In silico analysis of Omp25 and BLS Brucella melitensis antigens for designing subunit vaccine

Document Type : Original Articles

Authors

Department of Animal Science, Ferdowsi University of Mashhad, Mashhad, Iran

Abstract

Brucellosis is a well-known infection in domestic animals which caused by Brucella bacterium. Due to
serious economic and medical consequences of this disease, various efforts have been made to prevent the
infection through the use of recombinant vaccines based on Brucella outer membrane protein (OMP)
antigens. The objectives of the present study were cloning, sequencing and epitope prediction of Omp25
and BLS genes as two major Brucella melitensis antigens. The full-length open reading frame (ORF) of
Omp25 and BLS genes were amplified and cloned into pTZ57R/T vector. Phylogenetic analysis of
sequenced genes showed that both genes were nearly similar in different Brucella species. Several online
prediction softwares were used to predict B and T-cells epitopes, secondary and tertiary structures,
antigenicity ability and enzymatic degradation sites. Bioinformatic tools used in the current study were
confirmed by the results of three different experimental epitope predictions. Bioinformatic analysis
identified five and two B-cell and two and one T-cell epitopes for Omp25 and BLS antigens, respectively.
Finally, according to the antigenicity ability and proteosomal recognition site common B and T-cell epitope
was predicted for Omp25 (154-162 amino acids) and BLS (37-48 and 119-139 amino acids). Results of this
study might be useful for recombinant vaccine development.

Keywords

Main Subjects


Article Title [French]

Analyse in silico des antigènes Omp25 et BLS de Brucella melitensis pour l’élaboration d’un vaccin de sous-unité

Abstract [French]

La brucellose est une infection qui touche particulièrement les animaux domestiques et qui est causée par les bactéries du genre Brucella. En raison des conséquences économiques et médicales importantes de cette maladie, diverses efforts on été conduits dans l’élaboration de vaccins recombinants à partir de protéines antigéniques de la membrane externe (OMP). L’objectif de cette étude était le séquençage, le clonage et la prédiction des épitopes des gènes « Omp25 et BLS » codant les deux antigènes majeurs de Brucella melitensis. Le cadre de lecture ouvert (ORF) pleine longueur des gènes Omp25 et BLS a été amplifié et cloné dans le vecteur pTZ57R/T. L’analyse phylogénétique des séquences obtenues montrent que les deux gènes sont presque semblables dans les différentes espèces du genre Brucella. Plusieurs logiciels prévisionnels ont été utilisés pour prédire l’emplacement des épitopes des cellules B et T, les structures secondaires et tertiaires, le potentiel antigénique et les sites de clivage enzymatique des antigènes d’intérêt. Les résultats de nos tests bioinformatiques ont été confirmés par 3 méthodes différentes de prédiction expérimentale des épitopes. Ces analyses ont révélé que le nombre d’épitopes de cellules B était de 5 pour Omp25 et 2 pour BLS, alors que seulement 2 et 1 épitopes de cellules T ont été respectivement identifiés pour ces deux antigènes. Enfin, l’étude du potentiel allergénique et du protéasome des antigènes, nous a permis de prédire la présence d’épitopes de cellules B et T communs aux niveaux des acides aminés numéros 154 à 162 pour Omp25 et 37 à 48 ainsi que 119 à 139 pour BLS. Les résultats de ce travail pourraient être utiles dans l’élaboration future d’un vaccin recombinant.

Keywords [French]

