Enhanced potency of replicon vaccine using one vector to simultaneously co-express antigen and interleukin-4 molecular adjuvant

Figures & data

Figure 1. AHc-specific antibody responses in mice after i.m. vaccination with DNA vaccines. (A) Sera from each group of mice at 4 weeks after the last immunization were collected, and the specific anti-AHc total IgG titers and individual IgGs isotype (IgG1 and IgG2a) titers were analyzed by ELISA. Serum samples from individual mice were assayed and the group mean titer (GMT) was calculated for the group (n = 8). (B) The IgG1/IgG2 ratio of serum antibodies was determined to evaluate the type of response to immunization. Results for each group represent the average ratio ± SEM *p < 0.05; **p < 0.01.

Table 1. Survival and serum neutralization titers of mice after i.m. vaccination with DNA vaccines

Vaccine	Serum neutralization titer (IU/ml)	Number Alive^a
		10^3	10^4
pVAX1SAHc	< 0.16	4	0
pSCARSAHc	0.16	6^$	0
pVAX1SAHc+pVAX1-IL-4	0.32	8*	4
pSCARSAHc+pVAX1-IL-4	0.64	8*	7^#
pVAX1-IL-4	< 0.16	0	ND
pVAX1	< 0.16	0	ND

^a Balb/c mice alive (8 mice/group) after i.p. challenge with the indicated dose (10³ or 10⁴ LD₅₀) of BoNT/A at 4 weeks after the last vaccination. *p = 0.00016 < 0.001 indicates a significant survival difference between the IL-4-codelivered groups that vaccinated with pVAX1SAHc+pVAX1-IL-4 or pSCARSAHc+pVAX1-IL-4 and the negative control groups (pVAX1-IL-4 or pVAX1). ^#p = 0.0014 < 0.01 indicates a significant survival difference between groups vaccinated with pSCARSAHc+pVAX1-IL-4 and pSCARSAHc or pVAX1SAHc. ^$p = 0.007 < 0.01 indicates a significant survival difference between the pSCARSAHc-vaccinated groups and the negative control groups.

Figure 2. AHc-specific antibody responses in mice after i.m. vaccination with DNA replicon vaccines. Sera from each group of Balb/c (A) and C57/BL6 (B) mice immunized with different DNA replicon vaccines at 4 weeks after the last immunization were collected, and the specific anti-AHc total IgG titers and individual IgGs isotype (IgG1 and IgG2a) titers were analyzed by ELISA. Serum samples from individual mice were assayed and the group mean titer (GMT) was calculated for the group (n = 8). (C) The IgG1/IgG2a isotype ratio for each group represents the average ratio ± SEM *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the pSCARSAHc-vaccinated group without IL-4 molecular adjuvant.

Table 2. Survival and serum neutralization titers of mice after i.m. vaccination with plasmid DNA replicon vaccines

Vaccine	Serum neutralization titer (IU/ml)		Number Alive^a
	Balb/c	C57/BL6	10^3		10^4
			Balb/c	C57/BL6	Balb/c	C57/BL6
pSCARSAHc	0.16	0.16	5	6	0	0
pSCARSAHc+pSCAR-IL-4	0.64	0.64	8	8	6*	7^$
pSCARSAHc-26S-IL-4	1.28	2.56	8	8	8^#	8^#
pSCARSAHc-IRES-IL-4	1.28	2.56	8	8	8^#	8^#
pSCAR-IL-4	< 0.16	< 0.16	0	0	ND	ND
pSCAR	< 0.16	ND	0	ND	ND	ND

^a Balb/c and C57/BL6 mice alive (8 mice/group) after i.p. challenge with the indicated dose (10³ or 10⁴ LD₅₀) of BoNT/A at 4 weeks after the last vaccination. *p = 0.007 < 0.01 indicates a significant survival difference between the IL-4-codelivered group that vaccinated with pSCARSAHc+pSCAR-IL-4 and the pSCARSAHc-vaccinated group without IL-4 molecular adjuvant in Balb/c mice. ^#p = 0.00016 < 0.001 indicates a significant survival difference between the IL-4-coexpressed groups that vaccinated with pSCARSAHc-26S-IL-4 or pSCARSAHc-IRES-IL-4 and the pSCARSAHc-vaccinated group. ^$p = 0.0014 < 0.01 indicates a significant survival difference between the IL-4-codelivered group that vaccinated with pSCARSAHc+pSCAR-IL-4 and the pSCARSAHc-vaccinated group in C57/BL6 mice. ND, not done.

Figure 3. AHc-specific antibody responses in mice after s.c. vaccination with recombinant SFV particles (VRP) vaccines. Sera from each group of Balb/c (A) and C57/BL6 (B) mice immunized with different VRP vaccines at 4 weeks after the last immunization were collected, and the specific anti-AHc total IgG titers and individual IgGs isotype (IgG1 and IgG2a) titers were analyzed by ELISA. Serum samples from individual mice were assayed and the group mean titer (GMT) was calculated for the group (n = 8). (C) The IgG1/IgG2a isotype ratio for each group represents the average ratio ± SEM **p < 0.01 and ***p < 0.001 compared with the pSCARSAHc-vaccinated group without IL-4 molecular adjuvant.

Table 3. Survival and serum neutralization titers of mice after s.c. vaccination with recombinant SFV virus replicon particles (VRP) vaccines

Vaccine	Serum neutralization titer (IU/ml)		Number Alive^a
	Balb/c	C57/BL6	10^3		10^4
			Balb/c	C57/BL6	Balb/c	C57/BL6
VRP-AHc	0.32	0.32	8*	8*	2	3
VRP-AHc+VRP-IL-4	1.28	2.56	8*	8*	8^#	8^$
VRP-AHc-26S-IL-4	2.56	5.12	8*	8*	8^#	8^$
VRP-AHc-IRES-IL-4	2.56	2.56	8*	8*	8^#	8^$
VRP-IL-4	< 0.16	< 0.16	0	0	ND	ND
VRP (Control)	< 0.16	ND	0	ND	ND	ND

^a Balb/c and C57/BL6 mice alive (8 mice/group) after i.p. challenge with the indicated dose (10³ or 10⁴ LD₅₀) of BoNT/A at 4 weeks after the last vaccination. *p = 0.00016 < 0.001 indicates a significant survival difference between the VRP-AHc-vaccinated groups and the negative control groups (VRP-IL-4 or VRP). ^#p = 0.007 < 0.01 indicates a significant survival difference between the IL-4-codelivered or co-expressed groups and the VRP-AHc-vaccinated group without IL-4 molecular adjuvant in Balb/c mice. ^$p = 0.026 < 0.05 indicates a significant survival difference between the IL-4-codelivered or co-expressed groups and the VRP-AHc-vaccinated group without IL-4 molecular adjuvant in C57/BL6 mice. ND, not done.

Figure 4. Schematic diagram of expression plasmids used in DNA immunization. CMV, cytomegalovirus immediate early (CMV IE) enhancer/promoter. TT/pA, BGH transcription termination and polyadenylation signal. HDV, HDV antigenomic ribozyme sequence. 26S, the subgenomic promoter of SFV. S, Ig κ leader sequence. pSCARSAHc-26S-IL-4, a dual-expression plasmid DNA replicon vaccine that designed to co-express both AHc and IL-4 under the control of two separate 26s promoters. pSCARSAHc-IRES-IL-4, a plasmid DNA replicon vaccine that designed to expressed AHc and allow the simultaneous expression of IL-4 separately from the same RNA transcript using bicistronic IRES.