Production and evaluation of a recombinant subunit vaccine against botulinum neurotoxin serotype B using a 293E expression system

Figures & data

Figure 1. Analysis of purified recombinant BHc protein by SDS-PAGE (A) and immunoblot (B). Lane 1, the protein standards; lane 2, 1 μg of recombinant BHc expressed and purified in one experiment; lanes 3 and 4, 2 μg of recombinant BHc expressed and purified in another experiment. Arrows indicate the position of the recombinant BHc protein.

Figure 2. Enzyme-linked immunosorbent assay of binding activity of the recombinant BHc protein to ganglioside (GT1b). Wells were coated with different concentration of ganglioside (GT1b), and incubated with 50 μg/mL recombinant BHc protein or BSA. The same amount of BSA was coated on the plate as negative control. Values represent means from 4 separate experiments with bars representing standard mean of deviations.

Table 1. Survival, sera antibody titers, and neutralizing titers of mice following immunization with 1 or 10 μg recombinant BHc formulated with aluminum hydroxide adjuvant

	Number Alive^a			Number Alive^b		Log10 GMT(SD)^C			Sera neutralizing titer (IU/ml)^d
Vaccination	1^e	2^e	3^e	2^e	3^e	1^e	2^e	3^e	2^e	3^e
1 μg	0	8	8	3	8	2.67(0.27)	3.51(0.35)	4.49(0.34)	0.16	2.56
10 μg	4	8	8	8	8	2.90(0.32)	4.31(0.25)	5.01(0.22)	0.64	5.12
Control	0	0	0	ND	ND	< 2	< 2	< 2	< 0.16	< 0.16

^aBalb/c mice alive (8 mice/group) after i.p. challenge with a dose 10³ LD₅₀ of BoNT/B 3 wk after the last injection; ^bBalb/c mice alive (8 mice/group) after i.p. challenge with a dose 10⁴ LD₅₀ of BoNT/B 3 wk after the last injection; ^cSera antibody titers for individual mice after the last immunization were analyzed by ELISA and the geometric mean titer (GMT) for each group was determined. Antibody titers to BHc are shown in the table; ^dSera neutralizing titers against BoNT/B were shown in table; ^eNumber of vaccinations. Control mice were injected with PBS and Alhydrogel; ND, not done.

Figure 3. Immune sera antibodies blocked BHc binding to ganglioside. The sera from mice immunized with three doses of 1 or 10 μg recombinant BHc formulated with aluminum hydroxide adjuvant were used to block BHc binding to GT1b. Sera from mice injected with PBS and Alhydrogel were used as negative control. Values represent means from 4 separate sera samples of mice with bars representing standard mean of deviations.

Table 2. Survival of mice following vaccination once, twice or three times with different doses of recombinant BHc formulated with aluminum hydroxide adjuvant

	Number Alive^a			Number Alive^b
Vaccination Dose (μg)	1^c	2^c	3^c	2^c	3^c
0.04	0	6	8	0	4
0.2	0	8	8	2	8
1	1	8	8	4	8
5	4	8	8	8	8

^aBalb/c mice alive (8 mice/group) after i.p. challenge with a dose 10³ LD₅₀ of BoNT/B 3 wk after the last injection; ^bBalb/c mice alive (8 mice/group) after i.p. challenge with a dose 10⁴ LD₅₀ of BoNT/B 3 wk after the last injection; ^cNumber of vaccinations. Mice were vaccinated once, twice or three times at four different doses of BHc formulated with aluminum hydroxide adjuvant range from 0.04 to 5 μg.

Table 3. Survival and sera antibody titers following vaccination i.m. with pABE293SBHc or pVAX1SBHc

Vaccination group	Log10 GMT(SD) ^a	Number Alive ^b
pABE293SBHc	3.65(0.31) **	8^#	5^&
pVAX1SBHc	2.84(0.33)	2	0

^aSera antibody titers for individual mice after the last immunization were analyzed by ELISA and the geometric mean titer (GMT) for each group were determined; Antibody titers to BHc are shown in the table. **P < 0.01 indicates significant titer difference between pABE293SBHc and pVAX1SBHc-vaccinated groups; ^bBalb/c mice alive (8 mice/group) after i.p. challenge with the indicated dose of BoNT/B (10² or 10³LD₅₀) 3 wk after the last injection. ^#P = 0.007 < 0.01 or ^&P = 0.026 < 0.05 indicates significant survival difference between groups vaccinated with pH293SBHc and pVAX1SBHc.