Francisella tularensis is considered a potential bioterrorism agent due to its low infectious dose, high mortality rate, and ability to be spread via the aerosol route. We characterized the F. tularensis subspecies novicida mutant strain FTN0109 as a potential vaccine candidate against tularemia. This strain, which lacks an outer membrane lipoprotein, is attenuated in vitro and in vivo, as it exhibits reduced replication within murine J774 macrophages and has a pulmonary LD50 in BALB/c and C57BL/6 mice of >105 CFU (compared to WT parental strain U112, LD50 < 10 CFU). Intranasal immunization induced strong cellular responses (IFN-γ and IL-2 in splenocyte recall assays) as well as strong humoral responses. Vaccination with FTN0109 also conferred complete protection in BALB/c mice against subsequent pulmonary challenge with 10 LD50 (60,000 CFU) of the murine virulent Francisella strain LVS. We also have demonstrated partial protection (50%) against the highly human virulent subspecies tularensis strain SCHU S4 (25 LD50, 12,500 CFU) following intratracheal vaccination in the Fischer 344 rat, a second rodent model for tularemia. Overall, our results suggest that FTN0109 serves as a potential putative vaccine candidate against pulmonary tularemia.
References
[1]
Dennis, D.T., Inglesby, T.V., Henderson, D.A., Bartlett, J.G., Ascher, M.S., Eitzen, E., Fine, A.D., Friedlander, A.M., Hauer, J., Layton, M., Lillibridge, S.R., McDade, J.E., Osterholm, M.T., O’Toole, T., Parker, G., Perl, T.M., Russell, P.K. and Tonat, K., Working Group on Civilian Biodefense (2001) Tularemia as a Biological Weapon: Medical and Public Health Management. JAMA, 285, 2763-2773. http://dx.doi.org/10.1001/jama.285.21.2763
[2]
Saslaw, S., Eigelsbach, H.T., Prior, J.A., Wilson, H.E. and Carhart, S. (1961) Tularemia Vaccine Study. II. Respiratory Challenge. Archives of Internal Medicine, 107, 702-714. http://dx.doi.org/10.1001/archinte.1961.03620050068007
[3]
Schmerk, C.L., Duplantis, B.N., Wang, D., Burke, R.D., Chou, A.Y., Elkins, K.L., Ludu, J.S. and Nano, F.E. (2009) Characterization of the Pathogenicity Island Protein PdpA and Its Role in the Virulence of Francisella novicida. Microbiology, 155, 1489-1497. http://dx.doi.org/10.1099/mic.0.025379-0
[4]
Salomonsson, E., Kuoppa, K., Forslund, A.L., Zingmark, C., Golovliov, I., Sjostedt, A., Noppa, L. and Forsberg, A. (2009) Reintroduction of Two Deleted Virulence Loci Restores Full Virulence to the Live Vaccine Strain of Francisella tularensis. Infection and Immunity, 77, 3424-3431. http://dx.doi.org/10.1128/IAI.00196-09
[5]
Saslaw, S. and Carhart, S. (1961) Studies with Tularemia Vaccines in Volunteers. III. Serologic Aspects Following Intracutaneous or Respiratory Challenge in Both Vaccinated and Nonvaccinated Volunteers. American Journal of the Medical Sciences, 241, 689-699. http://dx.doi.org/10.1097/00000441-196106000-00001
[6]
Saslaw, S. and Carlisle, H.N. (1961) Studies with Tularemia Vaccines in Volunteers. IV. Brucella aggiutinins in Vaccinated and Nonvaccinated Volunteers Challenged with Pasteurella tularensis. American Journal of the Medical Sciences, 242, 166-172. http://dx.doi.org/10.1097/00000441-196108000-00004
[7]
Saslaw, S., Eigelsbach, H.T., Wilson, H.E., Prior, J.A. and Carhart, S. (1961) Tularemia Vaccine Study. I. Intracutaneous Challenge. Archives of Internal Medicine, 107, 689-701.
http://dx.doi.org/10.1001/archinte.1961.03620050055006
[8]
Signarovitz, A.L., Ray, H.J., Yu, J.J., Guentzel, M.N., Chambers, J.P., Klose, K.E. and Arulanandam, B.P. (2012) Mucosal Immunization with Live Attenuated Francisella novicida U112DiglB Protects against Pulmonary F. tularensis SCHU S4 in the Fischer 344 Rat Model. PLoS ONE, 7, e47639. http://dx.doi.org/10.1371/journal.pone.0047639
[9]
Ray, H.J., Chu, P., Wu, T.H., Lyons, C.R., Murthy, A.K., Guentzel, M.N., Klose, K.E. and Arulanandam, B.P. (2010) The Fischer 344 Rat Reflects Human Susceptibility to Francisella Pulmonary Challenge and Provides a New Platform for Virulence and Protection Studies. PLoS ONE, 5, e9952. http://dx.doi.org/10.1371/journal.pone.0009952
[10]
Chu, P., Cunningham, A.L., Yu, J.J., Nguyen, J.Q., Barker, J.R., Lyons, C.R., Wilder, J., Valderas, M., Sherwood, R.L., Arulanandam, B.P. and Klose, K.E. (2014) Live Attenuated Francisella novicida Vaccine Protects against Francisella tularensis Pulmonary Challenge in Rats and Non-Human Primates. PLoS Pathogens, 10, e1004439.
