The recombinant botulinum neurotoxin serotype A binding domain BoNT/A(Hc), SEB vaccine (STEBVax) and the fusion protein of F1 and V antigens (rF1-V) were prepared as previously described [10, 13, 16, 19]. The recombinant protective antigen (rPA) was obtained from List Laboratories (Wako, TX). Each vaccine was combined with AH adjuvant (Superfos Biosector, Kvistgård, Denmark), before administration using previously optimized ratios (unpublished observations) that in all cases resulted in delivery of < 1 mg of elemental aluminum per animal. Rhesus monkeys were obtained from Primate Products, Inc. (Woodside, CA) and quarantined for 30 d before study initiation. Just before vaccination, anesthetized (ketamine/acepromazine) monkeys were shaved on the deltoid/upper arm region or thigh using electric clippers, and the vaccines were administered i.d. on days 0, 28, and 56. On day 0 the vaccines were administered on the left arm, on day 28 the vaccines were administered on the right arm, and on day 56 the vaccines were administered on the left thigh. Vaccinated animals received 5 μg of the BoNT/A(Hc) vaccine, 150 μg of rF1-V, 50 μg of rPA, and 40 μg of STEBVax. Control animals received injection of AH adjuvant with no antigen. Two 100-μl i.d. injections of each vaccine were administered 2 cm apart with a stainless steel microneedle (1-mm exposed length, 76-μm inner diameter, 178-μm outer diameter) attached to a 1-ml syringe, as previously described .
Complete blood counts with white blood cell differential counts as well as serum concentrations of IgM and IgG were determined from blood collected on days 14, 42, and 70. Before each blood draw, animals were anesthetized by injection with ketamine/acepromazine. Antigen-specific serum antibody levels were determined by ELISA. Plastic plates (96 well) were coated (1 h, 37°C) with 100 μl/well of 2 μg/ml of BoNT/A(Hc), rF1-V, rPA, or STEBVax diluted in PBS (pH 7.4) for the sample unknowns, and purified monkey IgM or IgG was serially diluted threefold for the standard curve. The plates were washed three times with PBS/0.1% Tween and blocked (1 h, 37°C) with 0.2% casein/PBS (100 μl/well), washed as above, and then were incubated (1 h, 37°C) with 100 μl of diluted serum samples. Plates were then washed and incubated (1 h, 37°C) with 100 μl/well of goat anti-monkey IgG or goat anti-monkey IgM (1:10,000 dilutions) conjugated to horseradish peroxidase, washed, and developed (30 min, 22°C) with 100 μl of TMB peroxidase substrate (KPL, Gaithersburg, MD). Absorbance was measured at 650 nM and concentrations were determined by comparison to the absorbance of the standard curve.
Neutralizing antibody assays
For the anthrax toxin neutralization assay, 100 ng/ml LF and 200 ng/ml of PA, both in high-glucose DMEM with 7.5% fetal bovine serum (FBS), were mixed 1:1 with dilutions of sera and incubated for 1 h (37°C) before being added to J774 cells growing on a 96-well plate (63,000 cells/well in high-glucose DMEM, 7.5% FBS). The cells were incubated at 37°C for 4 h and cell viability was determined by ATP content (Vialight HS, Cambrex, Rockland, ME). The endpoint titer was determined as the serum dilution that gave a response three times greater than background. For the SEB neutralization assay, human peripheral blood mononuclear cells were isolated by density gradient centrifugation and added to a 96-well plate (100,000 cells/well in RPMI, 5% fetal calf serum). After plating, cells were allowed to rest for 2 h at 37°C. Dilutions of the test and control sera were prepared and SEB (200 ng/ml) was added to each dilution. Serum dilutions were then incubated for 1 h. at 37°C. The treatments (50 μl/well) were added to the cells and the plates were incubated at 37°C for 60 h. Finally, 1 μCi of [3H] thymidine (Sigma, St. Louis, MO) was added to each well, the plates were incubated for 9 h at 37°C, and incorporated radioactivity was measured by liquid scintillation. The antibody titer was determined as the highest serum dilution that significantly inhibited (Student's t-test) SEB-induced proliferation of the monocytes compared to the negative control. For the BoNT/A neutralization assay, dilutions of serum from animals in the BoNT/A challenge groups were mixed with 10 LD50 of toxin and incubated for 1 h at room temperature. Each dilution was injected intraperitoneally (IP) into four CD-1 mice. The mice were observed for 4 days and the number of deaths in each group was recorded. The neutralizing antibody titer was determined as the reciprocal of the serum dilution that protected 50% of the mice from intoxication with BoNT/A.
