Detection And Identification Of Periplasmic Protein BP26Epitopes Of Brucella Melitensis

Background:Brucellosis, also called Mediterranean fever, Malta fever, Undulant fever, is a highly contagious zoonosis caused by Brucellae. Brucella infections cause human arthritis, meningitis or endocarditis, which remains persistent and rarely cured. Brucellae cause orchitis and epididymitis in infected male animals and abortion, miscarriage, premature birth or stillbirth in pregnant animals, which is one of most important reason causing huge economic loss in animal husbandry. In recent years, the incidence and prevalence of Brucellosis is increasing in the world. More than500,000new cases of human brucellosis are reported, which may greatly underestimate according to the World Health Organization (WHO). While, over35,000human cases were identified in2010by Chinese Center for Disease Control and Prevention (CDC) in China.Brucellae are gram-negative intracellular bacteria with capluse but no spores and flagellums. Brucellae currently can be classified into nine species, including B. melitensis, B. abortus, B. suis, B. neotomae, B. ovis, B. canis, B. microti, B. ceti and B. pirmipedialis. In China brucellosis is prevalent in a mixed infection and mainly is caused by B. melitensis which responses for85%infections. An attenuated B. melitensis vaccine M5-90is currently used for vaccination of sheep and goats. However, there are two weaknesses in M5-90vaccine. One, 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; two, vaccine still remains residual virulence causing the abortion of pregnant animals. Therefore, it is necessary to develop a low virulence, high protective immunogenicity, molecular labeled vaccine.Modification of current vaccine by genetic engineering is one of better choices. In the previous studies, a mutant (CGV26) of B. melitensis Rev.1strain was constructed by deleting bp26gene and was found protective against the challenges with B.melitensis or B. ovis in BALB/c mice. However, a similar mutant (M5Abp26) derived from M5-90did not provide sufficient immune protection for mice when challenged with B. melitensis, which indicated that bp26played an important role in protective immunity of the vaccine.BP26, also called omp28or CP28, is 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. Excellent protective antigen and adjuvant activity were found with bp26that induced elevated anibody and cellular responses. In view of the potent role of bp26in vaccine M5-90, the deletion of this protein to create a M5-90mutant might not be an ideal approach for resolving the drawbacks of live vaccines. Here we hypothesize that by genetically modifying or/and molecularly labeling bp26within M5-90instead of knocking out the whole protein, the molecular modified candidate vaccine might maintain its original protective immunity and gain an antigenic marker allowing to differentiation of vaccinated from the wild-type Brucella infected animals.Aims:The study will extensively characterize the bp26epitopes of B. melitensis M5-90vaccine, which will be helpful for evaluation of bp26function within vaccine M5-90, or genetic modification of bp26for designing and developing a molecular labeling vaccine on the basis of M5-90.Materials and methods:Gene expression in prokaryotic cell E. coli was used to prepare recombinant BP26(rBP26) protein. As the antigen rBP26immunized BALB/c mice, and hybridoma cells by fusing mouse spleen and myeloma cells were used to produce monoclonal antibodies (mAbs) specific to BP26. Reactivity of mAbs were tested to a panel of BP26peptides, three truncated rBP26and native BP26containing membrane protein extracts (NMP) of B. melitensis M5-90in ELISA and Western-Blot. The epitopes recognized by mAbs were identified by overlapping peptides and mutated peptides in Peptide-ELISA. The reactivity of linear epitope peptides, rBP26and NMP was tested with sheep sera collected from immunized or infected sheep and goats by ELISAs, which was compared with Standard Tube Agglutination Test (SAT) and Rose Bengal Plate Test (RBPT). ELISPOT was used to measure the level of specific IFN-y secreted by peptides induced PBMCs from immunized sheep, which would initially screen T cell epitopes.Results:1. Recombinant BP26protein. The vectors expressing entire rBP26(1-250aa), truncated rBP26-1(29-250), rBP26-2(48-131aa) and rBP26-3(129-250aa) were constructed with plasmid pET-28a or pET-30a, and expressed in E. coli induced by IPTG. Soluble recombinant BP26proteins were purified and refolded in PBS from guanidine hydrochloride dissolved the inclusion bodies in sonicated bacteria. The purity of the rBP26was above80%by SDS-PAGE analysis.2. Monoclonal antibodies to BP26. BALB/c mice were immunized for three times by injection of100,50,25μg rBP26.The antibody titers were tested above1:102400. The best responded mice were sacrificed and the spleen was obtained for fusion with SP2/0myeloma cells. Reactive hybridomas were screened and cloned for4times. A total of29mAbs reactive to rBP26of B. melitensis were selected from screening of hybridomas by indirect-ELISA. Of29clones,18IgGl (k),1IgG2a (k),8IgG2b (k),1IgG3(k) and1IgA (k) were identified. Twenty-nine mAbs were tested reactivity to the native membrane protein extract (NMP) by Western-Blot and Dot-ELISA. The results showed that19mAbs reacted with the denatured native BP26of NMP in Western-Blot.16mAbs recognized non-denatured