Immunogenic Consensus Sequence T helper Epitopes for a Pan-Burkholderia Biodefense Vaccine

Background Biodefense vaccines against Category B bioterror agents Burkholderia pseudomallei (BPM) and Burkholderia mallei (BM) are needed, as they are both easily accessible to terrorists and have strong weaponization potential. Burkholderia cepaciae (BC), a related pathogen, causes chronic lung infections in cystic fibrosis patients. Since BPM, BM and BC are all intracellular bacteria, they are excellent targets for T cell-based vaccines. However, the sheer volume of available genomic data requires the aid of immunoinformatics for vaccine design. Using EpiMatrix, ClustiMer and EpiAssembler, a set of immunoinformatic vaccine design tools, we screened the 31 available Burkholderia genomes and performed initial tests of our selections that are candidates for an epitope-based multi-pathogen vaccine against Burkholderia species. Results Immunoinformatics analysis of 31 Burkholderia genomes yielded 350,004 9-mer candidate vaccine peptides of which 133,469 had perfect conservation across the 10 BM genomes, 175,722 had perfect conservation across the 11 BPM genomes and 40,813 had perfect conservation across the 10 BC genomes. Further screening with EpiMatrix yielded 54,010 high-scoring Class II epitopes; these were assembled into 2,880 longer highly conserved ‘immunogenic consensus sequence’ T helper epitopes. 100% of the peptides bound to at least one HLA class II allele in vitro, 92.7% bound to at least two alleles, 82.9% to three, and 75.6% of the binding results were consistent with the immunoinformatics analysis. Conclusions Our results show it is possible to rapidly identify promiscuous T helper epitopes conserved across multiple Burkholderia species and test their binding to HLA ligands in vitro. The next step in our process will be to test the epitopes ex vivo using peripheral leukocytes from BC, BPM infected humans and for immunogenicity in human HLA transgenic mice. We expect that this approach will lead to development of a licensable, pan-Burkholderia biodefense vaccine.

[1]  Anne S De Groot,et al.  Engineering immunogenic consensus T helper epitopes for a cross-clade HIV vaccine. , 2004, Methods.

[2]  D. Palmer,et al.  Melioidosis. Forgotten, but not gone! , 1991, Archives of internal medicine.

[3]  D. Weiner,et al.  Developing an epitope-driven tuberculosis (TB) vaccine. , 2005, Vaccine.

[4]  A. D. De Groot,et al.  An interactive Web site providing major histocompatibility ligand predictions: application to HIV research. , 1997, AIDS research and human retroviruses.

[5]  M. Urbanowski,et al.  A secreted regulatory protein couples transcription to the secretory activity of the Pseudomonas aeruginosa type III secretion system. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[6]  G. Dougan,et al.  A Mutant of Burkholderia pseudomallei, Auxotrophic in the Branched Chain Amino Acid Biosynthetic Pathway, Is Attenuated and Protective in a Murine Model of Melioidosis , 2002, Infection and Immunity.

[7]  Julie A McMurry,et al.  In silico-accelerated identification of conserved and immunogenic variola/vaccinia T-cell epitopes. , 2009, Vaccine.

[8]  Anne S De Groot,et al.  Reducing risk, improving outcomes: bioengineering less immunogenic protein therapeutics. , 2009, Clinical immunology.

[9]  P. Vandamme,et al.  Burkholderia cepacia: medical, taxonomic and ecological issues. , 1996, Journal of medical microbiology.

[10]  S. Raghavan,et al.  Secreted transcription factor controls Mycobacterium tuberculosis virulence , 2008, Nature.

[11]  O. Hashim,et al.  Identification of immunogenic proteins from Burkholderia cepacia secretome using proteomic analysis. , 2010, Vaccine.

[12]  K. Hughes,et al.  Sensing structural intermediates in bacterial flagellar assembly by export of a negative regulator. , 1993, Science.

[13]  W. Wiersinga,et al.  MELIOIDOSIS , 1975, The Lancet.

[14]  N. White,et al.  HALVING OF MORTALITY OF SEVERE MELIOIDOSIS BY CEFTAZIDIME , 1989, The Lancet.

[15]  S. Peacock,et al.  Management of melioidosis , 2006, Expert review of anti-infective therapy.

[16]  A. D. De Groot,et al.  Epitope-based vaccination against pneumonic tularemia. , 2009, Vaccine.

[17]  A. Cross.  Development of an anti-endotoxin vaccine for sepsis. , 2010, Sub-cellular biochemistry.

