Correlation between Lethal Toxin-Neutralizing Antibody Titers and Protection from Intranasal Challenge with Bacillus anthracis Ames Strain Spores in Mice after Transcutaneous Immunization with Recombinant Anthrax Protective Antigen

Abstract

Transcutaneous immunization of mice with recombinant protective antigen (rPA) of Bacillus anthracis resulted in significantly higher lethal toxin-neutralizing antibody titers than did intramuscular injection of alum-adsorbed rPA. Immunized mice were partially protected against intranasal challenge with 235,000 (10 50% lethal doses) Ames strain B. anthracis spores. A highly significant correlation was observed between toxin-neutralizing antibody titer and survival after challenge. Future experiments with rabbits and nonhuman primates should confirm the significance of protection by this vaccine strategy.Development of the mouse as a model animal for testing new types of vaccines to Bacillus anthracis has presented a problem in that spores from encapsulated strains, such as the Ames strain, exhibit much higher virulence than do unencapsulated strains, such as the STI and Sterne strains. Recent descriptions of the pulmonary infection model of B. anthracis in mice and the requirement of capsule formation by B. anthracis for dissemination in murine inhalation anthrax further support the need for a capsulated anthrax strain such as Ames in inhalation models (1,8). The extreme virulence of the Ames strain isolate has made it a logical choice as a challenge strain for examining the efficacy of proposed improved vaccines (2,8,13). Delivery of Ames strain spores intranasally instead of by the previously reported subcutaneous or intraperitoneal routes more closely mimics natural transmission pathways. In previous studies we and others demonstrated that transcutaneous immunization (TCI) with recombinant protective antigen (rPA) induced long-lasting neutralizing antibody titers in mice that were superior to those obtained with intramuscular (i.m.) injection of alumadsorbed rPA and completely protected the immunized mice against challenge with spores of the Sterne and STI strains, which are avirulent strains (6, 9). In the present study we demonstrate a clear improvement of TCI over injection with alum-adsorbed rPA for achieving protective efficacy against intranasally administered Ames strain spores. We also demonstrate a very strong correlation between protection and the quantitative level of toxin-neutralizing antibodies by TCI.Recombinant protective antigen was either purchased from List Biological Laboratories, Campbell, CA, or manufactured directly at NIH (12). Aluminum hydroxide gel (alum) (Rehydrogel) was provided as a gift by Reheis Inc., Berkeley Heights, N.J. Heatlabile Escherichia coli enterotoxin (HLT) was kindly supplied by John Clements at Tulane University. Female CBA/J mice (6 weeks old; 15/group) were purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained with food and water ad libitum. The backs of the mice were shaved, hydrated with 10% glycerol in 0.9% saline for 5 min, blotted dry, mildly abraded with fine-grade emery paper strips (GE Medical Systems, Milwaukee, WI), rehydrated for 5 min, and blotted dry. The rPA was mixed with 4 g of HLT in phosphate-buffered saline in a total volume of 5...

Multicomponent anthrax toxin display and delivery using bacteriophage T4

“…The amount of each antigen(s) was 1.25 μg displayed on 4 × 10 10 hoc -soc -phage particles in phosphate buffered saline without any adjuvant. The mice were bled every 2 weeks and sera from individual mice were assayed for the presence of PA, LF, and EF-specific IgG using an enzyme-linked immunosorbent assay (ELISA) [31]. Briefly, 96-well flat-bottomed Nunc Maxisorp plates (VWR, Bridgeport, NJ) coated with 0.1 μg/well of purified recombinant PA, LF or EF were blocked overnight with 0.5% gelatin in PBS, washed and incubated overnight with the test serum followed by HRP-labeled, affinity purified goat anti-mouse IgG (The Binding Site, San Diego, CA) and ABTS substrate (KPL, Gaithersburg, MD).…”

Effect of aluminum hydroxide adjuvant and formaldehyde in the formulation of rPA anthrax vaccine

“…Several studies have demonstrated the efficacy of PA in vaccines to protect against anthrax intoxication or infection [10][11][12][13]. The importance of anti-PA serum also has been shown in the identification of in vitro correlates of immunity [14][15][16][17] and in passive antibody studies [18][19][20]. This report evaluates the role of the aluminum hydroxide gel adjuvant and the excepient formaldehyde in the formulation of rPA vaccines in the rabbit model using in vitro surrogate markers (the quantitative anti-rPA IgG ELISA and toxin neutralizing antibody (TNA) assay) and efficacy studies.…”

“…We did not observe a decrease in the TNA assay ED 50 titers in the rabbit animal model at the highest concentration of aluminum tested (500 g). Both the anti-PA ELISA titer and toxin neutralizing antibody titers have been identified as serological correlates of immunity in rabbits and guinea pigs [13][14][15][16][17]25] and are thus important measurements in developing effective vaccine strategies. Various formulations have been tested in preparing anthrax vaccines based upon rPA for its ability to elicit optimal immunological responses.…”

Transcutaneous Immunization with Toxin-Coregulated Pilin A Induces Protective Immunity against Vibrio cholerae O1 El Tor Challenge in Mice

“…In this feasibility study, we did not evaluate various doses or immunization regimens; however, previous work has found induction of immune responses following transcutaneous immunization with as little as 1 g of antigen and 10 g of immunoadjuvant (13,29). Previous work has also shown that, when antigen is used alone, three or four transcutaneous applications of antigen are usually required to induce immune responses to topically applied antigens (10,12,14).…”