Table 1.
The immunisation groups in the sheep study.
Fig 1.
The morphology of HMSA observed by transmission electron microscopy.
Fig 2.
N2 adsorption-desorption isotherms of HMSA.
Fig 3.
(A): Evaluation by SDS-PAGE of Opti-E2/HMSA formulations after freeze-drying with different combinations of trehalose and PEG8000. Lane 1: Opti-E2 control (4 μg); Opti-E2/HMSA freeze-dried with Lane 2: 1% PEG8000; lane 3: 5% trehalose and 0.5% PEG8000; lane 4: 5% trehalose and 0.1% PEG8000. (B): Evaluation by SDS-PAGE of Opti-E2/HMSA formulations after freeze-drying with different combinations of trehalose and glycine. Lane 1: Opti-E2 control (4 μg); Opti-E2/HMSA freeze-dried with lane 2: 5% trehalose and 1% glycine; lane 3: 5% trehalose and 0.5% glycine; lane 4: 5% trehalose and 0.1% glycine.
Fig 4.
The morphology of Opti-E2/HMSA particles visualised by transmission electron microscopy following freeze-drying with 5% trehalose and 1% glycine.
Fig 5.
Group 1 received 500 μg Opti-E2 and 1 mg Quil-A; Group 2 received the non-freeze-dried E2 nanovaccine (500 μg Opti-E2 adsorbed to 6.2 mg HMSA), Group 3 received the freeze-dried (FD) E2 nanovaccine (500 μg Opti-E2 adsorbed to 6.2 mg HMSA), Group 4 received HMSA particles (6.2 mg) only.
(A) Average Opti-E2-specific ELISA antibody responses (n = 4) in sheep in pre-immune sera (PI), and after one (1st), two (2nd) and three (3rd) subcutaneous immunisations. (B) Opti-E2-specific ELISA antibody responses in sheep after three subcutaneous immunisations. The individual response for each sheep is shown two weeks after the third immunisation using a sera dilution of 1:200.
Fig 6.
(A) The long term immune memory response of sheep PBMC cells following stimulation to Opti-E2 antigen. IFN-γ secretion of PBMC cells obtained four months after immunisation was assessed by ELISPOT assay in response to Opti-E2 (10 ng/μL, blue bars) and compared to unstimulated cells (red bars). The Mean Spot SFU /million cells is shown for each animal (assayed in triplicate) in the treatment groups. The polyclonal activator, Concavalin A (dark blue bars, ConA), was used to confirm cell viability and functionality of the assay. The asterisk (*) indicates significant responses with p < 0.05 (unpaired t-test analysis) compared to the unstimulated controls. (B) ELISPOT plate of the long term immune memory response of sheep PBMC cells following stimulation to Opti-E2 antigen compared to unstimulated cells. The polyclonal activator, Concavalin A (Red Box), was used to confirm cell viability and functionality of the assay.