| Background:Brucellosis is a highly contagious zoonosis caused by Brucellae. Brucellosis is harmful as it can cause sterility or miscarriage in pregnant animals, as well as human acute Malta fever. The infection easily becomes chronic allergic diseases such as the hepatosplenimegaly and enlargement lymph nodes. Some clinical cases have been reported in vertical transmission. More than500,000peoples are infected by Brucella each year in the world, which leads an economic loss nearly3billion dollars. China is a severely affected area for the incidences of more than1/100000in populations. Brucelosis is prevalent most in twenty-eight provinces, including the higher edemic areas such as Ningxia, Xinjiang, Zhejiang, Shanxi, Gansu, Liaoning, Hebe and Jiangsu. According to Chinese Center for Disease Control and Prevention (CDC), the numbers of new cases of Brucellosis are increasing from18,416to30,002between2005and2008in China. Now over40,000human cases (with10%increasing each year) were identified in2012by Chinese CDC, which already becomes one of the most serious issues in public health.Brucellae are gram-negative intracellular bacteria with capsule but no spores and flagellums, which cause allergic reaction and it mostly parasite in the full-time and non-professional phagocytes. About sixty species of wild animals can be infected by Brucellae and have serological reactions to Brucellae infection, in which thirty species of animals carry Brucellae. Currently Brucellae are classified into nine species, including B. melitensis, B. abortus, B. suis, B. neotomae, B. ovis, B. canis, B. microti, B. ceti and B. pinnipedialis. Human brucellosis is prevalent over China in a mixed way, which is mainly caused by B. melitensis, B. abortus and B. suis. Among those infections, B. melitensis is counted for80%, B. abortus for10%, and B. suis for less than1%, respectively. It could be concluded that B. melitensis is a major pathogen for humans throught contacting with infected sheep and goats, or consuming their products.The clinical symptoms of Brucellosis are complex and difficult to differentiate from other common diseases with abortion or fever. Doctors can not diagnose Brucellosis by just relying on the history exposed to livestock. The infection easily progresses to chronic infection. Furthermore, the lack of effective treatment means and the resistences induced by a long-term of use for abundant antibiotics make it hard to control the infection of Brucellae. Therefore, the vaccination and quanrantine are the most realistic mean to control animal brucellosis. The effecay of vaccination and the accuracy of diagnosis are crtical issues in Brucella infection control.Brucella vaccine must effectively induce Thl cellular immune response for producing IFN-γ, especially CTL mediated cytotoxic effect. The attanuated live vaccine M5-90is currently used for vaccination of sheep and goats for preventing B. melitensis infection, which was developed in China previously. However, there are little data available for the efficacy of vaccine evaluated by cellular immune responses. There are two weaknesses for M5-90vaccine. First, current diagnostic assays can not distinguish the infected from the vaccinated animals in quarantine inspection practice, which largely limits the application of M5-90vaccine in farms; the sceond, the vaccine still remains residual virulence causing the abortion of pregnant animals. Therefore, it is necessary to develop a low virulence vaccine with high protective immunogenicity and molecular labeled as well.Molecular modification of Brucella vaccine by genetic engineering is considered to be a good choice. In the previous studies, a mutant (CGV26) of B. melitensis Rev.1strain was constructed by deleting bp26gene and found it remained the protection against the challenges with B. melitensis or B. ovis in BALB/c mice. However, a similar mutant (M5△bp26) derived from M5-90did not provide sufficient immune protection for mice when challenged with B. melitensis. In addition, the researches demonstrated the Rev.1strain OMP31had a similar situation to CGV26. All data indicated that BP26and OMP31played an important role in protective immunity.BP26is a periplasmic protein in Brucella bacteria. It is highly conserved among Brucella species. BP26protein is an immunodominant antigen in infected cattle, sheep, goats and humans, and it can be used as a diagnostic antigen for detecting over86%infections.OMP31is a membrane protein with98%homology among different species of Brucelae, except for deletion in B. abortus. In addition, nine nucleotide substitutions were detected in the OMP31genes encoding substitutional amino acids between Brucella ovis and Brucella melitensis. Consequently, the two species of bacteria can be identified by monoclonal antibody to OMP31. Here we hypothesize that the BP26and OMP31proteins are genetically modified or/and molecularly labelled within the M5-90instead of knocking out the both proteins, the candidate vaccine might maintain its original protective immunity and gain an antigenic marker, constructing a diagnosis of Peptide-ELISA to solve the problem of identification the vaccinated from the wild-type Brucellα infected animals.Aims:The study is to measure the humoral and celluar immune response from M5-90vaccinated sheep, to reveal the epitopes of major proteins BP26and OMP31of B. melitensis, to evaluate the immune responses for associating with protective efficacy of vaccine, and finally to present a map of epitopes relating to MHC