Development of Protective Immunity in New Zealand White Rabbits Challenged with Bacillus anthracis Spores and Treated with Antibiotics and Obiltoxaximab, a Monoclonal Antibody against Protective Antigen

Abstract

The recommended management of inhalational anthrax, a high-priority bioterrorist threat, includes antibiotics and antitoxins. Obiltoxaximab, a chimeric monoclonal antibody against anthrax protective antigen (PA), is licensed under the U.S. Food and Drug Administration's (FDA's) Animal Rule for the treatment of inhalational anthrax. Because of spore latency, disease reemergence after treatment cessation is a concern, and there is a need to understand the development of endogenous protective immune responses following antitoxin-containing anthrax treatment regimens. Here, acquired protective immunity was examined in New Zealand White (NZW) rabbits challenged with a targeted lethal dose of Bacillus anthracis spores and treated with antibiotics, obiltoxaximab, or a combination of both. Survivors of the primary challenge were rechallenged 9 months later and monitored for survival. Survival rates after primary and rechallenge for controls and animals treated with obiltoxaximab, levofloxacin, or a combination of both were 0, 65, 100, and 95%, and 0, 100, 95, and 89%, respectively. All surviving immune animals had circulating antibodies to PA and serum toxin-neutralizing titers prior to rechallenge. Following rechallenge, systemic bacteremia and toxemia were not detected in most animals, and the levels of circulating anti-PA IgG titers increased starting at 5 days postrechallenge. We conclude that treatment with obiltoxaximab, alone or combined with antibiotics, significantly improves the survival of rabbits that received a lethal inhalation B. anthracis spore challenge dose and does not interfere with the development of immunity. Survivors of primary challenge are protected against reexposure, have rare incidents of systemic bacteremia and toxemia, and have evidence of an anamnestic response.

Keywords: Bacillus anthracis; anthrax; antitoxin; immune memory; monoclonal antibodies; obiltoxaximab; protective antigen.

FIG 1

Kinetics of anti-PA antibodies in challenged and treated animals. NZW rabbits were aerosol challenged with targeted 200 LD₅₀ of B. anthracis spores and treated with obiltoxaximab (circles), levofloxacin (triangles), or a combination of both (squares) at 30 h PC. (A) Kaplan-Meier curves representing time to death from challenge and survival data for each group are shown. (B and C) Serum samples were collected at the indicated times relative to treatment (posttreatment [PT]) or challenge for a specific assessment of obiltoxaximab (B) or nonspecific assessment of all circulating anti-PA IgGs (C). (A) Each symbol represents individual animal values. (B) Vertical lines indicate means and standard error for each group at each indicated time point. (C) Vertical lines indicate means and standard deviations for each group at each indicated time point. Numbers of animals with measurable total anti-PA IgG levels (n) and the total numbers of animals surviving to each time point (N) are shown on the bottom. Total anti-PA pool is composed of endogenous rabbit antibodies to PA as well as circulating obiltoxaximab. Dotted lines indicate lower limit of quantitation for each assay. For statistical computations, levels below limit of detection were replaced with ½ LLOQ. The LLOQ was 50 ng/ml in each assay.

FIG 2

Toxin-neutralizing activity in serum of challenged and treated animals (phase 1). Serum samples were collected immediately prior to primary challenge or at the indicated times postchallenge for the assessment of TNA ED₅₀ (A) and NF₅₀ (B). Vertical lines indicate means and standard error. For statistical computations, TNA ED₅₀ and NF₅₀ levels below the limit of detection were replaced with 11.5 and 0.027 (½ of each respective LOD).

FIG 3

Survival and systemic disease development in immune animals following rechallenge. Nine months after primary challenge, phase 1 survivors and 12 additional naive controls were challenged with targeted 200 LD₅₀ of B anthracis spores and monitored for 21 days. (A) Kaplan-Meier curves representing time to death from challenge and survival data for each group are shown. (B and C) Whole-blood samples were collected prior to treatment (day [D] −7) or at indicated times postrechallenge (PC) for the assessment of quantitative bacteremia (B) and circulating free PA (C). Shown are the means and standard error of the mean (SEM) for bacteremia and free PA at each indicated time point. Dotted lines represent limit of detection (LOD) for bacteremia or limit of quantitation (LOQ) for PA. Numbers of animals with measurable levels for each parameter (n) and the total numbers of animals surviving to each time point (N) are indicated at the bottom. Shaded areas indicate that no animals survived to that time point. For statistical computations, bacteremia levels below the LOD were replaced with 2 CFU/ml (½ LOD), and PA levels below limit of quantitation (LOQ) were replaced with 4.84 ng/ml (½ LOQ).

FIG 4

Assessment of anti-PA IgG and TNA responses following rechallenge. Whole-blood samples were collected prior to rechallenge (D −7) or at indicated times postrechallenge (PC) for the assessment of circulating anti-PA IgG levels (A), TNA ED₅₀ (B), and TNA NF₅₀ (C). Shown are means and standard deviation (SD) (A) and SEM (B and C) at each indicated time point. Dotted lines represent the LLOQ (anti-PA IgG) and LOD (TNA ED₅₀ and NF₅₀).