http://dx.doi.org/10.1371/journal.ppat.1004439
[11]
Balonova, L., Mann, B.F., Cerveny, L., Alley Jr., W.R., Chovancova, E., Forslund, A.L., Salomonsson, E.N., Forsberg, A., Damborsky, J., Novotny, M.V., Hernychova, L. and Stulik, J. (2012) Characterization of Protein Glycosylation in Francisella tularensis subsp. holarctica: Identification of a Novel Glycosylated Lipoprotein Required for Virulence. Molecular and Cellular Proteomics, 11, M111.015016.
[12]
Su, J., Yang, J., Zhao, D., Kawula, T.H., Banas, J.A. and Zhang, J.R. (2007) Genome-Wide Identification of Francisella tularensis Virulence Determinants. Infection and Immunity, 75, 3089-3101.
http://dx.doi.org/10.1128/IAI.01865-06
[13]
Wehrly, T.D., Chong, A., Virtaneva, K., Sturdevant, D.E., Child, R., Edwards, J.A., Brouwer, D., Nair, V., Fischer, E.R., Wicke, L., Curda, A.J., Kupko III, J.J., Martens, C., Crane, D.D., Bosio, C.M., Porcella, S.F. and Celli, J. (2009) Intracellular Biology and Virulence Determinants of Francisella tularensis Revealed by Transcriptional Profiling Inside Macrophages. Cellular Microbiology, 11, 1128-1150. http://dx.doi.org/10.1111/j.1462-5822.2009.01316.x
[14]
Rockx-Brouwer, D., Chong, A., Wehrly, T.D., Child, R., Crane, D.D., Celli, J. and Bosio, C.M. (2012) Low Dose Vaccination with Attenuated Francisella tularensis Strain SchuS4 Mutants Protects against Tularemia Independent of the Route of Vaccination. PLoS ONE, 7, e37752. http://dx.doi.org/10.1371/journal.pone.0037752
[15]
Gallagher, L.A., Ramage, E., Jacobs, M.A., Kaul, R., Brittnacher, M. and Manoil, C. (2007) A Comprehensive Transposon Mutant Library of Francisella novicida, a Bioweapon Surrogate. Proceedings of the National Academy of Sciences of the United States of America, 104, 1009-1014. http://dx.doi.org/10.1073/pnas.0606713104
[16]
Cong, Y., Yu, J.J., Guentzel, M.N., Berton, M.T., Seshu, J., Klose, K.E. and Arulanandam, B.P. (2009) Vaccination with a Defined Francisella tularensis subsp. novicida Pathogenicity Island Mutant (ΔiglB) Induces Protective Immunity against Homotypic and Heterotypic Challenge. Vaccine, 27, 5554-5561.
http://dx.doi.org/10.1016/j.vaccine.2009.07.034
[17]
Powell, H.J., Cong, Y., Yu, J.J., Guentzel, M.N., Berton, M.T., Klose, K.E., Murthy, A.K. and Arulanandam, B.P. (2008) CD4+ T Cells Are Required during Priming but Not the Effector Phase of Antibody-Mediated IFN-γ-Dependent Protective Immunity against Pulmonary Francisella novicida Infection. Immunology and Cell Biology, 86, 515-522.
http://dx.doi.org/10.1038/icb.2008.31
[18]
Sjostedt, A., Eriksson, M., Sandstrom, G. and Tarnvik, A. (1992) Various Membrane Proteins of Francisella tularensis Induce Interferon-Gamma Production in both CD4+ and CD8+ T Cells of Primed Humans. Immunology, 76, 584-592.
[19]
Ray, H.J., Cong, Y., Murthy, A.K., Selby, D.M., Klose, K.E., Barker, J.R., Guentzel, M.N. and Arulanandam, B.P. (2009) Oral Live Vaccine Strain-Induced Protective Immunity against Pulmonary Francisella tularensis Challenge Is Mediated by CD4+ T Cells and Antibodies, Including Immunoglobulin A. Clinical and Vaccine Immunology, 16, 444- 452. http://dx.doi.org/10.1128/CVI.00405-08
[20]
Surcel, H.M., Syrjala, H., Karttunen, R., Tapaninaho, S. and Herva, E. (1991) Development of Francisella tularensis Antigen Responses Measured as T-lymphocyte Proliferation and Cytokine Production (Tumor Necrosis Factor Alpha, Gamma Interferon, and Interleukin-2 and -4) during Human Tularemia. Infection and Immunity, 59, 1948-1953.