Animals were split into four separate challenge groups, each containing two controls and six vaccinated monkeys. Each group was challenged with one agent: BoNT/A, Ames strain spores of B. anthracis, or SEB, all obtained from USAMRIID. Before challenge, monkeys were anesthetized with ketamine/acepromazine and their breathing rate was determined by plethysmography. For groups challenged with botulinum neurotoxin A (50 LD50), B. anthracis (200 LD50), or SEB (25 LD50), each animal was exposed to the agent for 10 min in a head-only exposure chamber. Animals were observed up to two months after challenge. On days 2, 4, and 6 postchallenge, blood was drawn and complete blood counts with white blood cell differential counts were performed on all samples and bacteremia was determined for samples from animals challenged with bacterial agents. Necropsies were performed on animals that did not survive to verify death was a result of exposure to the challenge agent.
Pathology and necropsy
A necropsy was performed on all animals, either as soon as death occurred from infection or intoxication or after humane euthanasia of terminally ill or moribund animals by established protocols. Samples of spleen, lymph nodes (mandibular, axillary, tracheobronchial, mesenteric), lung, trachea, mediastinum, and haired skin from the vaccine sites from each monkey were collected for histopathology. Additionally, brain tissue was collected from animals that succumbed due to infection with B. anthracis. All tissues were immersion-fixed in 10% neutral buffered formalin.
Histology and immunohistochemistry
Formalin-fixed tissues for histology were trimmed, processed, and embedded in paraffin according to established protocols . Histology sections were cut at 5–6 μm, mounted on glass slides, and stained with hematoxylin & eosin (H&E). Immunohistochemical staining was performed using the Envision+ method (DAKO, Carpinteria, CA). Briefly, sections were deparaffinized in Xyless, rehydrated in graded ethanol, and endogenous peroxidase activity was quenched in a 0.3% hydrogen peroxide/methanol solution for 30 min at room temperature. Slides were washed in distilled water, placed in a Tris-EDTA Buffer (10 mM Tris Base, 1 mM EDTA Solution, 0.05% Tween 20, pH 9.0) and heated in a vegetable steamer for 30 min. Sections were incubated in the primary antibody, rabbit anti-major histocompatibility complex class II polyclonal antibody (RGU, unpublished), diluted 1:500 for 1 h at room temperature. After the primary antibody incubation, sections were washed in PBS and incubated for 30 min with Envision + System HRP (horseradish peroxidase-labeled polymer conjugated to goat anti-rabbit immunoglobulins) at room temperature. Peroxidase activity was developed with 3,3'-diaminobenzidine (DAB), counterstained with hematoxylin, dehydrated, cleared in Xyless, and coverslips were applied with Permount.
Adjuvant visualization in tissues
Adjuvant was localized in tissue samples by detection of aluminum. Five micrometer sections were prepared from formalin fixed, paraffin-embedded tissue blocks, deparaffinized in Xyless, and rehydrated in graded alcohols. Slides were rinsed in distilled water then pretreated in a 1% aqueous solution of hydrochloric acid for 10 min. After rinsing the slides in distilled water for 5 min, we stained them in a 0.2% alcoholic Morin solution (Sigma, Atlanta, GA) for 10 min. After staining with Morin, the sections were incubated for 2 h at 37°C with a 1:20 dilution of Texas Red phalloidin and approximately 1 μg/ml of Hoechst-33258 (Molecular Probes, Eugene Oregon) in PBS. Sections were rinsed twice in PBS and once in water before coverslips were applied with Vecta Shield mounting medium (Vector Labs, Burlingame, CA).
Images were collected with a BioRad 2000 MP confocal system attached to a Nikon TE300 inverted microscope fitted with a 60× (1.20 N.A.) water-immersion objective lens. Morin fluorescence was detected with 488 nm laser excitation and a HQ515/30 emission filter. Texas Red phalloidin was imaged with 568 nm laser excitation and an E600LP emission filter. Hoechst dye was visualized with 800 nm 2-photon excitation and a HQ390/70 emission filter. Subsequent contrast enhancement of the resulting images was performed using Adobe PhotoShop software.
Analysis of variance was used to analyze serology data obtained at various time points after vaccine administration to determine if there were any statistical differences within or between the vaccinated and control groups. The data conformed with the assumptions of the test if plots of the residuals revealed no structure. Comparisons of antibody production and lymphocyte proliferation between vaccinated and control animals were performed using Student's t-test. The data conformed to the assumptions of the t-test if the normal probability plot was a straight line. Historical controls were used to increase the statistical power of the experiment. Uniform lethality was observed in more than 15 untreated control Rhesus exposed to the same strain and route of each agent used in the experiment. Efficacy was evaluated using Fishers exact test comparing the treated group to the control group for each agent consisting of 2 experimental controls and 15 historical controls.