A plant-produced protective antigen vaccine confers protection in rabbits against a lethal aerosolized challenge with Bacillus anthracis Ames spores

Figures & data

Figure 1. Flow diagram of pp-PA83 purification process.

Figure 2. Electrophoretic mobility and immunoblot analysis of purified pp-PA83. (A) Coomassie-stained SDS-PAGE. pp-PA83 was loaded at 1 µg/lane. (B and C) Immunoblot. pp-PA83 was loaded at 100 ng/lane. pp-PA83 was separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes and blotted with (B) a commercially available anti-4 × His mAb or (C) a pp-mAb^PA.

Figure 3. N-terminal sequencing of purified pp-PA83. Identification of the first five amino acids confirms that the signal peptide was processed correctly.

Figure 4. Representative SEC-MALLS profile for pp-PA83. pp-PA83 elutes from SEC with two main peaks at ~12 mL and ~13.6 mL, and a light scattering calculated average molecular weight of 84.9 ± 1.3 kDa.

Figure 5. Glycoform analysis of pp-PA83 by SDS-PAGE and western blotting. Protein bands were visualized by Coomassie staining (A) and western blotting using pp-mAb^PA (B). Separated bands are shown with arrowheads in (B). Western blot analysis of pp-PA83 with (+) and without (-) pre-treatment with PNGase F (C).

Figure 6. Stability of the pp-PA83 drug product. (A) Absorbance at 280 nm of pp-PA83 in saline with (♦) and without (■) Alhydrogel, as a function of time. (B) T_m of pp-PA83, determined by using the extrinsic fluorescence of SYPRO Orange, with (♦) and without (■) Alhydrogel, as a function of time.

Table 1A. Anti-PA IgG titers in mice immunized with pp-PA83 plus Alhydrogel

	GMT anti-PA IgG ± SE
Day	-1	20	42	70
45 µg pp-PA83 /Alhydrogel	50 ± 17	2540 ± 1176	4.28 × 10^5 ± 1.37 × 10^5	3.17 × 10^5 ± 1.45 × 10^5
15 µg pp-PA83 /Alhydrogel	50 ± 17	200 ± 245	7.13 × 10^4 ± 1.18 × 10^5	5.29 × 10^4 ± 1.22 × 10^5
3 µg pp-PA83 /Alhydrogel	79 ± 13	100 ± 0	809 ± 749	455 ± 543

GMT: Geometric mean titer. SE: Standard error.

Table 1B. LeTx neutralizing antibody titers in mice immunized with pp-PA83 plus Alhydrogel

	GMT ED50 ± SE		% Responders (Day 70)
Day	42	70
45 µg pp-PA83 /Alhydrogel	521 ± 784	1068 ± 776	100%
15 µg pp-PA83 /Alhydrogel	133 ± 162	159 ± 505	50%
3 µg pp-PA83 /Alhydrogel	58 ± 3	22 ± 5	0%

GMT: Geometric mean titer. ED₅₀: Effective dilution 50. SE: Standard error.

Table 2. Immunogenicity of formulated pp-PA83 plus Alhydrogel in mice

pp-PA83 dose		7.5 µg	7.5 µg	7.5 µg
0.3% Alhydrogel		1.5 mg/mL aluminum	1.5 mg/mL aluminum	None
Preparation		2 weeks at 4°C	Fresh	Fresh
GMT anti-PA IgG ± SE	Day -1	25 ± 0	25 ± 0	25 ± 0
	Day 20	230 ± 374	57 ± 18	25 ± 0
	Day 42	7.8 × 10^4 ± 1.5 × 10^4	3.4 × 10^4 ± 2.1 × 10^4	25 ± 0
	Day 70	1.5 × 10^4 ± 4.7 × 10^3	1.5 × 10^4 ± 4.7 × 10^3	25 ± 0
GMT ED50 ± SE	Day 20	13 ± 2	12 ± 2	13 ± 3
	Day 42	1185 ± 849	308 ± 386	10 ± 0
	Day 70	680 ± 611	200 ± 142	10 ± 0
% Responders (Day 70)		100%	80%	0%

GMT: Geometric mean titer. ED₅₀: Effective dilution 50. SE: Standard error.