native BP26of NMP in Dot-ELISA, and2mAbs were only reactive with recombinant BP26. In addition,28of overlaping peptides were tested with29mAbs in Peptide-ELISA and11mAbs reacted with peptides. According to the nature of antigen, the epitopes of natural periplasmic protein bp26were classified into three groups of linear, semi-conformational and conformational epitopes, which were recognized by11,8and8mAbs, respectively.3. B cell linear epitopes. To map mAb’s epitope recognitions,29mAbs were tested for reactivity with a panel of28of16mer overlapping peptides with7mer overlap which were designated as PO1to P28spanning the250amino acids of the entire bp26periplasmic protein. Eleven mAbs reacted with three peptides P11, P12or P13in peptide-ELISA. Of11mAbs reacted with peptides, three groups were classified. Group1, mAbs3D7,3H5and4D9were reactive with P11(aa87-102); group2, mAbs2A4,2H9,3H6and5F12were reactive with P12(aa96-111); group3, mAbs1G1,5A5,5B12and7C6were reactive with both P12and P13(aa105-120). The amino acid sequences indicated that P11and P12spanned the different linear epitopes, while P13shared a partly linear epitope with P12. To clearly separate epitopes within P11and P12, one of16mer peptide was syntheszied and designated as P12’(aa102-117), as it’s only the first amino acid overlapped with the C-terminal amino acid of P11. To map the epitopes of BP26accurately, twelve9mer peptides with8mer overlap shorten from P11and P12’ were synthesized and designated as P1101-06and P1201-06. MAb3H5,2A4and5A5were selected from three groups for further identification. The results showed that the binding of mAb3H5to P11was inhibited by the9mer peptide P1104and P11itself. The core motif of amino acid residues within P11recognized by mAb3H5was93DRDLQTGGI101(position93to101within BP26). The bindings of mAbs2A4were competitively inhibited by three9mer peptides P1202, P1203, P1204, and P12’ itself. The bindings of mAbs5A5were competitively inhibited by five9mer peptides P1202, P1203,P1204, P1205, P1206and P12itself. P1203and P1204strongly inhibited mAb5A5binding to P12’, while P1202, P1205and P1206inhibited weakly. Comparing strongly reactive sequences of9mer peptides and P12’, the core motif of amino acid residues within PI2’was104QPIYVYPD111. For further confirming those two linear epitopes of BP26, two mutated peptides MutP11and MutP12’ were synthesized by substituting two amino acids D93N and D95N from P11or substituting amino acids Y107F and Y109F from P12’,respectively. The binding abilities of MutPll and MutP12’to11mAbs were compared with P11or P12’. The reactivity of MutP11and MutP12’to mAbs was mostly lost or clearly decreased, which indicated that the two mutated sites were critical to the epitopes93DRDLQTGGI101within P11or104QPIYVYPD111within P12’ for maintaining the mAb’s recognitions.4. Location of semi-conformational and conformational epitopes of B cell. To localize the epitopes of BP26recognized, three truncated proteins of rBP26-1(aa29-250), rBP26-2(aa48-131) and rBP26-3(aa129-250) were tested by ELISA for reacting with16semi-conformational and conformational mAbs. All of those mAbs reacted with the truncated rBP26-1protein,5reacted with rBP26-2,1reacted with rBP26-3and1reacted with both rBP26-2and rBP26-3.5. Reactivity of linear epitopes to sheep antibodies. To evaluate the potential capacity of those two novel linear epitopes for raising antibody response in sheep, 137sheep sera (117random epidemic area and20vaccinated) were tested with P11-KLH and P12’-KLH conjugates, rBP26and native BP26containing membrane protein extracts (NMP) of M5-90by ELISAs. Sheep antibody responses to epitope peptides, rBP26or NMP were analyzed in line with the results obtained by combination of Standard Tube Agglutination Test (SAT) or Rose Bengal Plate Test (RBPT), and their ROC curves were plotted. Two linear epitope peptide conjugates (P11-KLH and P12’-KLH) were reactive to65-70%sheep sera in peptide-ELISA consistent with the standard serological tests, which indicated that both linear epitopes were immunodominant in M5-90vaccine elicited antibodies.6. T cell epitopes. PHA (Phytohaemagglutinin) or ConA (Concanavalin A) was used as a positive control and no peptide and un-related peptide were used as negative controls, the level of IFN-y secreted by BP26peptides induced PBMCs of M5-90immunized sheep was measured by ELISPOT assay. The value above the mean+2SD from negative controls was considered as positive. Five peptides were preliminarily detected highly reactive to stimulate T cell responses, which were15TIMLVGAFSLPAFAQE30,42VTGEGMMTASPDMAIL57,69REAMTANN EAM TKVLD84,105PIYVYPDDKNNLKEPT120and195LGRVVEISELSRPPMP210, respectively.Conclusion:1. Twenty-seven mAbs to native BP26of attenuated B. melitensis M5-90were produced, which were classified as three statuses of linear, semi-conformational and conformational epitopes.2. Two novel linear epitopes93DRDLQTGGI101and104QPIYVYPD111were identified, which were immunodominant epitopes in B. melitensis or M5-90vaccine elicited antibodies.3. Five T cell epitopes of BP26were preliminarily detected as 15TIMLVGAFSLPAFAQE30,42VTGEGMMTASPDMAIL57,69REAMTANNEAMT KVLD84,105PIYVYPDDKNNLKEPT120and195LGRVVEISELSRPPMP210, respectively, which stimulated the higher specific response of IFN-y.4. The results were helpful for evaluation and genetic modification of bp26antigen for developing a molecular labeling vaccine on the basis of M5-90.