Diphtheria is a fulminant bacterial disease caused by toxigenic strains of Corynebacterium diphtheriae whose local and systemic manifestations are due to the action of the diphtheria toxin (DT). The vaccine which is used to prevent diphtheria worldwide is a toxoid obtained by detoxifying DT. Although associated with high efficacy in the prevention of disease, the current anti-diphtheria vaccine, one of the components of DTP (diphtheria, tetanus and pertussis triple vaccine), may present post vaccination effects such as toxicity and reactogenicity resulting from the presence of contaminants in the vaccine that originated during the process of production and/or detoxification. Therefore, strategies to develop a less toxic and at the same time economically viable vaccine alternatives are needed to improve existing vaccines in use worldwide. In this study, the Moreau substrain of BCG which is used in Brazil as a live vaccine against human tuberculosis was genetically modified to carry and express the gene encoding for the diphtheria toxin fragment B (DTB). As such, the DNA sequence encoding the dtb gene was cloned into the pUS977 shuttle vector for cytoplasmic expression and successfully introduced into BCG cells by electroporation. Mice immunized with recombinant BCG expressing DTB showed seroconversion with the detection of specific antibodies against DTB. Also, rBCGs stably expressing DTB persisted up to 60 days in the absence of selective pressure in mice and cell viability did not change significantly during the period tested. Finally, immune sera from BALB/c mice vaccinated with rBCGpUS977 dtb PW8 were preliminarily tested for their capacity of neutralizing the diphtheria toxin in the Vero Cells assay.
KeywordsRecombinant BCGDiphtheria Toxin <i>dtb</i>GenePark Williams 8 (PW8)<i>Corynebacterium diphtheriae<
Mattos-Guaraldi, A.L., Formiga, L.C.D., Camello, T.C.F., Pereira, G.A., Hirata Jr., R. and Halpern, M. (2001) Corynebacterium diphtheriae Threats in Cancer Patients. Revista Argentina de Microbiologia, 33, 96-100.
Hirata Jr., R., Pereira, G.A., Filardy, A.A., Gomes, D.L.R., Damasco, P.V., Rosa, A.C. P., Nagao, P.E., Pimenta, F.P. and Mattos-Guaraldi, A.L. (2008) Potential Pathogenic Role of Aggregative Adhering Corynebacterium diphtheriae of Different clonal Groups in Endocarditis. Brazilian Journal of Medical and Biological Research, 41, 986-991. https://doi.org/10.1590/S0100-879X2008001100007
Kimura, Y., Watanabe, Y., Suga, N., Suzuki, N., Maeda, K., Suzuki, K., Kitagawa, W., Miura, N., Morita, H. and Imai, H. (2011) Acute Peritonitis Due to Corynebacterium ulcerans in a Patient Receiving Continuous Ambulatory Peritoneal Dialysis: A Case Report and Literature Review. Clinical and Experimental Nephrology, 15, 171-174. https://doi.org/10.1007/s10157-010-0346-4
Pappenheimer Jr., A.M. (1993) The Story of a Toxic Protein, 1888-1992. Protein Science, 2, 292-298. https://doi.org/10.1002/pro.5560020218
Wang, J. and London, E. (2009) The Membrane Topography of the Diphtheria Toxin T Domain Linked to the Chain Reveals a Transient Transmembrane Hairpin and Potential Translocation Mechanisms. Biochemistry, 48, 10446-10456. https://doi.org/10.1021/bi9014665
Man, P., Montagner, C., Vitrac, H., Kavan, D., Pichard, S., Gillet, D., Forest, E. and Forge, V. (2010) Accessibility Changes within Diphtheria Toxin T Domain When in the Functional Molten Globule State, as Determined Using Hydrogen-Deuterium Exchange Measurements. FEBS Journal, 277, 653-662. https://doi.org/10.1111/j.1742-4658.2009.07511.x
