculovirus System, and the kinetics of protein expression were analyzed using various insect cell lines in suspension cultures, different cell densities in suspension cultures, multiplicities of infection and recombinant virus replication times. Trichoplusia ni cells expressed only BA HtrA, but Spodoptera frugiperda (Sf9) cells expressed all three recombinant proteins. The best GroEL expression was achieved by infecting 2x10(6) Sf9 cells/ml with an MOI 10 of recombinant virus and harvesting the cells after 96h of virus replication. GroES and HtrA were best expressed when infecting 2x10(6) Sf9 cells/ml with an MOI 1 of recombinant viruses and harvesting the cells after 120h of virus replications. Under these conditions BA recombinant HSPs were expressed as follows: GroEL at 16% of the total cellular proteins (105microg/ml concentration); GroES 2% (15.25microg/ml); and HtrA 8% (84.48microg/ml). This is the first report of cloning and expression of BA genes in the baculovirus system.     

Kinetics of in vitro bovine lymphocyte immunostimulation with a Brucella abortus antigen.

A Brucella abortus-soluble antigen was investigated, using in vitro assay of lymphocyte immunostimulation, to determine which concentration of this antigen and which period of incubation of the lymphocyte cultures would induce maximum specific lymphocyte immunostimulation as an additional method for further study of B abortus infection in cattle. Soluble antigen was prepared from autoclaved cells of B abortus strain 1119-3. Peripheral blood lymphocytes were obtained from cattle infected with B abortus and from healthy control cattle not infected with B abortus. The lymphocytes were prepared by the Ficoll-Hypaque density gradient technique, suspended in RPMI 1640 medium (1.5 X 10(6)/ml), cultured with several dilutions of soluble antigen, and incubated. Prior to termination of incubation, cultures were labeled with 1 muCi of [3H]thymidine and, after harvesting, assayed for [3H]thymidine incorporation in DNA by a liquid scintillation spectrometer. Maximum specific immunostimulation of lymphocytes from B abortus-infected cattle was induced in this assay system with 6 days' incubation and 22 microgram of protein/ml/1.5 X 10(6) lymphocytes, using protein content to express concentration of soluble antigen in this system.     

Enzyme-linked immunosorbent assay for detecting antibodies to Brucella abortus in bovine milk and serum

An enzyme-linked immunosorbent assay (ELISA) method was evaluated for the detection of antibodies to Brucella abortus in cows milk. Milk samples from seropositive or -negative cows were sed to determine the distribution of absorbance values to classify milk as ELISA positive or ELISA negative. Brucella abortus was isolated from milk samples from 10 (45%) of the 22 cows whose milk and serum were ELISA positive. The ELISA was evaluated and determined to be an appropriate method for detecting antibodies to B abortus in bovine milk.     

Immunogenicity of Brucella abortus salt-extractable proteins

The immunogenic properties of salt-extractable proteins and chromatographic fractions thereof from Brucella abortus were evaluated in lemmings (Dicrostonyx rubricatus). The efficacy of the Brucella proteins as immunogens was determined after challenge with virulent B. abortus strain 2308 and was based on protection against clinical signs and gross lesions of brucellosis, as well as on numbers of viable Brucella in the spleen. Vaccination of lemmings with as little as 0.1 microgram of salt-extractable proteins (CSP) suppressed splenic infection, resulting in reduced numbers of viable organisms per spleen of 5-6 logs compared to non-vaccinated controls. Protein fractions separated by column chromatography were generally effective in reducing splenic infection, and contained proteins with molecular weights of 30,000, 20,000 and 12,000. Vaccines containing chemically modified dodecanoyl-CSP offered no additional advantage over unmodified CSP vaccines.     

Enhanced efficacy of recombinant Brucella abortus RB51 vaccines against B. melitensis infection in mice

