The potential use of B. anthracis
spores for biowarfare has led to the need for a safe and efficient vaccine to protect humans against inhalational anthrax. Current licensed anthrax vaccines, based on PA, have serious drawbacks: they confer protection for only a limited duration and require frequent booster injections to maintain sufficient immunity. Furthermore, such PA-based vaccines, acting on toxins, are less effective than live attenuated vaccines, suggesting that additional antigens may make a significant contribution to protective immunity, especially by containing septicemia. Coimmunization with PA and FIS has previously been shown to confer protection against experimental cutaneous anthrax, whereas immunization with either PA alone or FIS alone was not protective in mice and only partially protective in guinea pigs (10
). Here, we assessed the efficacy of the PA-FIS vaccine candidate against pulmonary anthrax in experimental rodent models of inhalational anthrax.
s.c. immunization with PA-FIS elicited 100% protection of guinea pigs against i.n. challenge with 50 LD50 of spores of the virulent strain 9602 of B. anthracis but failed to elicit any protection of mice against i.n. challenge with 9 LD50 of the same strain.
The difference in vaccine efficacy between mice and guinea pigs may be partly due to the γDPGA capsule being the primary virulence factor in mice (87
) but less important in guinea pigs (and also rabbits and nonhuman primates).
Indeed, i.n. challenge with the nontoxinogenic 9602P strain, a derivative of 9602 carrying a pagA
gene deletion, the virulence of which is due solely to its multiplication (10
), was as lethal as 9602 i.n. challenge in naive mice (Y. P. Gauthier, unpublished results). PA-FIS immunization similarly failed to protect mice against i.n. challenge with 9602P, indicating that septicemia was not prevented (Gauthier, unpublished). Other studies have demonstrated the dominance of the capsule component as a virulence factor in mice (22
): most mouse strains are very sensitive to infection by even low doses of toxin-producing encapsulated stains of B. anthracis
). However, susceptibility to the toxin component varies greatly between mouse strains, from resistant (e.g., CBA/J, BALB/cJ, and C57L/J mice) to susceptible (e.g., A/J and DBA/2J mice) to infection with the unencapsulated toxin-producing Sterne strain (86
). These observations demonstrate the diversity of the protective activity of PA-based vaccines in mice, varying from moderate in CBA/J mice to very weak in A/J mice (45
). Thus, vaccine efficacy may vary greatly depending on the mouse strain, the B. anthracis
challenge strain and the route of infection (30
). More generally, there is no direct correlation between anti-PA titers and protection in mice or hamsters (27
), whereas both the anti-PA IgG titer and toxin neutralization correlate with protection in rabbits (50
). Brossier et al. demonstrated that s.c. immunization with PA alone is unable to protect either Swiss mice or guinea pigs against s.c. lethal challenge with the encapsulated nontoxinogenic strain 9602P, whereas immunization with FIS alone elicits partial protection of mice (50% survival) and total protection (100% survival) of guinea pigs against this strain (10
). These findings and our results highlight the large differences between mice and guinea pigs with regard to their ability to raise protective immunity against B. anthracis
. Nevertheless, we observed that s.c. immunization with PA-FIS partially protected mice against i.n. challenge with B. anthracis
strain 17JB, in contrast to its inefficacy against challenge with strain 9602. Brossier et al. showed that s.c. immunization with PA-FIS elicits total protection of Swiss mice against s.c. challenge with 17JB, whereas mice immunized with either PA or FIS alone were only partially protected (10
In an effort to improve the protective immune response against pulmonary anthrax, we immunized mice and guinea pigs with PA-FIS via the i.n. route to stimulate a mucosal immune response. Administration of vaccines via the nasal route can, unlike parenteral immunization, efficiently induce both mucosal and systemic antibody responses against infectious diseases. This is a consequence of the large amounts of associated lymphoid tissue and antigen-presenting cells in the nasal mucosa, which is also the site of pathogen entry (56
Mice immunized with PA-FIS through the i.n. route (i.n.×2), but not those immunized s.c., survived significantly longer after i.n. challenge with strain 9602 than did both mice immunized with PA alone and control mice. This suggests potentially synergistic protective activity of antispore and anti-PA immune responses against anthrax spore infection at mucosal surfaces.
We measured anti-PA and antispore antibody titers elicited by PA-FIS in lung mucosal secretions (BAL fluids) from both mice and guinea pigs. As expected, total anti-PA and antispore IgG values for mouse BAL fluid were significantly higher in i.n.-immunized mice than in s.c.-immunized animals. Also, TNA titers were significantly greater in i.n.×3-immunized animals than in s.c.×2-immunized mice.
Surprisingly, in contrast to mice, both anti-PA and TNA titers were significantly higher in BAL from s.c.-immunized guinea pigs than in BAL from i.n.-immunized animals.
Nasal immunization of mice with either rPA or purified PA coadministered with either CT (8
) or other adjuvants (7
) yields high levels of both plasma and mucosal anti-PA, as well as neutralizing anti-PA. Anti-PA and antispore responses in lung mucosal secretions of mice and guinea pigs immunized either i.n. or s.c. have not previously been compared. Oral vaccination (mucosal immunization) of guinea pigs with a nontoxinogenic nonencapsulated B. anthracis
live spore vaccine expressing rPA has been reported to elicit very high specific humoral and mucosal responses and generate long-lasting protective immunity (2
). Nevertheless, the proportion of guinea pigs that develop measurable anti-PA antibodies and protective immunity is lower after per os immunization than after s.c. immunization. Furthermore, although s.c. vaccination with effective spore doses elicits protection of all immunized guinea pigs against a high lethal challenge, oral vaccination is consistently only partially protective (2
). A clinical study showed that i.n. administration of live attenuated measles vaccine to humans elicited immune responses that were very much lower than those generated by s.c. administration (69
); this observation is in agreement with our observations.