[18]  J. Goldberg,et al.  Strategies toward vaccines against Burkholderia mallei and Burkholderia pseudomallei , 2008, Expert review of vaccines.

[19]  P. Sansonetti,et al.  A secreted anti‐activator, OspD1, and its chaperone, Spa15, are involved in the control of transcription by the type III secretion apparatus activity in Shigella flexneri , 2005, Molecular microbiology.

[20]  J. Lipuma.  Update on the Burkholderia cepacia complex , 2005, Current opinion in pulmonary medicine.

[21]  M. Ho,et al.  Structural characterization of the lipopolysaccharide O antigens of Burkholderia pseudomallei , 1995, Infection and immunity.

[22]  R. Strugnell,et al.  CONSTRUCTION AND CHARACTERIZATION OF AN UNMARKED AROC DELETION MUTANT OF BURKHOLDERIA PSEUDOMALLEI STRAIN A 2 , 2009 .

[23]  R. Ulrich,et al.  Aerogenic vaccination with a Burkholderia mallei auxotroph protects against aerosol-initiated glanders in mice. , 2005, Vaccine.

[24]  R. Doolittle,et al.  A simple method for displaying the hydropathic character of a protein. , 1982, Journal of molecular biology.

[25]  J. Mekalanos,et al.  ExsE, a secreted regulator of type III secretion genes in Pseudomonas aeruginosa , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[26]  A. Cheng,et al.  Melioidosis: Epidemiology, Pathophysiology, and Management , 2005, Clinical Microbiology Reviews.

[27]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[28]  H. Reijonen,et al.  Use of HLA class II tetramers in tracking antigen-specific T cells and mapping T-cell epitopes. , 2003, Methods.

[29]  M F del Guercio,et al.  Several common HLA-DR types share largely overlapping peptide binding repertoires. , 1998, Journal of immunology.

[30]  R. Strugnell,et al.  Construction and characterization of an unmarked aroC deletion mutant of Burkholderia pseudomallei strain A2. , 2009, The Southeast Asian journal of tropical medicine and public health.

[31]  R. Titball,et al.  Progress toward development of vaccines against melioidosis: A review. , 2010, Clinical therapeutics.

[32]  A. Haque,et al.  A live experimental vaccine against Burkholderia pseudomallei elicits CD4+ T cell-mediated immunity, priming T cells specific for 2 type III secretion system proteins. , 2006, The Journal of infectious diseases.

[33]  L. Sprague,et al.  Serodiagnosis of Burkholderia mallei infections in horses: state-of-the-art and perspectives. , 2005, Journal of veterinary medicine. B, Infectious diseases and veterinary public health.

[34]  D. Dance.  Melioidosis: the tip of the iceberg? , 1991, Clinical Microbiology Reviews.

[35]  A. Krogh,et al.  Prediction of lipoprotein signal peptides in Gram‐negative bacteria , 2003, Protein science : a publication of the Protein Society.

[36]  N. Ketheesan,et al.  Development of protective immunity in a murine model of melioidosis is influenced by the source of Burkholderia pseudomallei antigens , 2007, Immunology and cell biology.

[37]  A. Krogh,et al.  A combined transmembrane topology and signal peptide prediction method. , 2004, Journal of molecular biology.

[38]  D. DeShazer,et al.  Nonviable Burkholderia mallei Induces a Mixed Th1- and Th2-Like Cytokine Response in BALB/c Mice , 2002, Infection and Immunity.

[39]  K. Brown,et al.  The identification of surface proteins of Burkholderia pseudomallei. , 2007, Vaccine.

[40]  M. Valvano,et al.  Nasal Immunization with Burkholderia multivorans Outer Membrane Proteins and the Mucosal Adjuvant Adamantylamide Dipeptide Confers Efficient Protection against Experimental Lung Infections with B. multivorans and B. cenocepacia , 2007, Infection and Immunity.

[41]  Julie A McMurry,et al.  Diversity of Francisella tularensis Schu4 antigens recognized by T lymphocytes after natural infections in humans: identification of candidate epitopes for inclusion in a rationally designed tularemia vaccine. , 2007, Vaccine.

[42]  P. Dřevínek,et al.  Burkholderia cenocepacia in cystic fibrosis: epidemiology and molecular mechanisms of virulence. , 2010, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[43]  J. Lipuma,et al.  Induction of immune response to the 17 kDa OMPA Burkholderia cenocepacia polypeptide and protection against pulmonary infection in mice after nasal vaccination with an OMP nanoemulsion-based vaccine , 2010, Medical Microbiology and Immunology.