Ladokhin, A.S. (2013). Ph-Triggered Conformational Switching along the Membrane Insertion Pathway of the Diphtheria Toxin T-Domain. Toxins (Basel), 5, 1362-1380. https://doi.org/10.3390/toxins5081362
Lee, C.W., Halperin, S.A., Morris, A. and Lee, S.F. (2005) Expression of Diphtheria Toxin in Streptococcus mutans and Induction of Toxin Neutralizing Antisera. Canadian Journal of Microbiology, 51, 841-846. https://doi.org/10.1139/w05-078
Chellapandi, P., Sakthishree, S. and Bharathi, M. (2013) Phylogenetic Approach for Inferring the Origin and Functional Evolution of Bacterial ADP-Ribosylation Superfamily. Protein and Peptide Letters, 20, 1054-1065. https://doi.org/10.2174/0929866511320090012
Bloom, B.R. and Fine, P.E.M. (1994) The BCG Experience: Implications for Future Vaccines Against Tuberculosis. In: Bloom, B.R., Ed., Tuberculosis: Pathogenesis, Protection, and Control, ASM Press, Washington DC, 531-558. https://doi.org/10.1128/9781555818357.ch31
/i>
rDTB<
sub>
PW8<
/sub>
pUS977 Vector
Benévolo-de-Andrade, T.C., Monteiro-Maia, R., Cosgrove, C. and Castello-Branco, L.R. (2005) BCG Moreau Rio de Janeiro An Oral Vaccine against Tuberculosis Review. Memórias do Instituto Oswaldo Cruz, 100, 459-465. https://doi.org/10.1590/S0074-02762005000500002
Winter, N., Lagranderie, M., Gangloff, S., Leclerc, C., Gheorghiu, M. and Gicquel, B. (1995) Recombinant BCG Strains Expressing the SIVmac251 nef Gene Induce Proliferative and CTL Responses against nef Synthetic Peptides in Mice. Vaccine, 13, 471-478.
Chapman, R., Shephard, E., Stutz, H., Douglass, N., Sambandamurthy, V., Garcia, I., Ryffel, B., Jacobs, W. and Williamson, A.L. (2012) Priming with a Recombinant Pantothenate Auxotroph of Mycobacterium bovis BCG and Boosting with MVA Elicit HIV-1 Gag Specific CD8+ T Cells. PLoS ONE, 7, e32769. https://doi.org/10.1371/journal.pone.0032769
Grode, L., Ganoza, C.A., Brohm, C., Weiner, J., Eisele, B. and Kaufmann, S.H.E. (2013) Safety and Immunogenicity of the Recombinant BCG Vaccine VPM1002 in a Phase 1 Open-Label Randomized Clinical Trial. Vaccine, 31, 1340-1348.
Ohara, N. and Yamada, T. (2001) Recombinant BCG Vaccines. Vaccine, 19, 4089-4098.
Matsuo, K. and Yasutomi, Y. (2011) Mycobacterium bovis Bacille Calmette-Guerin as a Vaccine Vector for Global Infectious Disease Control. Tuberculosis Research and Treatment, 2011, Article ID: 574591. https://doi.org/10.1155/2011/574591
Yasutomi, Y., Koenig, S., Haun, S.S., Stover, C.K., Jackson, R.K. and Conard, P. (1993) Immunization with Recombinant BCG-SIV Elicits SIV-Specific Cytotoxic T Lymphocytes in Rhesus Monkeys. The Journal of Immunology, 150, 3101-3107.
Langermann, S., Palaszynski, S.R., Burlein, J.E., Koenig, S., Hanson, M.S., Briles, D.E. and Stover, C.K. (1994) Protective Humoral Response against Pneumococcal Infection in Mice Elicited by Recombinant Bacilli Calmette-Guérin Vaccines Expressing Pneumococcal Surface Protein. The Journal of Experimental Medicine, 180, 2277-2286. https://doi.org/10.1084/jem.180.6.2277
Langermann, S., Palaszynski, S.R., Sadziene, A., Stover, C.K. and Koenig, S. (1994) Systemic and Mucosal Immunity Induced by BCG Vector Expressing an Outer-Surface Protein A of Borrelia burgdorferi. Nature, 372, 552-555. https://doi.org/10.1038/372552a0
Kaufmann, S.H.E. (2010) Foresight: Novel Tuberculosis Vaccination Strategies Based on Understanding the Immune Response. Journal of Internal Medicine, 267, 337-353. https://doi.org/10.1111/j.1365-2796.2010.02216.x
Da Cruz, F.W., McBride, A.J., Conceição, F.R., Dale, J.W., McFadden, J. and Dellagostin, O.A. (2001) Expression of the B-Cell and T-Cell Epitopes of the Rabies Virus Nucleoprotein in Mycobacterium bovis BCG and Induction of an Humoral Response in Mice. Vaccine, 20, 731-736.
