Tularemia is a severe, zoonotic disease caused by a gram-negative bacterium, Francisella tularensis We have previously shown that rabbits are a good model of human pneumonic tularemia when exposed to aerosols containing a virulent, type A strain, SCHU S4. We further demonstrated that the live vaccine strain (LVS), an attenuated type B strain, extended time to death when given by scarification. Oral or aerosol vaccination has been previously shown in humans to offer superior protection to parenteral vaccination against respiratory tularemia challenge. Both oral and aerosol vaccination with LVS were well tolerated in the rabbit with only minimal fever and no weight loss after inoculation. Plasma antibody titers against F. tularensis were higher in rabbits that were vaccinated by either oral or aerosol routes compared to scarification. Thirty days after vaccination, all rabbits were challenged with aerosolized SCHU S4. LVS given by scarification extended time to death compared to mock-vaccinated controls. One orally vaccinated rabbit did survive aerosol challenge, however, only aerosol vaccination extended time to death significantly compared to scarification. These results further demonstrate the utility of the rabbit model of pneumonic tularemia in replicating what has been reported in humans and macaques as well as demonstrating the utility of vaccination by oral and respiratory routes against an aerosol tularemia challenge.

Tularemia, also known as rabbit fever, is a severe zoonotic disease in humans caused by the gram-negative bacterium Francisella tularensis (Ft). While there have been a number of attempts to develop a vaccine for Ft, few candidates have advanced beyond experiments in inbred mice. We report here that a prime-boost strategy with aerosol delivery of recombinant live attenuated candidate Ft S4ΔaroD offers significant protection (83% survival) in an outbred animal model, New Zealand White rabbits, against aerosol challenge with 248 cfu (11 LD50) of virulent type A Ft SCHU S4. Surviving rabbits given two doses of the attenuated strains by aerosol did not exhibit substantial post-challenge fevers, changes in erythrocyte sedimentation rate or in complete blood counts. At a higher challenge dose (3,186 cfu; 139 LD50), protection was still good with 66% of S4ΔaroD-vaccinated rabbits surviving while 50% of S4ΔguaBA vaccinated rabbits also survived challenge. Pre-challenge plasma IgG titers against Ft SCHU S4 corresponded with survival time after challenge. Western blot analysis found that plasma antibody shifted from predominantly targeting Ft O-antigen after the prime vaccination to other antigens after the boost. These results demonstrate the superior protection conferred by a live attenuated derivative of virulent F. tularensis, particularly when given in an aerosol prime-boost regimen.

Francisella tularensis is a gram negative non-motile coccobacillus that is the causative agent of tularemia. There is concern that F. tularensis could be used as a biological weapon and development of vaccines is a high priority. Type A strains are highly virulent but the current live vaccine strain (LVS) is based on a type B strain and is only partially protective against aerosol challenge with SCHU S4, a type A strain. A series of live attenuated vaccine candidates were derived from the SCHU S4 strain containing specific deletion mutations in target genes. After initial studies in mice, these attenuated strains were evaluated in rabbits. Rabbits were vaccinated by scarification which induced a mild fever and eschar formation at the inoculation site but no other clinical signs of disease. Serum IgG responses could be detected in all vaccinated rabbits as early as day 7 and continued to increase through day 21 before declining slightly on day 28. CD4+ T cells but not CD8+ T cells proliferated when cultured in vitro with heat-killed SCHU S4. Three of the vaccine strains conferred partial protection against death (ranging from 27-40%) and prolonged time to death in animals that did succumb. Vaccination with LVS prolonged survival by 1-2 days but did not protect against death. Type A-derived vaccines are immunogenic and provide better protection than LVS in the rabbit model against a robust aerosol challenge. These candidates warrant further study and evaluation.