To sum up, i.n. immunization of mice elicited both a better mucosal immune response to PA-FIS and better protection against inhalational anthrax than could be obtained with s.c. immunization. In contrast, s.c. immunization was more effective than nasal immunization in guinea pigs as assessed by either the specific antibody response or protection. These observations show that the induction of an efficient mucosal response is dependent on the animal species tested and suggest that it may be beneficial to combine the two routes of immunization for a species-independent optimized protective immunization protocol.
Combining both the s.c. and the i.n. routes of immunization, the optimized (s.c.+i.n.)×3 PA-FIS immunization schedule elicited a significantly higher anti-PA and antispore mucosal response in mice than either s.c.×2 or i.n.×3 immunization. No such difference was observed in mouse sera. However, this optimized immunization did not elicit a better mucosal response than s.c.×2 immunization in guinea pigs. Unlike the findings for mice, anti-PA, antispore, and TNA titers were significantly higher in the BAL fluids and sera from (s.c.+i.n.)×3-immunized guinea pigs than in i.n.×3-immunized animals. These various observations demonstrate the differences between mice and guinea pigs regarding the mucosal immune response to PA-FIS after mucosal and s.c. immunization.
Nevertheless, despite the differences between mice and guinea pigs regarding the comparative efficacy of i.n. and s.c. immunization, the optimized (s.c.+i.n.)×3 immunization with PA-FIS elicited 100% protection in both species against experimental inhalational anthrax with B. anthracis
spores of strains 17JB and 9602, respectively. Flick-Smith et al. reported that, despite differences in rPA-specific antibody titers, A/J mice vaccinated with rPA by a combined intramuscular (i.m.-i.n.) schedule were more consistently protected against both injected and aerosol lethal challenges with B. anthracis
STI spores than after either i.m. or i.n. immunization alone (29
). These results and data emphasize the importance of stimulating both the mucosal and systemic immune systems to elicit full protection against inhalational anthrax, because the respiratory tract epithelium is the initial site of infection after inhalation of spores.
Despite the combined s.c.-i.n. schedule, alhydrogel-adjuvanted PA alone failed to elicit protection of guinea pigs against i.n. or aerosol challenge with B. anthracis 9602.
PA-based vaccines are more effective in guinea pigs than in mice (7
). However, vaccination of guinea pigs with PA alone generally confers only limited protection against aerosol or i.n. lethal challenge with B. anthracis
). Furthermore, guinea pigs are commonly described as being difficult to protect consistently with alum-adjuvanted PA (26
). Nevertheless, in our study guinea pigs immunized with the alum-containing PA-FIS vaccine were fully protected against aerosol and i.n. lethal challenge with 75 and 100 LD50
, respectively, of the highly virulent B. anthracis
strain 9602. Protection was elicited by combined s.c. plus i.n. immunization, as well as by s.c. immunization alone. It has been reported that immunization with FIS alone elicits total protection of guinea pigs against s.c. infection with the PA-deficient strain 9602P (10
), indicating that the sensitivity of guinea pigs to anthrax disease is not solely due to toxemia and that antibodies to PA are not sufficient to confer protection in this species. This suggests that antibodies specific to other B. anthracis
antigens (spore or others), stimulation of cell-mediated immunity, or both are needed for protection.
Marcus et al. demonstrated that primary immunization with threshold levels of PA could induce a long-term T-cell immunological memory response in guinea pigs without inducing detectable anti-PA antibodies. However, protection is attained only after boosting, concomitant with the detection of neutralizing antibodies in the circulation (52
). Other authors have confirmed that, despite the absence of correlation between protective immunity and anti-PA titers determined by ELISA in the guinea pig model, neutralizing antibodies to PA are a major component of the protective immunity of guinea pig against anthrax (63
). Nevertheless, vaccination of guinea pigs with PA combined with Ribi adjuvant (MPL-TDM-CWS) is more effective against both aerosol (53
) and parenteral (i.m.) (45
) challenge with the Ames strain than immunization with either PA adsorbed onto Al(OH)3
) or the licensed human anthrax vaccine (45
), thereby suggesting the possible role of cell-mediated immunity in protection against anthrax (53
). Similar observations have been reported for mice (85
In conclusion, as demonstrated for mice (36
), although the production of antibody is necessary, a cell-mediated immune response may be required for protection of guinea pigs against anthrax infection. The notion that spore antigen(s) exerts a protective role through eliciting a cell-mediated immune response in guinea pig is strengthened by recent reports that mouse immunization with FIS induces a protective cellular immune response by gamma interferon-producing CD4 T lymphocytes, whereas humoral immunity is not protective (36
In summary, our study clearly shows that the addition of killed spores to PA antigen greatly enhances the protective efficacy of the PA-based vaccine and elicits total protection against inhalational anthrax. Our findings emphasize the importance of stimulating both the mucosal and systemic immune systems to elicit full protection against inhalational anthrax: the best protection of mice against inhalational anthrax was elicited with a combined s.c.-i.n. immunization schedule and increased protection of mice correlated with higher antispore, anti-PA, and TNA titers in lung mucosal secretions than those elicited by s.c. immunization alone. Nevertheless, no such clear correlation was observed in guinea pigs, and s.c. immunization alone is strongly protective in this model. These findings and other reported observations demonstrate that there is no absolute correlation between specific antibody titers and protection and implicate cell-mediated immunity in protection against inhalational anthrax.