A Francisella tularensis Live Vaccine Strain (LVS) Mutant with a Deletion in capB , Encoding a Putative Capsular Biosynthesis Protein, Is Significantly More Attenuated than LVS yet Induces Potent Protective Immunity in Mice against F. tularensis Challenge

Abstract

Francisella tularensis, the causative agent of tularemia, is in the top category (category A) of potential agents of bioterrorism. The F. tularensis live vaccine strain (LVS) is the only vaccine currently available to protect against tularemia; however, this unlicensed vaccine is relatively toxic and provides incomplete protection against aerosolized F. tularensis, the most dangerous mode of transmission. Hence, a safer and more potent vaccine is needed. As a first step toward addressing this need, we have constructed and characterized an attenuated version of LVS, LVS ⌬capB, both as a safer vaccine and as a vector for the expression of recombinant F. tularensis proteins. LVS ⌬capB, with a targeted deletion in a putative capsule synthesis gene (capB), is antibiotic resistance marker free. LVS ⌬capB retains the immunoprotective O antigen, is serum resistant, and is outgrown by parental LVS in human macrophage-like THP-1 cells in a competition assay. LVS ⌬capB is significantly attenuated in mice; the 50% lethal dose (LD 50 ) intranasally (i.n.) is >10,000-fold that of LVS. Providing CapB in trans to LVS ⌬capB partially restores its virulence in mice. Mice immunized with LVS ⌬capB i.n. or intradermally (i.d.) developed humoral and cellular immune responses comparable to those of mice immunized with LVS, and when challenged 4 or 8 weeks later with a lethal dose of LVS i.n., they were 100% protected from illness and death and had significantly lower levels (3 to 5 logs) of LVS in the lung, liver, and spleen than sham-immunized mice. Most importantly, mice immunized with LVS ⌬capB i.n. or i.d. and then challenged 6 weeks later by aerosol with 10؋ the LD 50 of the highly virulent type A F. tularensis strain SchuS4 were significantly protected (100% survival after i.n. immunization). These results show that LVS ⌬capB is significantly safer than LVS and yet provides potent protective immunity against virulent F. tularensis SchuS4 challenge.

Members of the Francisella tularensis Phagosomal Transporter Subfamily of Major Facilitator Superfamily Transporters Are Critical for Pathogenesis

“…We also observed that vaccination doses as low as 200 to 500 CFU were completely protective against a challenge dose of 5.1 ϫ 10 3 CFU. This is important to note, since many of the strains previously reported to be attenuated and protective either were not challenged with such a high dose, required a higher vaccination dose, or required multiple vaccinations to achieve the same level of protection (15,16,18,20,26,30,32,36). There has been one other LVS mutant reported to provide full protection against an LVS challenge of 10 5 CFU with a vaccination dose of only 10 3 CFU (37).…”

Subversion of Host Recognition and Defense Systems by Francisella spp

“…However, to date, no poly-D-glutamic acid has been identified in Francisella. Although Francisella capB mutants are attenuated during murine infection, an LVS capB mutant did not exhibit increased sensitivity to serum (110,213). Furthermore, it is not known whether CapB is required for production of the O-antigen capsule, the glycoprotein capsule-like complex, or a different bacterial envelope structure.…”

“…Interestingly, the Francisella genome is known to contain an operon with similarity to the Bacillus species capBCA genes necessary for the production of a poly-D-glutamic acid capsule (71,110,153). However, to date, no poly-D-glutamic acid has been identified in Francisella.…”

“…infection of host cells in culture, while "in vivo" refers to infection of mammalian hosts such as mice) (110,130,153,232). This is important to note, since recent evidence has suggested that the O antigen and capsule of Francisella are structurally similar, with the capsule consisting of a polymer of O-antigen tetrasaccharide subunits (9) as well as a glycoprotein component termed a "capsulelike complex" (18).…”

Live Attenuated Mutants of Francisella tularensis Protect Rabbits against Aerosol Challenge with a Virulent Type A Strain

“…Recent efforts to develop a tularemia vaccine have focused on live attenuated vaccines using site-specific deletion of virulence genes (3,(19)(20)(21)(22)(23)(24)(25)(26)(27). Studies have identified a number of mutations that attenuate F. tularensis to various degrees.…”