[21]
Balagopal, A., MacFarlane, A.S., Mohapatra, N., Soni, S., Gunn, J.S. and Schlesinger, L.S. (2006) Characterization of the Receptor-Ligand Pathways Important for Entry and Survival of Francisella tularensis in Human Macrophages. Infection and Immunity, 74, 5114-5125. http://dx.doi.org/10.1128/IAI.00795-06
[22]
Barel, M., Hovanessian, A.G., Meibom, K., Briand, J.P., Dupuis, M. and Charbit, A. (2008) A Novel Receptor—Ligand Pathway for Entry of Francisella tularensis in Monocyte-Like THP-1 Cells: Interaction between Surface Nucleolin and Bacterial Elongation Factor Tu. BMC Microbiology, 8, 145. http://dx.doi.org/10.1186/1471-2180-8-145
[23]
Schulert, G.S. and Allen, L.A. (2006) Differential Infection of Mononuclear Phagocytes by Francisella tularensis: Role of the Macrophage Mannose Receptor. Journal of Leukocyte Biology, 80, 563-571.
http://dx.doi.org/10.1189/jlb.0306219
[24]
Nallaparaju, K.C., Yu, J.J., Rodriguez, S.A., Zogaj, X., Manam, S., Guentzel, M.N., Seshu, J., Murthy, A.K., Chambers, J.P., Klose, K.E. and Arulanandam, B.P. (2011) Evasion of IFN-γ Signaling by Francisella novicida Is Dependent upon Francisella Outer Membrane Protein C. PLoS ONE, 6, e18201.
http://dx.doi.org/10.1371/journal.pone.0018201
[25]
Bitsaktsis, C., Rawool, D.B., Li, Y., Kurkure, N.V., Iglesias, B. and Gosselin, E.J. (2009) Differential Requirements for Protection against Mucosal Challenge with Francisella tularensis in the Presence versus Absence of Cholera Toxin B and Inactivated F. tularensis. Journal of Immunology, 182, 4899-4909. http://dx.doi.org/10.4049/jimmunol.0803242
[26]
Collazo, C.M., Meierovics, A.I., De Pascalis, R., Wu, T.H., Lyons, C.R. and Elkins, K.L. (2009) T Cells from Lungs and Livers of Francisella tularensis-Immune Mice Control the Growth of Intracellular Bacteria. Infection and Immunity, 77, 2010-2021. http://dx.doi.org/10.1128/IAI.01322-08
[27]
Sanapala, S., Yu, J.J., Murthy, A.K., Li, W., Guentzel, M.N., Chambers, J.P., Klose, K.E. and Arulanandam, B.P. (2012) Perforin-and Granzyme-Mediated Cytotoxic Effector Functions Are Essential for Protection against Francisella tularensis Following Vaccination by the Defined F. tularensis subsp. Novicida ΔfopC Vaccine Strain. Infection and Immunity, 80, 2177-2185. http://dx.doi.org/10.1128/IAI.00036-12
[28]
Cowley, S.C., Meierovics, A.I., Frelinger, J.A., Iwakura, Y. and Elkins, K.L. (2010) Lung CD4-CD8- Double-Negative T Cells Are Prominent Producers of IL-17A and IFN-γ during Primary Respiratory Murine Infection with Francisella tularensis Live Vaccine Strain. Journal of Immunology, 184, 5791-5801.
http://dx.doi.org/10.4049/jimmunol.1000362
[29]
Elkins, K.L., Cowley, S.C. and Bosio, C.M. (2007) Innate and Adaptive Immunity to Francisella. Annals of the New York Academy of Sciences, 1105, 284-324. http://dx.doi.org/10.1196/annals.1409.014
[30]
Rohmer, L., Fong, C., Abmayr, S., Wasnick, M., Larson Freeman, T.J., Radey, M., Guina, T., Svensson, K., Hayden, H.S., Jacobs, M., Gallagher, L.A., Manoil, C., Ernst, R.K., Drees, B., Buckley, D., Haugen, E., Bovee, D., Zhou, Y., Chang, J., Levy, R., Lim, R., Gillett, W., Guenthener, D., Kang, A., Shaffer, S.A., Taylor, G., Chen, J., Gallis, B., D’Argenio, D.A., Forsman, M., Olson, M.V., Goodlett, D.R., Kaul, R., Miller, S.I. and Brittnacher, M.J. (2007) Comparison of Francisella tularensis Genomes Reveals Evolutionary Events Associated with the Emergence of Human Pathogenic Strains. Genome Biology, 8, R102. http://dx.doi.org/10.1186/gb-2007-8-6-r102
[31]
Larsson, P., Elfsmark, D., Svensson, K., Wikstrom, P., Forsman, M., Brettin, T., Keim, P. and Johansson, A. (2009) Molecular Evolutionary Consequences of Niche Restriction in Francisella tularensis, a Facultative Intracellular Pathogen. PLoS Pathogens, 5, e1000472. http://dx.doi.org/10.1371/journal.ppat.1000472
