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B. anthracis 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 (recombinant Protective Antigen of anthrax) mixed in nanoemulsion adjuvant can provide protective immunity against respiratory or cutaneous forms of anthrax. We have evaluated 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.

The nanoemulsion droplets retained their stability after mixing with rPA antigen

Figure 1. The nanoemulsion droplets retained their stability after mixing with rPA antigen.

We have tested the nanoemulsion and antigen concentrations that are most effective in the development of specific mucosal responses against protective antigen of B. anthracis in mice and guinea pigs after intranasal administration. Our data indicated that after only two intranasal administrations of the rPA/nanoemulsion vaccines containing 10mg, 50mg and 100 mg of PA the guinea pigs became seropositive with significant and durable levels of anti-PA IgG present in serum. Immunization with the antigen alone did not produce antibody 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.

Intradermal challenge

Figure 2.  Intradermal challenge. 
At 6 months after nasal rPA/NE vaccination guinea pigs were injected with intraderma1 dose of 1000 x  LD50 of Ames spores.  All vaccinate animals survived, while control guinea pigs succumbed to infection and died. Insert presents LeTx (lethal toxin) neutralization with the immune sera. 

Sera from the rPA⁄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 concentrations of nanoemulsion in the vaccine. The analysis of the antigen-specific activation of splenic lymphocytes in vitro and anti-PA IgG subclass indicated Th1 polarization of the cellular response.
Challenge studies with live B. anthracis Aimes strain spores documented that nasal rPA/nanoemulsion vaccination can protect against inhalation or cutaneous infection with anthrax.

Intranasal Challenge

Figure 3.  Intranasal Challenge.
At 7 weeks after nasal rPA/NE vaccination guinea pigs were infected with intranasal instillation of 10 x  LD50 of Ames spores. All vaccine concentrations yielded 70% survival of animals with none of controls surviving the challenge.

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.

These studies have been published in Infection & Immununity, 2007: May 14:
Mucosal Immunization with a Novel Nanoemulsion-based Recombinant Anthrax Protective Antigen Vaccine Protects against B. anthracis Spore Challenge.
Bielinska AU, Janczak KW, Landers JJ, Makidon P, Sower LE, Peterson JW, Baker JR.)



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