This Is AuburnElectronic Theses and Dissertations

Development and Efficacy Assessment of Equine Source Hyper-Immune Plasma against Bacillus anthracis




Caldwell, James

Type of Degree





The objective of the studies described here was to develop an equine source immune plasma against Bacillus anthracis and test its efficacy in two in vitro applications; as well as determine its capacity for passive protection in an infection model in mice. Initially, a safe and reliable immunization protocol for producing equine source hyper-immune plasma against B. anthracis was developed. Six Percheron horses were hyper-immunized with either the B. anthracis Sterne strain vaccine, recombinant protective antigen (rPA) homogenized with Freund’s incomplete adjuvant, or a combination of both vaccines. Multiple routes of immunization, dose (antigen mass) and immunizing antigens were explored for safety. A modified automated plasmapheresis process was then employed for the collection of plasma at a maximum target dose of up to 22 ml of plasma/kg of donor bodyweight to establish the proof-of-concept that large volumes of plasma could be safely collected from horses for large scale production of immune plasma. All three immunization protocols were found to be safe and repeatable in horses and three pheresis events were performed with the total collection of 168.36 L of plasma and a mean collection volume of 18.71 L (± 0.302 L) for each event. Once the hyper-immune plasma had been successfully harvested, two in vitro potency tests, an indirect enzyme-linked immunosorbent assay (ELISA) were developed to determine the titer of anti-PA antibodies and a toxin neutralization assay to determine the titer of protective antibodies against lethal toxin. In addition, the performance characteristics of the assay were validated in concordance with standards set forth by the International Conference on Harmonics (ICH) and the Center for Biologics Evaluation and Research (CBER) for analytical procedures. A working range for this assay was established (73-1581 EU/ml) on the bases of the following parameters: linearity (25 and 1,662 EU/ml, R2 = 0.999, p < 0.001), accuracy (94.8 - 105.4 %, recovery within the range of 25 and 1,662 EU/ml), precision (≤ 17.6%CV, repeatability; ≤ 15.7 and ≤ 13.1 % CV, intermediate precision per day and per analyst, respectively), limit of detection (2.25 EU/ml) and limit of quantitation (25 EU/ml). The assay was also demonstrated to be specific for the evaluation of anti-PA antibodies. Based on these performance characteristics it was determined that this assay is adequate for use in B. anthracis immunogenicity testing in horses. Antibody titers against PA were found to be as high as 1:512,000 in some horses. The geometric mean titers for horses hyper-immunized with spore vaccine alone, for horses hyper-immunized with the spore vaccine then hyper-immunized with rPA, for horses hyper-immunized with the spore vaccine then hyper-immunized with twice the volume of spore vaccine, and for horses hyper-immunized with rPA alone was 1:43,031 (SEM±3995, CI 95% 1:35,675 – 1:51,906), 1:213,027 (SEM±32,252, CI 95% 1:152,849 – 1:296,897), 1:83,912 (SEM±16,818, CI 95% 1:54,046 – 1:129,971) and 1:34,912 (SEM±13,961, CI 95% 1:15,035 – 1:81,066), respectively. Neutralizing antibodies were assessed using mouse macrophage J774A.1 cells in an LF-induced cytotoxicity toxin neutralization assay. Several horses developed neutralizing titers as high 1:1,024. The geometric mean neutralizing titers for horses immunized with the Sterne strain spore vaccine alone, for horses immunized with the spore vaccine then immunized with rPA, for horses immunized with the spore vaccine then immunized with twice the volume of spore vaccine, and for horses immunized with rPA alone was 1: 130 (SEM±2.26, CI 95% 1:126 – 1:135), 1: 964 (SEM±56, CI 95% 1:849 – 1:1,094), 1: 683 (SEM ± 171, CI 95% 1:408 – 1:1,143) and 1:478 (SEM ± 5.5, CI 95% 1:9.5 – 1:68), respectively. Overall, this demonstrates that plasma derived from horses immunized against B. anthracis Sterne strain and rPA provides strong in vitro correlates of protection. As a final assessment of the efficacy, hyper-immune plasma and affinity purified immunoglobulins harvested from horses hyper-immunized with B. anthracis Sterne strain vaccine was evaluated in the treatment of A/J strain mice intranasal (IN) and subcutaneous (SC) challenge with B. anthracis Sterne strain. The treatment of native (un-concentrated) hyper-immune plasma at time 0 hour and 24 hour post-infection following both an IN and SC challenge had no effect on rate of survival to the end of the study period, but did significantly increase mean time to death (p < 0.0001) compared to mice treated with naïve equine plasma. Mice treated with concentrated immunoglobulins at both time points following IN and SC challenge demonstrated significantly different survival rates (p < 0.001) compared to mice treated with naïve plasma. B. anthracis colony forming units/gram of lung, liver and spleen tissue were also assessed and were not significantly different in mice treated with hyper-immune plasma but were reduced by 4 fold and completely cleared in some cases after treatment with concentrated immunoglobulins (p < 0.0001).