DNA immunization with fusion genes encoding different regions of hepatitis C virus E2 fused to the gene for hepatitis B surface antigen elicits immune responses to both HCV and HBV

Table 1 Sero-conversion Ratios and Titres of Anti-HBsAg in DNA Immunized Mice

Weeks	DNA Immunogen
	pcDNA3S	pcDNA3SE2-A	pcDNA3SE2-B	pcDNA3SE2-C	pcDNA3SE2-D	pcDNA3SE2-E
0a	(0/5)	(0/5)	(0/5)	(0/5)	(0/5)	(0/5)
2	(5/5)	(1/5)	(1/5)	(1/5)	(1/5)	(2/5)
4b	1280d (5/5c)	1280 (4/5)	640 (2/5)	320 (2/5)	320 (2/5)	320 (4/5)
6	10240 (5/5)	10240 (5/5)	5120 (4/5)	5120 (4/5)	5120 (5/5)	2560 (5/5)
8	5120 (5/5)	5120 (5/5)	5120 (5/5)	5120 (5/5)	5120 (5/5)	5120 (5/5)
10	5120 (5/5)	5120 (5/5)	2560 (4/5)	2560 (5/5)	2560 (5/5)	5120 (5/5)
16	1280 (5/5)	1280 (5/5)	640 (4/5)	320 (5/5)	640 (5/5)	80 (5/5)
24	1280 (5/5)	1280 (5/5)	640 (4/5)	320 (5/5)	640 (5/5)	80 (5/5)

Note: Groups of mice (n = 5) were injected i.m. with the respective plasmids, and anti-HBsAg antibody titers were measured at different time points by ELISA. Sera from mice immunized with pcDNA3 vector were used as negative controls and showed no reactivity with any coated antigens (data not shown). For sero-conversion, 1:20 starting dilution were assayed and the sera giving positive reading were taken as converted one. To deter-mine antibody titers, serial two-fold dilutions of pooled sero-converted immune sera were incubated in antigen-coated wells. A positive result was defined as an absorbance value of greater than twice the absorbance of the negative control with a cutoff of 0.050 (also see Materials and Methods). At week 2 only sero-conversion testing was performed at 1:20 dilution.

^athe first immunization;

^bthe second immunization (boost);

^c(n/n); sero-conversion ratio, numbers of sero-converted mice relative to the total number tested;

^dtiter; reciprocal of the two-fold dilution factor at end point; pools of sero-converted sera from test groups were tested.

Table 2 Sero-conversion Ratios and Titres of Anti-HCV-E2 in DNA Immunized Mice

Weeks	DNA Immunogen
	pcDNA3S	pcDNA3SE2-A	pcDNA3SE2-B	pcDNA3SE2-C	pcDNA3SE2-D	pcDNA3SE2-E
0a	(0/5)	(0/5)	(0/5)	(0/5)	(0/5)	(0/5)
2	(1/5)	(2/5)	(1/5)	(1/5)	(1/5)	(1/5)
4b	40d (2/5c)	320 (3/5)	160 (1/5)	320 (3/5)	320 (2/5)	320 (1/5)
6	160 (5/5)	640 (5/5)	640 (5/5)	2560 (5/5)	2560 (5/5)	5120 (4/5)
8	880 (5/5)	640 (5/5)	320 (5/5)	5120 (5/5)	5120 (5/5)	5120 (4/5)
10	80 (5/5)	160 (5/5)	320 (5/5)	5120 (5/5)	5120 (5/5)	5120 (4/5)
16	80 (5/5)	80 (5/5)	160 (5/5)	160 (5/5)	320 (5/5)	80 (4/5)
24	40 (5/5)	40 (5/5)	80 (5/5)	80 (5/5)	160 (5/5)	40 (4/5)

Note: Groups of mice (n = 5) were injected i.m. with the respective plasmids, and sero-conversion ratio and anti-HCV E2 antibody titers were measured at different time points by ELISA. With the exception of coating antigen (E2 (385-565)), all experimental procedures were the same as described in Table 1.

^athe first immunization;

^bthe second immunization (boost);

^c(n/n); sero-conversion ratio, numbers of sero-converted mice relative to the total number tested;

^dtiter; reciprocal of the two-fold dilution factor at end point, pools of positive sera from test groups were tested.