Effect of reduced dose schedules and intramuscular injection of anthrax vaccine adsorbed on immunological response and safety profile: a randomized trial.

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.

Effects of a reduced dose schedule and intramuscular administration of anthrax vaccine adsorbed on immunogenicity and safety at 7 months: a randomized trial.

CONTEXT: In 1999, the US Congress directed the Centers for Disease Control and Prevention to conduct a pivotal safety and efficacy study of anthrax vaccine adsorbed (AVA). OBJECTIVE: To determine the effects on serological responses and injection site adverse events (AEs) resulting from changing the route of administration of AVA from subcutaneous (s.q.) to intramuscular (i.m.) and omitting the week 2 dose from the licensed schedule. DESIGN, SETTING, AND PARTICIPANTS: Assessment of the first 1005 enrollees in a multisite, randomized, double-blind, noninferiority, phase 4 human clinical trial (ongoing from May 2002). INTERVENTION: Healthy adults received AVA by the s.q. (reference group) or i.m. route at 0, 2, and 4 weeks and 6 months (4-SQ or 4-IM; n = 165-170 per group) or at a reduced 3-dose schedule (3-IM; n = 501). A control group (n = 169) received saline injections at the same time intervals. MAIN OUTCOME MEASURES: Noninferiority at week 8 and month 7 of anti-protective antigen IgG geometric mean concentration (GMC), geometric mean titer (GMT), and proportion of responders with a 4-fold rise in titer (%4 x R). Reactogenicity outcomes were proportions of injection site and systemic AEs. RESULTS: At week 8, the 4-IM group (GMC, 90.8 microg/mL; GMT, 1114.8; %4 x R, 97.7) was noninferior to the 4-SQ group (GMC, 105.1 microg/mL; GMT, 1315.4; %4 x R, 98.8) for all 3 primary end points. The 3-IM group was noninferior for only the %4 x R (GMC, 52.2 microg/mL; GMT, 650.6; %4 x R, 94.4). At month 7, all groups were noninferior to the licensed regimen for all end points. Solicited injection site AEs assessed during examinations occurred at lower proportions in the 4-IM group compared with 4-SQ. The odds ratio for ordinal end point pain reported immediately after injection was reduced by 50% for the 4-IM vs 4-SQ groups (P < .001). Route of administration did not significantly influence the occurrence of systemic AEs. CONCLUSIONS: The 4-IM and 3-IM regimens of AVA provided noninferior immunological priming by month 7 when compared with the 4-SQ licensed regimen. Intramuscular administration significantly reduced the occurrence of injection site AEs. Trial Registration clinicaltrials.gov Identifier: NCT00119067.

Standardized, mathematical model-based and validated in vitro analysis of anthrax lethal toxin neutralization.

Quantification of anthrax lethal toxin (LTx) neutralization activity (TNA) is pivotal in assessing protective antibody responses to anthrax vaccines and for evaluation of immunotherapies for anthrax. We have adapted and redesigned the TNA assay to establish a unifying, standardized, quantitative and validated technology platform for LTx neutralization in the J774A.1 murine cell line. Critical design features of this platform are 1) the application of a free-form or constrained 4 parameter logistic (4-PL) function to model neutralization responses within and between boundary limits of 100% cell survival and 95% cell lysis and 2) to exploit innovative assay curve recognition algorithms for interpretive endpoints. The assay was validated using human serum ED50 (dilution of serum effecting 50% neutralization) as the primary reportable value (RV). Intra-operator and intermediate precision, expressed as the coefficient of variation (%CV), were high at 10.5-15.5%CV and 13.5-14.5%CV respectively. TNA assay dilutional linearity was demonstrated for human sera using linear regression analysis of log(10) transformed data with slope=0.99, intercept=-0.03 and r(2)=0.985. Assay accuracy, inferred from the precision and linearity data and using a spike-recovery approach, was high with a percent error (%E) range of only 3.4-20.5%E. The lower limit of detection (LLOD) was ED50=12 and the lower limit of quantification (LLOQ) was ED50=36. The cell-based assay was robust, tolerating incubation temperatures from 35 to 39 degrees C, CO(2) concentrations from 3% to 7% and reporter substrate (MTT) concentrations of 2.5-7.5 mg/ml. Strict assay quality control parameters were met for up to 25 cell culture passages. The long term (50 month) assay stability, determined using human reference standards AVR414 and AVR801, indicated high precision, consistent accuracy and no detectable assay drift. A customized software program provided two additional assay metrics, Quantification Titer (QT) and Threshold Titer (TT), both of which demonstrate acceptable accuracy, precision and dilutional linearity. The TT was also used to establish the assay reactivity threshold (RT). The application of the assay to sera from humans, Rhesus macaques and rabbits was demonstrated separately and by aggregate dilutional linearity analysis of the ED50 (slope=0.98, intercept=0.003, r(2)=0.989). We propose this TNA assay format with a qualified standard reference serum and customized interpretive software as a unifying platform technology for determination of functional serologic responses to anthrax vaccines and for evaluation of anthrax immunotherapeutics.

Immune responses to Bacillus anthracis protective antigen in patients with bioterrorism-related cutaneous or inhalation anthrax.

Anti-protective antigen (PA) immunoglobulin (Ig) G, toxin neutralization, and PA-specific IgG memory B cell responses were studied in patients with bioterrorism-related cutaneous or inhalation anthrax and in a patient with laboratory-acquired cutaneous anthrax. Responses were determined for >1 year after the onset of symptoms. Eleven days after the onset of symptoms (15 days after likely exposure), anti-PA IgG was detected in 16 of 17 patients with confirmed or suspected clinical anthrax who were tested. Anti-PA IgG remained detectable 8-16 months after the onset of symptoms in all 6 survivors of inhalation anthrax and in 7 of 11 survivors of cutaneous anthrax who were tested. Anti-PA IgG levels and serum toxin neutralizing activity were strongly associated (R2=0.83). PA-specific IgG memory B cells were detectable in all 6 survivors of inhalation anthrax but in only 2 of 7 patients with cutaneous anthrax who were tested. Anti-PA IgG is an important diagnostic marker of anthrax, a predictor of serum anti-toxin activity, and a marker of immunological memory against anthrax.

Specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin G antibodies to anthrax toxin protective antigen.

The bioterrorism-associated human anthrax epidemic in the fall of 2001 highlighted the need for a sensitive, reproducible, and specific laboratory test for the confirmatory diagnosis of human anthrax. The Centers for Disease Control and Prevention developed, optimized, and rapidly qualified an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum. The qualified ELISA had a minimum detection limit of 0.06 micro g/mL, a reliable lower limit of detection of 0.09 micro g/mL, and a lower limit of quantification in undiluted serum specimens of 3.0 micro g/mL anti-PA IgG. The diagnostic sensitivity of the assay was 97.8%, and the diagnostic specificity was 97.6%. A competitive inhibition anti-PA IgG ELISA was also developed to enhance diagnostic specificity to 100%. The anti-PA ELISAs proved valuable for the confirmation of cases of cutaneous and inhalational anthrax and evaluation of patients in whom the diagnosis of anthrax was being considered.