  • Brucella melitensis
  • Omp25
  • BLS
  • Analyse bioinformatique
  • Vaccin recombinant
Berzofsky, J.A., 1985. Intrinsic and extrinsic factors in protein antigenic structure. Science 229, 932-940.
Buus, S., Lauemoller, S.L., Worning, P., Kesmir, C., Frimurer, T., Corbet, S., Fomsgaard, A., Hilden, J., Holm, A., Brunak, S., 2003. Sensitive quantitative predictions of peptide-MHC binding by a 'Query by Committee' artificial neural network approach. Tissue Antigens 62, 378-384.
Cassataro, J., Estein, S.M., Pasquevich, K.A., Velikovsky, C.A., de la Barrera, S., Bowden, R., Fossati, C.A., Giambartolomei, G.H., 2005. Vaccination with the recombinant Brucella outer membrane protein 31 or a derived 27-amino-acid synthetic peptide elicits a CD4+ T helper 1 response that protects against Brucella melitensis infection. Infect Immun 73, 8079-8088.
Chen, P., Rayner, S., Hu, K.H., 2011. Advances of bioinformatics tools applied in virus epitopes prediction. Virol Sin 26, 1-7.
Cloeckaert, A., Verger, J.M., Grayon, M., Zygmunt, M.S., Grepinet, O., 1996. Nucleotide sequence and expression of the gene encoding the major 25-kilodalton outer membrane protein of Brucella ovis: Evidence for antigenic shift, compared with other Brucella species, due to a deletion in the gene. Infect Immun 64, 2047-2055.
Cutler, S.J., Whatmore, A.M., Commander, N.J., 2005. Brucellosis--new aspects of an old disease. J Appl Microbiol 98, 1270-1281.
Delpino, M.V., Estein, S.M., Fossati, C.A., Baldi, P.C., Cassataro, J., 2007. Vaccination with Brucella recombinant DnaK and SurA proteins induces protection against Brucella abortus infection in BALB/c mice. Vaccine 25, 6721-6729.
Donnes, P., Elofsson, A., 2002. Prediction of MHC class I binding peptides, using SVMHC. BMC Bioinformatics 3, 25.
Edmonds, M.D., Cloeckaert, A., Elzer, P.H., 2002. Brucella species lacking the major outer membrane protein Omp25 are attenuated in mice and protect against Brucella melitensis and Brucella ovis. Vet Microbiol 88, 205-221.
Geourjon, C., Deleage, G., 1995. SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci 11, 681-684.
Gu, J., Bourne, P.E., 2009. Structural Bioinformatics, Wiley.
Gupta, V.K., Vohra, J., Kumari, R., Gururaj, K., Vihan, V.S., 2012. Identification of Brucella isolated from goats using PstI sitepolymorphism at Omp2 gene loci. Indian J Anim Sci 82, 240–243.
Hopp, T.P., Woods, K.R., 1981. Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci U S A 78, 3824-3828.
Karthik, K., Rathore, R., Verma, A.K., Tiwari, R., Dhama, K., 2013. Brucellosis – still it stings? Livestock Technol 2, 8-10.
Li, Y., Liu, X., Zhu, Y., Zhou, X., Cao, C., Hu, X., Ma, H., Wen, H., Ma, X., Ding, J.B., 2013. Bioinformatic prediction of epitopes in the Emy162 antigen of. Exp Ther Med 6, 335-340.
Noguchi, H., Kato, R., Hanai, T., Matsubara, Y., Honda, H., Brusic, V., Kobayashi, T., 2002. Hidden Markov model-based prediction of antigenic peptides that interact with MHC class II molecules. J Biosci Bioeng 94, 264-270.
Pappas, G., Papadimitriou, P., Christou, L., Akritidis, N., 2006. Future trends in human brucellosis treatment. Expert Opin Investig Drugs 15, 1141-1149.
Rajagunalan, S., Kumari, G., Gupta, S.K., Kumar, A., Agarwal, R.K., Rawool, D.B., Singh, D.K., 2013. Molecular characterization of Omp31 gene of Indian field Isolates of Brucella melitensis. Indian  J Anim Sci 83 673–677.
Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Simon, G.G., Hu, Y., Khan, A.M., Zhou, J., Salmon, J., Chikhlikar, P.R., Jung, K.O., Marques, E.T., August, J.T., 2010. Dendritic cell mediated delivery of plasmid DNA encoding LAMP/HIV-1 Gag fusion immunogen enhances T cell epitope responses in HLA DR4 transgenic mice. PLoS One 5, e8574.
Steere, A.C., Drouin, E.E., Glickstein, L.J., 2011. Relationship between immunity to Borrelia burgdorferi outer-surface protein A (OspA) and Lyme arthritis. Clin Infect Dis 52 Suppl 3, s259-265.
Toes, R.E., Nussbaum, A.K., Degermann, S., Schirle, M., Emmerich, N.P., Kraft, M., Laplace, C., Zwinderman, A., Dick, T.P., Muller, J., Schonfisch, B., Schmid, C., Fehling, H.J., Stevanovic, S., Rammensee, H.G., Schild, H., 2001. Discrete cleavage motifs of constitutive and immunoproteasomes revealed by quantitative analysis of cleavage products. J Exp Med 194, 1-12.
Wass, M.N., Sternberg, M.J., 2009. Prediction of ligand binding sites using homologous structures and conservation at CASP8. Proteins 77 Suppl 9, 147-151.
Zhang, W., Liu, J., Zhao, M., Li, Q., 2012 . Predicting linear B-cell epitopes by using sequence-derived structural and physicochemical features. Int J Data Min Bioinform 6, 557-569.