Parish, T. and Stoker, N.G. (1998) Electroporation of Mycobacteria. Mycobacteria Protocols. Method in Molecular Biology, 120-144. https://doi.org/10.1385/0-89603-471-2:129
Baulard, A., Jourdan, C., Mercenier, A. and Locht, C. (1992) Rapid Mycobacterial Plasmid Analysis by Electroduction between Mycobacterium spp. and Escherichia coli. Nucleic Acids Research, 20, 4105. https://doi.org/10.1093/nar/20.15.4105
Varaldo, P.B., Leite, L.C.C., Dias, W.O., Miyaji, E.M., Torres, F.I.G., Gebara, V.C., Armóa, G.R.G., Campos, A.S., Matos, D.C.S., Winter, N., Gicquel, B., Vilar, M.M., McFadden, J., Almeida, M.S., Tendler, M. and McIntosh, D. (2004) Recombinant Mycobacterium bovis BCG Expressing the Sm14 Antigen of Schistosoma mansoni Protects Mice from Cercarial Challenge. Infection and Immunity, 72, 3336-3343. https://doi.org/10.1128/IAI.72.6.3336-3343.2004
World Health Organization (WHO) (1993) Laboratory Methods for the Testing for Potency of Diphtheria (D), Tetanus (T), Pertussis (P) and Combined. 1-105.
Miyamura, K., Nishio, S., Ito, A., Murata, R. and Kono, R. (1974) Micro Cell Culture Method for Determination of Diphtheria Toxin and Antitoxin Titres Used VERO Cells. I Studies on Factors Affecting the Toxin and Antitoxin Titration. Journal of Biological Standardization, 2, 189-201.
Miyaji, E.N., Mazzantini, R.P., Waldely, O.D., Nascimento, A.L.T.O. and Gupta, R.K. (2001) Induction of Neutralizing Antibodies against Diphtheria Toxin by Priming with Recombinant Mycobacterium bovis BCG Expressing CRM197, a Mutant Diphtheria Toxin. Infection and Immunity, 69, 869-874. https://doi.org/10.1128/IAI.69.2.869-874.2001
Jacobs, W.R., Tuckman, M. and Bloom, B.R. (1987) Introduction of Foreign DNA into Mycobacteria using a Shuttle Plasmid. Nature, 327, 532-535. https://doi.org/10.1038/327532a0
Medeiros, M.A., Delagostin, O.A., Armóa, G.R., Degrave, W.M., De Mendonça-Lima, L., Lopes, M.Q., Costa, J.F., Mcfadden, J. and McIntosh, D. (2002) Comparative Evaluation of Mycobacterium vaccae as a Surrogate Cloning Host for Use in the Study of Mycobacterial Genetics. Microbiology, 148, 1999-2009. https://doi.org/10.1099/00221287-148-7-1999
Medeiros, M.A., Armôa, G.R.G., McIntosh, D. and Delagostin, A.O. (2005) Diferential Humoral Immune Response Induced in Mice Immunized with Two Strains of Recombinant Mycobacterium bovis BCG Expressing the S1 Subunit of Bordetella pertussis Toxin. Cannadian Journal Microbiology, 51, 1-6. https://doi.org/10.1139/w05-095
Rezende, C.A.F., De Moraes, M.T.B., Matos, D.C.S., McIntosh, D. and Armôa, G.R.G. (2005) Humoral Response and Genetic Stability of Recombinant BCG Expressing Hepatitis B Surface Antigens. Journal of Virological Methods, 125, 1-9.