Brucella abortus strain RB51 is an attenuated rough strain, currently being used as the official live vaccine for bovine brucellosis in the USA and several other countries. In strain RB51, the wboA gene, encoding a glycosyltransferase required for the O-side chain synthesis, is disrupted by an IS711 element. Recently, we have demonstrated that strain RB51WboA, RB51 complemented with a functional wboA gene, remains rough but expresses low quantities of O-side chain in the cytoplasm. Mice vaccinated with strain RB51WboA develop greatly enhanced resistance against challenge with B. abortus virulent strain 2308. We have also demonstrated that overexpression of Cu/Zn superoxide dismutase (SOD) in strain RB51 (RB51SOD) significantly increases its vaccine efficacy against strain 2308 challenge. In this study, we constructed a new recombinant strain, RB51SOD/WboA, that over expresses SOD with simultaneous expression of O-side chain in the cytoplasm. We tested the vaccine potential of strains RB51SOD, RB51WboA, RB51SOD/WboA against challenge with virulent Brucella melitensis 16M and B. abortus 2308 in mice. In comparison with strain RB51, strain RB51SOD induced better protection against strain 2308, but not strain 16M, challenge. Similar to strain RB51WboA, vaccination with strain RB51SOD/WboA resulted in complete protection of the mice from infection with strain 2308. When challenged with strain 16M, mice vaccinated with either strain RB51WboA or strain RB51SOD/WboA were significantly better protected than those vaccinated with strain RB51 or RB51SOD. These results suggest that strains RB51WboA and RB51SOD/WboA are good vaccine candidates for inducing enhanced protection against B. melitensis infection.     

The use of immunochemically purified anti-brucella antibody in a direct fluorescent antibody test for Brucella abortus

Antibodies specific for Brucella abortus were purified from the serum of hyperimmunized sheep using immunochemical procedures. They were conjugated to fluorescein isothiocyanate and used in a fluorescent antibody (FA) test for B. abortus. The conjugate did not stain any heterologous bacterial or fungal species tested and background fluorescence associated with its use on smears and sections of abortion materials was particularly low. Of 239 cases of abortion examined fluorescent microscopy demonstrated B. abortus in all 12 cases in which the organism was isolated. A few areas of fluorescence typical of B. abortus were also seen in 3 cases from which the organism was not cultured. B. abortus was demonstrated in lymph nodes from 6 of 36 Brucella reactor cows by culture and 7 by the FA test. However, only very low numbers of B. abortus were isolated or seen and sampling errors would have been significant. Use of the FA test allows diagnosis of Brucellosis to be made in 2 hours, compared to 6 days by the usual cultural procedures.     

Immunogenic proteins of Brucella abortus to minimize cross reactions in brucellosis diagnosis

To overcome the limitations of serological diagnosis, including false positive reactions caused by other pathogens, specific antigens for diagnosis of brucellosis other than LPS have been required. The present study was conducted to separate and identify immuno-dominant insoluble proteins of Brucella abortus against the antisera of cattle infected with B. abortus, or/and Yersinia enterocolitica, or the sera of non-infected cattle. After separating insoluble proteins of B. abortus by two dimensional electrophoresis (2-DE), their immuno-reactivity was determined by western blotting. A portion of the immunogenic spots against the positive antisera of B. abortus that have the potential for use as specific antigens were identified by MS/MS analysis. Overall, 18 immunogenic insoluble proteins of B. abortus 1119-3 showed immuno-reactivity against only the positive antisera of B. abortus, but failed to have immunogenicity toward both the positive sera of Y. enterocolitica and the negative sera of B. abortus. Identification of these proteins revealed the following: F0F1 ATP synthase subunit β, solute-binding family 5 protein, 28 kDa OMP, Leu/Ile/Val-binding family protein, Histidinol dehyddrogenase, Hypothetical protein, Twin-arginine translocation pathway signal sequence domain-containing protein, Dihydroorotase, Serine protease family protein, β-hydroxyacyl-(acyl-carrier-protein) dehydratase FabA, Short-chain dehydrogenase-/reductase carbonic anhydrase, Orinithine carbamoyltransferase, Leucyl aminopeptidase, Cold shock DNA-binding domain-containing protein, Cu/Zn superoxide dismutase, and Methionine aminopeptidase. The 18 immunogenic proteins separated in the present study can be considered candidate antigens to minimize cross reaction in the diagnosis of brucellosis and useful sources for Brucella vaccine development.     

A serological comparison of complement fixation reactions using Brucella abortus and B. melitensis antigens in B. abortus infected cattle

Brucella abortus and B. melitensis antigens were used in parallel on the National Standard Brucella abortus antiserum and on field sera coming from cattle where practically exclusively B. abortus biotypes 1 and 2 have been isolated over the last 11 years. With the National Standard serum the titres to B. melitensis were consistently lower than those to B. abortus antigen. Most were 1 dilution (twofold) lower. Although a similar trend was seen with the field sera, there were 7/346 sera which had twofold or higher titres to B. melitensis antigen. Although this may be due to the vagaries of the test it also warrants closer investigation of the animals concerned to see whether M-antigen predominant Brucella biotypes are possibly present. The use of the dual antigens could identify herds which are infected only with A-antigen predominant brucellae but would not be reliable for classifying individual animals.