To improve surge testing capability for a response to a release of Bacillus anthracis, the CDC anti-Protective Antigen (PA) IgG Enzyme-Linked Immunosorbent Assay (ELISA) was re-designed into a high throughput screening format. The following assay performance parameters were evaluated: goodness of fit (measured as the mean reference standard r2), accuracy (measured as percent error), precision (measured as coefficient of variance (CV)), lower limit of detection (LLOD), lower limit of quantification (LLOQ), dilutional linearity, diagnostic sensitivity (DSN) and diagnostic specificity (DSP). The paired sets of data for each sample were evaluated by Concordance Correlation Coefficient (CCC) analysis. The goodness of fit was 0.999; percent error between the expected and observed concentration for each sample ranged from -4.6% to 14.4%. The coefficient of variance ranged from 9.0% to 21.2%. The assay LLOQ was 2.6 mug/mL. The regression analysis results for dilutional linearity data were r2 = 0.952, slope = 1.02 and intercept = -0.03. CCC between assays was 0.974 for the median concentration of serum samples. The accuracy and precision components of CCC were 0.997 and 0.977, respectively. This high throughput screening assay is precise, accurate, sensitive and specific. Anti-PA IgG concentrations determined using two different assays proved high levels of agreement. The method will improve surge testing capability 18-fold from 4 to 72 sera per assay plate.

Protective antigen (PA)-specific antibody and cell mediated immune (CMI) responses to annual and alternate booster schedules of Anthrax Vaccine Adsorbed (AVA, BioThrax(R)) were characterized in humans over 43 months. Study participants received 1 of 6 vaccination schedules: 3-dose intramuscular (IM) priming series (0, 1, 6 months) with a single booster at 42 months (4-IM); 3-dose IM priming with boosters at 18 and 42 months (5-IM); 3-dose IM priming with boosters at 12, 18, 30 and 42 months (7-IM); the 1970 licensed priming series of 6 doses (0, 0.5, 1, 6, 12, 18 months) and two annual boosters (30, 42 months) administered either subcutaneous (SQ) (8-SQ) or IM (8-IM); or saline placebo control at all eight time-points.Antibody response profiles included serum anti-PA IgG levels, subclass distributions, avidity, and lethal toxin neutralization activity (TNA). CMI profiles included frequencies of IFN-gamma and IL-4 secreting cells and memory B cells (MBCs), lymphocyte proliferation indices (SI) and induction of IFN-gamma, IL-2, IL-4, IL-6, IL-1beta and TNF-alpha mRNA levels.All active schedules elicited high avidity PA-specific IgG, TNA, MBCs and T cell responses with a mixed Th1/Th2 profile and Th2 dominance. Anti-PA IgG and TNA were highly correlated (e.g. Month 7, r2 = 0.86, p < 0.0001, log10 transformed) and declined in the absence of boosters. Boosters administered IM generated the highest antibody responses. Increasing time intervals between boosters generated faster and statistically superior antibody responses to the final Month 42 vaccination. CMI responses to the 3-dose IM priming remained elevated up to 43 Months.

OBJECTIVE: We evaluated an alternative administration route, reduced schedule priming series, and increased intervals between booster doses for anthrax vaccine adsorbed (AVA). AVA's originally licensed schedule was 6 subcutaneous (SQ) priming injections administered at months (m) 0, 0.5, 1, 6, 12 and 18 with annual boosters; a simpler schedule is desired. METHODS: Through a multicenter randomized, double blind, non-inferiority Phase IV human clinical trial, the originally licensed schedule was compared to four alternative and two placebo schedules. 8-SQ group participants received 6 SQ injections with m30 and m42 "annual" boosters; participants in the 8-IM group received intramuscular (IM) injections according to the same schedule. Reduced schedule groups (7-IM, 5-IM, 4-IM) received IM injections at m0, m1, m6; at least one of the m0.5, m12, m18, m30 vaccine doses were replaced with saline. All reduced schedule groups received a m42 booster. Post-injection blood draws were taken two to four weeks following injection. Non-inferiority of the alternative schedules was compared to the 8-SQ group at m2, m7, and m43. Reactogenicity outcomes were proportions of injection site and systemic adverse events (AEs). RESULTS: The 8-IM group's m2 response was non-inferior to the 8-SQ group for the three primary endpoints of anti-protective antigen IgG geometric mean concentration (GMC), geometric mean titer, and proportion of responders with a 4-fold rise in titer. At m7 anti-PA IgG GMCs for the three reduced dosage groups were non-inferior to the 8-SQ group GMCs. At m43, 8-IM, 5-IM, and 4-IM group GMCs were superior to the 8-SQ group. Solicited injection site AEs occurred at lower proportions in the IM group compared to SQ. Route of administration did not influence the occurrence of systemic AEs. A 3 dose IM priming schedule with doses administered at m0, m1, and m6 elicited long term immunological responses and robust immunological memory that was efficiently stimulated by a single booster vaccination at 42 months. CONCLUSIONS: A priming series of 3 intramuscular doses administered at m0, m1, and m6 with a triennial booster was non-inferior to more complex schedules for achieving antibody response.