Michelon, A., Conceição, F. R., Binsfeld, P.C., da Cunha, C.W., Moreira, A.N., Argondizzo, A.P., McIntosh, D., Armôa, G.R., Campos, A.S., Faber, M., Mcfadden, J. and Dellagostin, O.A. (2006) Immunogenicity of Mycobacterium bovis BCG Expressing Anaplasma marginale MSP1 a Antigen. Vaccine, 11, 6332-6339.
Varaldo, P.B., Miyaji, E.N., Vilar, M.M., Campos, A.S.D., Dias, W.O., Armôa, G.R., Tendler, M., Leite, L.C. and McIntosh, D. (2006) Mycobacteria Codon Optimization of the Gene Encoding the Sm14 Antigen of Schistossoma mansoni in Recombinant mycobacterium bovis Bacille Calmette-Guerin Enhances Expression but not Protection against Cercarial Challenge in Mice. FEMS Immunology and Medical Microbiology, 48, 132-139. https://doi.org/10.1111/j.1574-695X.2006.00133.x
Santangelo, M.P., McIntosh, D., Bigi, F., Armôa, G.R., Campos, A.S.D., Ruybal, P., Dellagostin, O.A., McFadden, J., Mendum, T., Gicquel, B., Winter, N., Farber, M. and Cataldi, A. (2007) Mycobacterium bovis BCG as a Delivery System for the RAP-1 Antigen from Babesia bovis. Vaccine, 25, 1104-1113.
Nascimento, I.P., Dias, W.O., Quintilio, W., Christ, A.P., Moraes, J.F. and Vancetto M.D. (2008) Neonatal Immunization with a Single Dose of Recombinant BCG Expressing Subunit S1 from Pertussis Toxin Induces Complete Protection against Bordetella pertussis Intracerebral Challenge. Microbes and Infection, 10, 198-202.
Barbieri, J.T. and Collier, R.J. (1987) Expression of a Mutant, Full-Length Form of Diphtheria Toxin in Escherichia coli. Infection and Immunity, 55, 1647-1651.
Bishai, W.R., Miyanohara, A. and Murphy, J.R. (1987) Cloning and Expression in Escherichia coli of Three Fragments of Diphtheria Toxin Truncated within Fragment B. Journal of Bacteriology, 169, 1554-1563. https://doi.org/10.1128/jb.169.4.1554-1563.1987
Cabiaux, V., Phalipon, A., Wattiez, R., Falmagne, P., Ruysschaert, J.M. and Kaczorek, M. (1988) Expression of a Biologically Active Diphtheria Toxin Fragment B in Escherichia coli. Molecular Microbiology, 2, 339-346. https://doi.org/10.1111/j.1365-2958.1988.tb00037.x
Orr, N., Galen, J.E. and Levine, M.M. (1999) Expression and Immunogenicity of a Mutant Diphtheria Toxin Molecule, CRM197, and Its Fragments in Salmonella typhi Vaccine Strain CVD 908-htr A. Infection and Immunity, 67, 4290-4294.
Lee, C.W., Lee, S.F. and Halperin, S.A. (2004) Expression and Immunogenicity of a Recombinant Diphtheria Toxin Fragment A in Streptococcus gordonii. Applied and Environmental Microbiology Application, 70, 4569-4574. https://doi.org/10.1128/AEM.70.8.4569-4574.2004
Nascimento, D.V., Dellagostin, A.O., Hirata, J.R., Pereira, G.M.B., Mattos-Guaraldi, A.L. and Armôa, G.R.G. (2013) Plasmid Instability when the hsp60 Gene Promoter Is Used to Express the Protective Non-Toxic Fragment B of the Diphtheria Toxin in Recombinant BCG. American Journal of Molecular Biology, 3, 81-86. https://doi.org/10.4236/ajmb.2013.32011
Murray, A., Winter, N., Lagranderie, M., Hill, D.F., Rauzier, J., Timm, J., Leclerc, C., Moriarty, K.M., Gheorghiu, M. and Gicquel, B. (1992) Expression of Escherichia coli β-Galactosidase in Mycobacterium bovis BCG Using an Expression System from Mycobacterium Paratuberculosis which Induced Humoral and Cellular Immune Responses. Molecular Microbiology, 6, 3331-3342. https://doi.org/10.1111/j.1365-2958.1992.tb02201.x