B. anthrasis Adjuvant Vaccine

Currently licensed English and American human anthrax vaccines (AVA, BioTrax) consist of the culture supernatant from a toxigenic strain of B. anthracis adsorbed on aluminum hydroxide. To develop and maintain protective immunity in humans, this vaccine requires at least six subcutaneous administrations over a period of 18 months, followed by yearly boosters. Unfortunately, clinical evidence indicates a significant incidence of acute side effects as well as only partial protection from some strains of B. anthracis. Given the limitations of BioTrax, expedited development of a safe, efficacious, and well characterized anthrax vaccine is a priority.

Evidence from both the current vaccine and experimental trials with protective antigen of B. anthracis (PA) indicates that anti-PA responses are critical for the development of protective immunity against inhalation or injection challenge with either live bacilli or spores. As parenteral vaccines do not induce mucosal responses, which may help to protect against aerosolized anthrax, it is important to determine whether intra-nasal immunization would be effective to induce protective immunity.

We have developed a new antimicrobial nanoemulsion composed of soybean oil, emulsifying agents and ethanol, which has proven to be an effective adjuvant for intranasal immunization for live viral pathogens such as influenza A, as well as recombinant proteins, including PA, HIV protein gp120 and Hepatitis B surface antigen. When this nanoemulsion was mixed with influenza virus and placed into the nares of animals, it produced rapid and intense immune responses that protected animals from subsequent virus challenge.

We therefore set out to determine whether the mucosal immune responses to rPA mixed in nanoemulsion adjuvant can provide protective immunity against respiratory or cutaneous forms of anthrax. We are evaluating nanoemulsion adjuvant⁄rPA formulations as well as the addition of various immuno-stimulants, varying the concentrations and the schedule of vaccination to optimize the development of immune responses.

We have tested the nanoemulsion concentrations that are most effective in the development of specific mucosal responses against protective antigen of B. anthracis in mice after intranasal administration. Our preliminary data indicated that after only two administrations of the rPA/nanoemulsion vaccines containing 0.1%, 0.5%, 1%, and 2% nanoemulsion, the mice became seropositive with substantial levels of anti-PA IgG present in the mice serum. Animals immunized with the antigen alone did not have this response. Detailed analysis of the immune responses and titration of the antibodies showed that in this rapid and effective course of vaccination, the induction of the anti-PA IgGs correlated with the increase of nanoemulsion concentration in the vaccine.

Sera from the PA ⁄ nanoemulsion-immunized animals had a high titer of the lethal toxin neutralizing antibodies, which prevented anthrax lethal toxin-mediated killing of the RAW264 macrophage cells in vitro. The bronchial lavage fluid contained significant levels of anti-PA IgA class antibodies, indicating a robust mucosal response. As with IgGs, the highest concentrations of anti-PA IgA were detected in bronchial lavage fluid from animals vaccinated with higher (1% and 2%) concentrations of nanoemulsion in the vaccine. The analysis of the antigen-specific activation of splenic lymphocytes in vitro indicated Th1 polarization of the cellular response.

These studies are ongoing. Apart from characterizing and documenting the elements of the anti-B. anthracis immune responses (humoral, cellular, neutralizing antibodies), we will determine whether PA/nanoemulsion immunization can prevent or attenuate experimental inhalation or cutaneous infection with <B. anthracis spores.

This work has been supported by the National Institutes of Health via the Region V Great Lakes Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research.