Development and evaluation of candidate subunit vaccine against botulinum neurotoxin serotype B

Figures & data

Figure 1. Analysis of purified recombinant BHc protein during various purification steps by SDS-PAGE and western bolt. The soluble fraction and purified BHc were subjected to 10% SDS-PAGE and Western blot analysis using hyperimmune anti-neurotoxin B serum. Lane 1, pTIG-Trx vector transformed cell lysates; Lane 2, pTIG-Trx-BHc transformed BL21 cell lysates; Lane 3, MMC column purified products; Lane 4, Source 30s column purified products; Lane 5, Western blot analysis; M, protein markers 180, 130, 95, 72(red), 55, 43, 34, 26 and 17 KDa (from top to bottom); An arrow indicates the position of the BHc.

Table 1. Survival, group antibody ELISA titers, and serum neutralization titers of mice after vaccination with BHc and mBHC antigens.

	Number alive ^a		Number alive ^b	Log10 GMT(SD) ^c		Serum neutralization titer(IU/ml) ^d
Vaccination dose	1^e	2^e	2^e	1^e	2^e	2^e
1μg of BHc	5	5	5	3.22(0.05)	4.90(0.05)*	0.8
10μg of BHc	5	5	5	3.40(0.16)	5.04(0.08)*	2.33
1μg of mBHc	5	5	5	3.17(0.25)	4.37(0.07)	0.73
10μg of mBHc	5	5	5	3.07(0.02)	4.56(0.21)	1.25
Control	0	0	0	< 2	< 2	< 0.125

^aMice alive (5 mice/group) after i.p. challenge with a dose 1000 LD₅₀ of BoNT/B 3 weeks after last injection.

^bMice alive (5 mice/group) after i.p. challenge with a dose 10,000 LD₅₀ of BoNT/B 3 weeks after last injection.

^cSera obtained 2 days before challenge from individual mice was assayed and their mean was used to calculate the Log₁₀ GMT for each group. Standard deviations (SD) of the geometric mean titer (GMT) are in parenthesis.

^dPolled sera from each group of mice were diluted and assayed for serum neutralization titers. Serum form each group were pooled due to the limited amount of serum available from each mouse. The results represent the average neutralization titers of each group.

^eNumber of vaccinations. Mice were vaccinated either once or twice of two different dosages of BHc or mBHc.

*P < 0.05, compared to mBHc-immunized group.

Table 2. Survival, group antibody ELISA titers, and serum neutralization titers of mice after vaccination with BHc vaccine.

	Number alive ^a	Number alive ^b	Log10 GMT(SD) ^c		Serum neutralization titer (IU/ml) ^d
Vaccination dose	1^e	2^e	1^e	2^e	2^e
0 (Control)	0	0	< 2	< 2	< 0.125
0.0039 μg	0	0	2.49(0.16)	2.71(0.26)	< 0.125
0.0156 μg	0	4	2.39(0.07)	2.87(0.19)	< 0.125
0.0625 μg	0	5	2.46(0.12)	3.45(0.35)	0.25
0.25 μg	3	5	3.14(0.04)	3.83(0.06)	0.83
1 μg	5	5	3.17(0.01)	4.28(0.08)	1.33
4 μg	5	5	3.20(0.05)	4.56(0.16)	2.66

^aMice alive (5 mice/group) after i.p. challenge with a dose 1000 LD₅₀ of BoNT/B 3 weeks after last injection.

^bMice alive (5 mice/group) after i.p. challenge with a dose 10,000 LD₅₀ of BoNT/B 3 weeks after last injection.

^cSera obtained 2 days before challenge from individual mice was assayed and their mean was used to calculate the Log₁₀ GMT for each group. Standard deviations (SD) of the geometric mean titer (GMT) are in parenthesis.

^dPolled sera from each group of mice were diluted and assayed for serum neutralization titers. Serum form each group were pooled due to the limited amount of serum available from each mouse. The results represent the average neutralization titers of each group.

^eNumber of vaccinations. Mice were vaccinated either once or twice of BHc range from 0.0039 μg to 4 μg formulated with aluminum hydroxide adjuvant.

Figure 2. Survival of mice immunized with BHc vaccine. Mice alive after challenge with a dose of 1000 LD₅₀ of BoNT/B 3 weeks after one injection or 10,000LD₅₀ of BoNT/B 3 weeks after two injections.

Figure 3. Anti-BHc antibody titers in mice vaccinated with one or two doses of BHc formulated with aluminum hydroxide adjuvant. Mice were immunized one or two doses with 0.039 μg to 4 μg of BHc formulated with aluminum hydroxide adjuvant. Sera form individual mice from each group (n = 5) three weeks after final immunization were collected and specific antibody titer were analyzed by ELISA. Serum sample from individual mice sere assayed and the geometric mean titer (GMT) was calculated for each group.