Abstract
Francisella tularensis is a facultative intracellular pathogen, and is the causative agent of a fatal human disease known as tularemia. F. tularensis is classified as a Category A Biothreat agent by the CDC based on its use in bioweapon programs by several countries in the past and its potential to be used as an agent of bioterrorism. No licensed vaccine is currently available for prevention of tularemia. In this study, we used a novel approach for development of a multivalent subunit vaccine against tularemia by using an efficient tobacco mosaic virus (TMV) based delivery platform. The multivalent subunit vaccine was formulated to contain a combination of F. tularensis protective antigens: OmpA-like protein (OmpA), chaperone protein DnaK and lipoprotein Tul4 from the highly virulent F. tularensis SchuS4 strain. Two different vaccine formulations and immunization schedules were used. The immunized mice were challenged with lethal (10xLD100) doses of F. tularensis LVS on day 28 of the primary immunization and observed daily for morbidity and mortality. Results from this study demonstrate that TMV can be used as a carrier for effective delivery of multiple F. tularensis antigens. TMV-conjugate vaccine formulations are safe and multiple doses can be administered without causing any adverse reactions in immunized mice. Immunization with TMV-conjugated F. tularensis proteins induced a strong humoral immune response and protected mice against respiratory challenges with very high doses of F. tularensis LVS. This study provides a proof-of-concept that TMV can serve as a suitable platform for simultaneous delivery of multiple protective antigens of F. tularensis. Refinement of vaccine formulations coupled with TMV-targeting strategies developed in this study will provide a platform for development of an effective tularemia subunit vaccine as well as a vaccination approach that may broadly be applicable to many other bacterial pathogens.
Highlights
Francisella tularensis is the causative agent of a fatal human disease known as tularemia [1,2]
F. tularensis SchuS4 dnaK (FTT1269c), ompA (FTT0831c), and tul4 (FTT0901) genes were expressed in E. coli as N-terminal 6X-His tagged proteins and purified by metal affinity chromatography
Modified tobacco mosaic virus (TMV) which has a surface exposed Lysine was conjugated with purified DnaK, OmpA-like protein (OmpA) and Tul4 proteins of F. tularensis SchuS4 collectively in a single reaction for generation of TMV-monoconjugate vaccine (Fig 4A) or individually in multiple conjugation reactions to generate TMV-multiconjugate vaccine (Fig 4B, 4C and 4D)
Summary
Francisella tularensis is the causative agent of a fatal human disease known as tularemia [1,2]. The bioweapon potential of F. tularensis is on account of its extreme virulence, low infectious dose, ease of aerosol dissemination and capacity to cause severe illness and death in a very short period of time [7]. Considering the bioweapon potential of F. tularensis and repercussions of 2001 anthrax attack in the USA, there has been an increased interest in development of vaccine and effective countermeasures against bioterror agents. An ideal solution for prevention of tularemia occurring naturally or consequent to the use of Francisella as a bioweapon or an act of bioterrorism is to develop a safe and effective vaccine capable of inducing long lasting protection in a relatively short period of time [10]
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