Table 3A. Anti-PA IgG titers in rabbits immunized with pp-PA83 plus Alhydrogel

	GMT anti-PA IgG ± SE
Day	-1	21	35	56
135 µg pp-PA83	25 ± 0	400 ± 1207	1.11 × 10^4 ± 1.89 × 10^4	1.64 × 10^5 ± 2.92 × 10^5
67.5 µg pp-PA83	25 ± 0	303 ± 355	1.47 × 10^4 ± 4703	6.25 × 10^4 ± 0
22.5 µg pp-PA83	25 ± 0	57 ± 73	4850 ± 1.96 × 10^4	4.53 × 10^4 ± 5.61 × 10^4
7.5 µg pp-PA83	25 ± 0	33 ± 15	696 ± 5017	4.53 × 10^4 ± 1.0 × 10^4
Saline/Alhydrogel	25 ± 0	25 ± 0	25 ± 0	105 ± 85

GMT: Geometric mean titer. SE: Standard error.

Table 3B. LeTx neutralizing antibody titers in rabbits immunized with pp-PA83 plus Alhydrogel

pp-PA83 dose		135 µg	67.5 µg	22.5 µg	7.5 µg	0 µg
0.1% Alhydrogel		0.5 mg/mL	0.5 mg/mL	0.5 mg/mL	0.5 mg/mL	0.5 mg/mL
		aluminum	aluminum	aluminum	aluminum	aluminum
GMT	Day 21	28 ± 19	13 ± 5	10 ± 0	17 ± 6	17 ± 6
ED50 ± SE	Day 35	525 ± 526	719 ± 224	208 ± 407	46 ± 117	10 ± 0
	Day 56	1688 ± 768	867 ± 107	743 ± 337	758 ± 247	10 ± 0
% Responders		100%	100%	100%	100%	0%
(Day 56)

GMT: Geometric mean titer. ED₅₀: Effective dilution 50. SE: Standard error.

Table 4A. Anti-PA IgG titers in rabbits immunized with pp-PA83 plus Alhydrogel prior to challenge

	GMT anti-PA IgG ± SE
Day	-1	20	41
135 µg pp-PA83 /Alhydrogel	30 ± 9	951 ± 752	1.0 × 10^5 ± 4.1 × 10^4
67.5 µg pp-PA83 /Alhydrogel	25 ± 0	200 ± 180	4.3 × 10^4 ± 1.6 × 10^4
22.5 µg pp-PA83 /Alhydrogel	30 ± 9	168 ± 1032	3.0 × 10^4 ± 4.8 × 10^4
7.5 µg pp-PA83 /Alhydrogel	25 ± 0	50 ± 46	2263 ± 2928
Saline/Alhydrogel	25 ± 0	25 ± 0	25 ± 0

GMT: Geometric mean titer. SE: Standard error.

Table 4B. LeTx neutralizing antibody titers in rabbits immunized with pp-PA83 plus Alhydrogel prior to challenge

pp-PA83		135 µg	67.5 µg	22.5 µg	7.5 µg	0 µg
0.1% Alhydrogel		0.5 mg/mL aluminum	0.5 mg/mL aluminum	0.5 mg/mL aluminum	0.5 mg/mL aluminum	0.5 mg/mL aluminum
GMT ED50 ± SE	Day 20	19 ± 13	10 ± 0	18 ± 13	11 ± 1	13 ± 3
	Day 41	351 ± 100	154 ± 66	78 ± 149	20 ± 26	10 ± 0
% Responders (Day 41)		88%	88%	38%	13%	0%

GMT: Geometric mean titer. ED₅₀: Effective dilution 50. SE: Standard error.

Figure 7. Percent survival of pp-PA83-immunized rabbits after inhalation challenge with B. anthracis. New Zealand White rabbits were vaccinated with three doses of pp-PA83 plus Alhydrogel on study days 0, 21 and 42, and then challenged intranasally with 368 LD₅₀ of aerosolized B. anthracis Ames strain on study day 53.