Fig. 2. Anti-HBsAg antibody serum levels 30 days following i.p. inoculation with three different doses of the recombinant
BioHepB vaccine. A significant difference between the dose of 0.2 mcg compared with 0.4 and 0.8 mcg P B 0.05 and P B
0.03, respectively was seen. No significant difference between 0.4 and 0.8 mcg doses was observed P = 0.20.
was used for quantitative analysis of anti-HBs by RIA, utilizing the Hollinger formula and ex-
pressed in mIUml. Mice in all experimental and control groups were followed for anti-HBs anti-
body titers 30 days following each inoculation of the BioHepB vaccine, throughout the study.
2
.
6
. Statistical analysis Results were analyzed using the standard Stu-
dent’s t-test.
3. Results
3
.
1
. Induction of anti HBV immune response Three groups of mice were followed for anti-
HBs antibody titers post-inoculation with three different doses of the BioHepB vaccine. A signifi-
cant difference between a dose of 0.2 mcg com- pared with 0.4 and 0.8 mcg was observed.
Vaccination with 0.2 mcg induced anti-HBs titers of 126.5 9 72.8 mIUml compared with 570.5 9
358.5 and 762.0 9 423.1 mIUml, following inocu- lation with 0.4 and 0.8 mcg, respectively P B 0.05
and P B 0.03 for 0.4 and 0.8 versus 0.2, respec- tively, and P = 0.2 for the difference between 0.4
and 0.8 mcg; Fig. 2. Consequently, a vaccination dose of 0.4 mcg, administered i.p. was used in the
study.
3
.
2
. E6aluation of the effect of oral administration of HBV-en6elope proteins on
anti-HBs titers Oral administration of HBV-envelope proteins
without immunization had no effect on anti-HBs antibody titers. Mice in Group H that were fed
HBV envelope proteins had undetectable anti- HBs serum levels 30 days after vaccination.
3
.
3
. E6aluation of the effects of tolerance induction on anti-6iral humoral immune response
Induction of anti-HBV peripheral immune tol- erance was evaluated by measuring anti-HBsAg
antibody production at monthly intervals in mice istration of HBV envelope protein BioHepB to
mice in experimental group F, or of BSA to mice in control group G. Mice were followed for anti-
HBs titers before oral tolerance induction on day 30, and following tolerance induction on day 60
Fig. 1. To determine the effect of oral adminis- tration of HBV envelope proteins BioHepB on
anti-HBs titers, mice in group H received five oral doses of HBV antigens without vaccination. Mice
were followed 30 days later for development of anti-HBs serum levels.
2
.
5
. Assessment of anti-HBs humoral immune response
Antibodies to HBsAg were detected by a com- mercial solid phase radioimmunoassay RIA,
Ausab, Abbott Laboratories, North Chicago, IL. A World Health Organization reference serum
fed prior to HBV vaccination. Administration of HBV proteins prior to BioHepB vaccination
markedly downregulated the antiviral humoral im- mune response. Thirty days following the first
inoculation of the vaccine, serum anti-HBs anti- body levels were 385 9 154 versus 709 9 290 and
777 9 346 mIUml in tolerized, HBV-envelope proteins fed group B, compared with non-toler-
ized non-fed controls group A, and BSA-fed group C, controls, respectively P B 0.013, Fig.
3A. Moreover, 30 days following the second inoculation with the BioHepB vaccine, anti-HBs
antibody
titers were
66 495 9 44 007 versus
95 257 9 58 320 and 123 607 9 76 130 mIUml, in tolerized versus non-tolerized controls groups A
and C, P B 0.05, Fig. 3B.
3
.
4
. E6aluation of the effect of oral tolerance induction on preexisting anti-HBV immune
response The effect of tolerance induction towards HBV
proteins on pre-existing anti-viral immune response was evaluated by measuring anti-HBs antibody
titers in anti-HBV immunized mice orally treated with BioHepB antigens or BSA. The feeding of
HBV antigens immediately after exposure to HBV vaccination, reduced anti-HBs titers to 70 572 9
32 030 mIUml in tolerized group D compared with 95 257 9 58 320 and 115 198 9 40 715 mIUml
in non-tolerized controls groups A and E, 60 days following BioHepB inoculation P B 0.05 com-
pared to control groups A and E, Fig. 4.
Fig. 3. The effect of oral administration of HBV proteins on tolerance induction towards viral antigens: Mice were fed with HBV envelope proteins followed by two inoculation of the BioHepB vaccine on days 10 and 40. Anti-HBs antibody levels were measured
30 days following each injection, on days 40 A and 70 B. Marked reduction in anti-HBs antibody titers was observed in tolerized HBV-envelope-proteins fed mice group B, black bar versus non-tolerized non-fed controls and BSA-fed controls groups A and C,
respectively, white bars on day 40 Fig. 3A, P B 0.013, and on day 70 Fig. 3B, P B 0.05
Fig. 4. The effect of oral tolerance induction towards hepatitis B virus in mice with preexisting immunity towards HBV: Mice
were immunized with the BioHepB vaccine followed by oral tolerance induction. A second inoculation of the vaccine was
administered on day 30, and serum antibody levels were measured 30 days later on day 60. A reduction in anti-HBs
serum levels was observed in tolerized HBV-envelope proteins fed mice group D, black bar versus non-tolerized BSA-fed
controls group E, white bar on day 60 P B 0.05.
tolerized non-fed controls group A, and BSA- fed controls group G, respectively as measured
on day 60 P B 0.01, Fig. 5B.
4. Discussion
