Intranasal vaccination induces protective immunity against intranasal infection with virulent Francisella tularensis biovar A.

Tularemia is caused by infection with the small gram-negative coccobacillus Francisella tularensis. Pneumonic tularemia is the most serious form of tularemia and is caused by inhalation of either of the two pathogenic F. tularensis subspecies, F. tularensis subsp. tularensis (biovar A) or F. tularensis subsp. holarctica (biovar B) (8). The incidence of naturally occurring pneumonic tularemia is relatively low and is generally associated with activities such as farming and gardening that cause F. tularensis to be aerosolized and dispersed. However, the potential that F. tularensis may be prepared as a bioweapon and intentionally released as an aerosol to cause high mortality rates in the general public has led the Centers for Disease Control and Prevention of the U.S. government to list it as a category A bioterrorism agent. F. tularensis subsp. tularensis is the more virulent of the two pathogenic F. tularensis subspecies and has been estimated to cause >90% of the detected tularemia cases in North America. It is thought that the inhalation of fewer than 25 CFU of biovar A bacteria causes fulminant disease in humans (31, 34). The disease appears abruptly 3 to 5 days after exposure and can rapidly progress to severe pneumonia, respiratory failure, and death (32). Antibiotic treatments are highly effective but must be initiated early in disease development. However, diagnosis and therapeutic intervention are often delayed because of the variable clinical features of pneumonic tularemia. Preventative vaccination appears to be the best approach against pneumonic tularemia. However, there is no vaccine currently available that has been shown to completely protect humans from this disease. There is also very little information from vaccine studies of animal models to guide future vaccine development. The few vaccine studies to date have examined only the protective effects of subcutaneous (s.c.) vaccination with the highly attenuated live vaccine strain (LVS). A small study with 18 human volunteers showed that vaccination by scarification with LVS does not provide complete protection against aerosol challenge with the biovar A strain SCHU S4, and two of the vaccinated volunteers developed symptoms as severe as those in volunteers without vaccination (31). This result suggests that vaccination by scarification will likely have an unacceptably high rate of failure to protect against respiratory infections by virulent F. tularensis. The success rate may be improved by delivering the vaccine directly into the respiratory tract, like vaccines against other respiratory pathogens such as influenza virus (28) and Mycobacterium tuberculosis (2, 22). In fact, aerogenic LVS vaccination protected guinea pigs and monkeys better than s.c. LVS vaccination against respiratory infection by the biovar A strain SCHU S5 (11). However, these studies were limited in size and detail because of the animal models used, and therefore, additional studies will be required to establish the effectiveness of vaccination by the respiratory route. Mouse models are ideal for evaluating the protective effects generated by respiratory vaccination and for quickly dissecting the pulmonary immune response mediating protection. Inbred mouse strains such as BALB/c and C57BL/6 mice frequently respond differently to respiratory infections (18, 20, 24, 26, 35) and can be used to determine the mechanisms and genetic bases of resistance and susceptibility. Mouse models are also more useful for detailed analyses of the pulmonary immune responses to respiratory infections than other animal models because of the availability of transgenic and knockout mouse strains as well as analytical tools. However, mice are highly susceptible to intranasal (i.n.) (14) and aerogenic (16) LVS infections, with estimated 50% lethal dose (LD50) values of 1 × 102 and 1.5 × 103 CFU, respectively, and no murine model has been developed thus far to evaluate the protective effects of vaccination through these routes. Most studies have instead vaccinated mice intradermally (i.d.) or s.c. Two of these studies specifically examined protection against an aerosol challenge with biovar A strains and obtained conflicting results: Hodge et al. found that s.c. vaccination protected mice against SCHU S5 for at least 30 days (16), whereas Chen et al. reported that i.d. vaccination failed to protect mice against biovar A strain 33 (5). These inconsistencies highlight the need to further investigate not only the protective effects generated by s.c. or i.d. vaccination but also those generated by respiratory vaccination. A more extensive understanding of pulmonary immunity against F. tularensis biovar A will greatly improve new vaccine development. The purpose of the present study was to determine whether i.n. LVS vaccination induces effective protection in mice against an i.n. challenge with F. tularensis biovar A strains. We report that i.n. vaccination generated T-cell-dependent immunity in BALB/c mice but not in C57BL/6 mice which provided substantial resistance to i.n. and s.c. challenges with an F. tularensis biovar A strain.