restriction. The data will provide a helpful information for genetic modification or molecular labeling of M5-90vaccine.Methods:Tube agglutination test (SAT) and Rose Bengal plate agglutination test (RBPT) were used to evaluate humoral responses to B. melitensis vaccine M5-90through monitoring the antibody titer changes in0,0.5,1,2,2.5,7.5months at befor or after vaccination of sheep. ELISpot was used to assess the cellular immune response through detecting the levels of T cells for secreting IFN-γ. Specific IFN-γ secretion of PBMCs from vaccinated sheep stimulated by fifty-six BP26and OMP31overlapping peptides was detected by ELISpot for screening of T cell epitopes.Recombinant OMP31(rOMP31) protein was produced for immunizing BALB/c mice. Hybridoma cells were obtained by fusing mouse spleen and myeloma cells to produce monoclonal antibodies (mAbs) specific to OMP31. Reactivity of mAbs was tested with a panel of OMP31peptides, rOMP31and native OMP31containing membrane protein extracts (NMP) or sonicated supernatant of B. melitensis M5-90in ELISA and Western-Blot. The epitopes recognized by mAbs were identified by overlapping peptides in Peptide-ELISA. Results:1. Evaluation of immune responses from vaccinated sheep. Chinese Merino (Cm) and Kazak (Ka) sheep were injected by vaccine M5-90. Humoral and cellular responses of vaccinated animals were detected. The vaccine M5-90strain could raise the antibody response in sheep. The top level of SAT antibody was reached in0.5month after the first vaccination and the quickly deceased without improment by second and third boosts. However, the antibody detected by ELISA was maintained constantly at high level. Similarly, M5-90induced the specific T cell response for IFN-y secretion by stimulation of BP26and OMP31peptides. Even though T cell response responded strongly to some dominant epitope peptides, while the response was generally weak, which might be related with activation and profilication of Treg cells in M5-90vaccinated sheep.2. Screening of T cell epitopes of BP26and OMP31. By ELISpot with overlapping peptides derived from BP26and OMP31, nineteen peptides were detected reactive for inducing specific secretion of IFN-y in PBMCs of vaccinated sheep. Among those peptides,10peptides were high frequent common or dominant epitopes including:BP26-06:42VTGEGMMTASPDMAIL57, BP26-08:60SVLRQAKTAREAMTAN75, BP26-11:87KKAGIEDRDLQTGGIN102, BP26-12:96LQTGGINIQPIYVYPD111, OMP31-09:69EQVSGSLDVTAGGFVG84, OMP31-14:114SISAGASGLEGKAETK129, OMP31-17:141GYTATERLMVYGTGGL156, OMP31-18:150VYGTGGLAYGKVKSAF165, OMP31-23:195INNNWTLKSEYLYTDL210, and OMP31-26:222FLESKVNFHTVRVGLN237. These dominant epitope peptides induced specific T cell response from most two species of vaccinated sheep.Five peptides were species specific epitopes including BP26-10:78AMTKVLDAMKKAGIED93, BP26-18:150LGVNQGGDLNLVNDNP174, BP26-21: 177VANAIAKAKTLADAAG192, BP26-22:186TLADAAGVGLGRVVEI201and OMP31-12:96NGVVLGAETDFQGSSV111. These T cell epitope peptides were reactive to one of species of animals.Four peptides were individuals specific epitopes including BP26-15:123GYSVSTSLTVRVRELA138, BP26-23:195LGRVVEISELSRPPMP210, OMP31-06:42GGYIGINAGYAGGKFK57and OMP31-21:177WSDKTKAGWTLGAGAE192, which stimulated secretion of IFN-γ from vaccinated individual sheep.3. Screening of B cell epitopes of OMP31. Recombination protein OMP31was produced for immunizing mice. Hybridoma cells were generated by fusing spleen cells with myeloma cells and antibody producing hybridomas were selected. Twenty-two monoclonal antibodies (mAbs) recognizing the linear, semi-conformational and conformational epitopes of OMP31were produced, which were typed as11IgG2a,5IgG1and6IgM. Five novel linear B cell epitopes were discovered by overlapping peptides, including OMP31-05:33APVDTFSWTGGYIGIN48,OMP31-11:87QAGYNWQLDNGVVLGA102, OMP31-20:168GDDASALHTWSDKTKA183, OMP31-21:177WSDKTKAGWTLGAGAE192, and OMP31-24:204EYLYTDLGKRNLVDVD219. OMP31-06and21were also T cell epitopes identified above.4. T and B cell epitope map for BP26and OMP31. Taken together with B cell and T cell epitopes for BP26and OMP31identified by sheep or mice in the laboratory, the epitope map was primarily outlined.Conclusion:1. The level of SAT antibody raised by M5-90was low and lasted shorter, while The level of ELISA antibody stayed in high and persistent.2. M5-90induced specific secretion of IFN-γ by T cells but generally low, which reached at top in one month after first vaccination, then quickly decreased in short term.3. Simultaneously M5-90induced activation and profilication of Treg cells, which inhibited protective cellular immune response and might be one of reasons for lower efficacy of the vaccine.4. Ten common or dominant T cell epitope peptides of BP26and OMP31, five species specific epitope peptides and two individual specific epitope peptides were detected.5. Five groups of MHC-Ⅰ (A to E) and6groups of MHC-Ⅱ DRB1-exon2(a to f) alleles were clustered from11vaccinated sheep according to the polymorphisms of full-length nucleic acid sequences, which were correlated with reactive T cell epitope pepetides.6. Twenty-two mAbs to OMP31were produced, in which50%were IgG2a and recognized for the linear, semi-conformational and conformational B cell epitopes.7. Five B cell linear epitopes of OMP31were revealed.8. T and B cell epitope map for BP26and OMP31was primarily outlined.9. Data obtained here provided helpful information for molecular modification of vaccine M5-90. |
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