The development of safe and efficacious adjuvants is crucial to produce new mucosal vaccines to protect against inhalation anthrax. In general, antigens delivered via the mucosa are poorly immunogenic and are easily degraded by mucosal enzymes. This has led to concerns that this approach may induce a state of tolerance instead of protective immunity (18
). In this study, we present a novel mucosal adjuvant for anthrax vaccination that is both potent and free of toxicity. These stable and convenient preparations of NEs mixed with the rPA of B. anthracis
have induced significant mucosal and systemic immune responses. The NE adjuvant is effective over a broad range of antigen concentrations, and only a single application (in guinea pigs) or two vaccinations (in mice) are necessary to induce protective immune responses. Despite the nasal route of immunization, significant protection against either intradermal or intranasal spore challenge was achieved.
The guinea pig is a primary model for testing anthrax vaccine efficacy (19
). In our intranasal rPA-NE studies, complete protection was obtained against intradermal challenge with 1,000× LD50
of B. anthracis
spores 5 months after booster immunization. This suggests that a durable anti-PA IgG response with neutralizing antibody titers of greater than 102
provides protection from dermal anthrax exposure. In comparison, intramuscular immunization with the commercial anthrax vaccine resulted in overall survival rates of 53% to 63% in guinea pigs after intramuscular challenges with 10× to 1,000× LD50
of Ames strain spores, and IgG titers alone did not predict protection (19
). A high concentration (NC50
) of the LeTx-neutralizing antibodies present in the sera of mice and guinea pigs after rPA-NE immunization indicates that mixing with NE adjuvant did not denature essential protective epitopes in the antigen that are important for protection against infection with live B. anthracis
). Inhalational challenge of guinea pigs intranasally vaccinated with rPA-NE yielded 70% and 40% survival for 10× and 100× LD50
of Ames strain spores, respectively, while the neutralizing antibody titers were comparable (or higher) to these in fully protected intradermally challenged guinea pigs. These results were similar to those for other anthrax vaccines (21
) and suggest that anti-PA IgA, IgG, and neutralizing antibodies are not fully predictive for protection against inhalation anthrax. In addition, the intranasal challenge results indicate a complex mechanism of protection against inhalation infection with anthrax (50
). However, even when not providing complete survival, rPA-NE immunization significantly extended the TTD (approximately 3 to 5 days) after intranasal challenge. This may provide potential therapeutic advantages for when mucosal rPA-NE immunization is used for postexposure anthrax prophylaxis in combination with antibiotics or anti-PA monoclonal antibodies (38
Although it is difficult to directly compare titers of anti-PA IgG antibodies achieved with various adjuvants and routes of administration, the NE appeared to generate immune responses at least equivalent to those with other adjuvants. CT increased the effectiveness of the rPA-based vaccine (13
). However, unlike for the NEs, NIAID has raised safety concerns about CT, because when delivered intranasally it can transit the cribiform plate via the olfactory nerve and can cause inflammation in the olfactory region of the brain (12
). In comparable studies using the same guinea pig intranasal challenge model (and performed at the same facility with the same protocol), intramuscular immunization with rPA adsorbed on Alhydrogel and immunization with the commercial AVA vaccine provided 100 and 70% protection, respectively, when challenge was with 5× LD50
of anthrax Ames strain spores (37
). We used higher challenge doses and obtained equivalent to better protection, suggesting similar immunogenicity. Also, our intranasal vaccinations with rPA-NE vaccine typically resulted in at least 104
- to 105
-fold-higher titers of anti-PA antibodies than immunization with nonadjuvanted rPA. Therefore, NE adjuvants that both demonstrate high effectiveness and are made from “generally recognized as safe” materials and vegetable oils could be considered for use as safe and effective candidates for adjuvants in mucosal vaccines (17
Development of a protective humoral anti-PA response in humans or in various experimental animal models requires multiple immunizations with either parenteral or mucosal anthrax vaccines (6
). This cumbersome administration schedule and the short duration of protective immunity are serious disadvantages for anthrax immunization in response to bioterrorist attacks. A recent study reported that two intramuscular vaccinations of macaques with rPA bound to Alhydrogel produced higher serum IgG responses than the licensed AVA vaccine. However, the IgG levels in those animals significantly decreased by 6 to 10 weeks after immunization (54
). The reasons for the limited duration of immunity with these vaccines are unclear, and a direct comparison is complicated by differences in experimental models, but it is possible that the inherent instability of rPA leads to the degradation of critical antigenic epitopes (15
). We found that the rPA-NE vaccine was effective in inducing high titers of anti-PA IgG after only two intranasal administration and produced durable, long-term (6-month) neutralizing immunity. Incubation with NE seemed to decrease degradation of rPA, possibly stabilizing its antigenicity (data not shown). Thus, the increased stability of the antigen may play a role in the enhanced adjuvant activity observed with NE.
Most studies of anthrax vaccines focus on serum IgG concentration as the primary marker of protective immunity (2
). However, the NE adjuvant also induced mucosal immunity to PA. Significant concentrations of anti-PA IgA and IgG antibodies in mucosal secretions (BAL fluid) were detected in mice immunized with rPA-NE, although the value of mucosal immunity in protection against inhalation anthrax is unclear. rPA administered with either CpG ODN or MPL A immunostimulatory adjuvant (4
) was not effective in inducing a mucosal immune response. However, inclusion of MPL A into the NE-based vaccines resulted in a more rapid induction of serum anti-PA IgG but no change in the overall titer of anti-PA antibodies (data not shown). MPL A has proved to be an effective inducer of Th1 polarization of the immune response (48
). An examination of the PA-specific cytokine secretion pattern in splenocytes after rPA-NE immunization and the prevalence of IgG2a and IgG2b subtype antibody suggest that the NE-based vaccine may result in a similar Th1 polarization (6
). In contrast, the animals immunized with rPA-Alu had elevated levels of IgE and poor IgG and IgA responses, suggesting that their cellular immunity is biased toward a Th2 type of response (56
). Thus, rPA-NE yields a serum and systemic adjuvant activity similar to that with MPL A, which may be why the combination of these adjuvants did not appear to be synergistic when administered intranasally.
In summary, our study indicates that NEs appear to be an effective mucosal adjuvant for a rPA anthrax vaccine. These formulations induce long-lasting, robust, and specific humoral and cellular responses; appear to lack adverse effects; and have the ability to stabilize the antigen.