A 3 dose (0, 1, 6 months) intramuscular (3-IM) priming series of a human dose (HuAVA) and dilutions up to 1:10 of anthrax vaccine adsorbed (AVA) provided statistically significant levels of protection (60-100%) against inhalation anthrax for up to 4 years in rhesus macaques.Serum anti-protective antigen (PA) IgG and lethal toxin neutralization activity (TNA) were detectable following a single injection of HuAVA or 1:5 AVA, or two injections of diluted vaccine (1:10, 1:20, 1:40 AVA). Anti-PA and TNA were highly correlated (overall r(2) = 0.89 for log(10) transformed data). Peak responses were at 6.5 months (mo). In general, with the exception of animals receiving 1:40 AVA, serum anti-PA and TNA responses remained significantly above control levels at 28.5mo (last time point measured for 1:20 AVA) and through 50.5mo in HuAVA, 1:5 and 1:10 AVA groups (p <0.05).PA-specific IFN-gamma and IL-4 CD4(+) cell frequencies and T cell stimulation indices were sustained to 50.5mo (last time point measured). PA-specific memory B cell frequencies were highly variable, but in general were detectable in PBMC by 2mo, significantly above controls by 7mo, and remained detectable in the HuAVA, 1:5 and 1:20 AVA groups to 42mo (last time point measured).HuAVA and diluted AVA elicited a combined Th1/Th2 response and robust immunological priming with sustained production of high avidity PA-specific functional antibody, long term immune cell competence and immunological memory (30mo for 1:20 AVA; 52mo for 1:10 AVA). Vaccinated animals surviving inhalation anthrax developed high magnitude anamnestic anti-PA IgG and TNA responses.

Accurate, reliable and standardized quantification of anti-protective antigen (PA) IgG antibody levels is essential for comparative analyses of anti-toxin immune responses in anthrax cases, recipients of PA-based anthrax vaccines and for evaluation of anti-PA based immunotherapies. We have previously reported the early performance characteristics and application of a quantitative anti-PA IgG enzyme linked immunosorbent assay. The principal application of this assay was in a Phase 4 human clinical trial of anthrax vaccine adsorbed (AVA, BioThrax), the central component of the CDC Anthrax Vaccine Research Program (AVRP) and in humans following bioterrorism associated Bacillus anthracis infection (Quinn et al., 2002; Quinn et al., 2004; Marano et al., 2008). The objective of the AVRP was to determine the feasibility of reducing the number of priming series and booster doses of the licensed Anthrax Vaccine Adsorbed (AVA) (BioThrax(R); Emergent BioSolutions, Lansing, MI) and changing the route of administration from subcutaneous (SC) to intramuscular (IM) (Marano et al., 2008). In this paper we report the validation and long term performance characteristics of the assay during its six year application in the AVRP (2002-2008). The critical features are 1) extensive validation of the assay using two standard reference sera; 2) long term stability and 3) consistency of the data for quantitative analysis of human long term anti-PA IgG responses. The reportable value (RV) of the assay was expressed as anti-PA IgG concentration (mcg/ml). Accuracy of the assay was high with a percent error (%ER) range of 1.6-11.4%. Overall intra-operator and intermediate precision were high with Coefficients of Variation (%CVs) of 2.5-15.4% and 6.3-13.2%, respectively. The assay demonstrated excellent dilutional linearity for human sera using log(10) transformed data with the slope=0.95 to 0.99, intercept=0.02 to 0.06 and r(2)=0.980-0.987. The assay was robust, tolerating changes in serum incubation temperatures from 35 to 39 degrees C, serum incubation times from 55 to 65min and changes in key reagents. The long-term assay stability over 6years using consecutive reference sera AVR414 and AVR801 demonstrated sustained high accuracy and precision for the assay, confirming its suitability for long term studies of PA protein-based anthrax vaccines.

A two-stage, multilevel assay quality control (QC) system was designed and implemented for two high stringency QC anthrax serological assays; a quantitative anti-PA IgG enzyme-linked immunosorbent assay (ELISA) and an anthrax lethal toxin neutralization activity (TNA) assay. The QC system and the assays were applied for the congressionally mandated Centers for Disease Control and Prevention (CDC) Phase 4 human clinical trial of anthrax vaccine adsorbed (AVA, BioThrax). A total of 57,284 human serum samples were evaluated by anti-PA enzyme-linked immunosorbent assay (ELISA) and 11,685 samples by anthrax lethal toxin neutralization activity (TNA) assay. The QC system demonstrated overall sample acceptance rates of 86% for ELISA and 90% for the TNA assays respectively. Monitoring of multiple assay and test sample variables showed no significant long term trends or degradation in any of the critical assay reagents or reportable values for both assays. Assay quality control data establish the functionality of the quality control system and demonstrates the reliability of the serological data generated using these assays.