Department of Virology, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan
Correspondence
Kimiyasu Shiraki
kshiraki{at}toyama-mpu.ac.jp
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ABSTRACT |
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MAIN TEXT |
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Some vaccinees respond to the VZV skin test on day 4 after vaccination and patients infected with VZV respond once the rash appears (Asano et al., 1985). The cutaneous reaction to gH : gL appears in guinea pigs on day 3 after Oka varicella vaccine inoculation (Sato et al., 1998
). Oka varicella vaccine replicates near the inoculation site and then the immune response, which might be created mainly by dendritic cells and infiltrating lymphocytes, results in the induction of DTH (Finkelman, 1995
; Abbas et al., 1996
; Moser & Murphy, 2000
). We hypothesized that the immune response induced by Oka varicella vaccine may recognize HBsAg to create the Th1-dominant immune response, as assessed by DTH, after its co-immunization with HBsAg. Thus, we have immunized HBsAg with Oka varicella vaccine and observed a Th1-dominant immune response to HBsAg in the experimental model, confirming the novel adjuvant activity of Oka varicella vaccine.
HBsAg and alum-conjugated HBsAg vaccine (10 µg) derived from yeast were supplied from the Research Foundation for Microbial Diseases of Osaka University, Suita, Japan. VZV gE : gI was purified by the application of an Oka varicella vaccine-infected cell lysate to an affinity column coupled with a monoclonal antibody against gE (Kamiyama et al., 2000; Shiraki et al., 1997
, 2001
). Oka varicella vaccine was prepared from infected human embryonic lung cells (Kamiyama et al., 2000
; Sato et al., 1998
; Shiraki et al., 1984
, 2001
). The mock vaccine was prepared similarly but without infection. The dose of Oka varicella vaccine was 1x104 p.f.u. for immunization. The dose for the heat-inactivated vaccine was also 1x104 p.f.u.; heat inactivation was at 56 °C for 30 min.
Male guinea pigs (Hartley strain) weighing about 280 g (Sankyo Labo Service) were immunized subcutaneously with various vaccines (Sato et al., 1998; Shiraki et al., 1984
). For the HBsAg dose-finding experiment, Oka varicella vaccine and 0, 1, 10, 20 or 40 µg of HBsAg were mixed and five guinea pigs from each group were immunized. After 3 weeks, humoral and cell-mediated immune responses were evaluated by ELISA and the skin test to gE : gI and HBsAg.
From the results of the dose-finding experiment, the amount of HBsAg was determined to be 20 µg per dose. To determine the specificity of the live, combined vaccine, the immune responses to gE : gI and HBsAg were compared among the seven immunization groups. When the live or heat-inactivated vaccine was mixed with HBsAg, the mixture was inoculated to the right back. When the live or heat-inactivated vaccine and HBsAg were inoculated separately, the live or heat-inactivated vaccine and HBsAg were inoculated on the right and left backs, respectively. Five guinea pigs were immunized under the following vaccination schemes: a mixture of live vaccine and HBsAg; a mixture of heat-inactivated vaccine and HBsAg; a mixture of mock vaccine and HBsAg; a live vaccine and HBsAg inoculated at separate sites; a heat-inactivated vaccine and HBsAg inoculated at separate sites; a mock vaccine and HBsAg inoculated at separate sites; and a control (PBS alone). Immune responses to gE : gI and HBsAg were evaluated 3 weeks after vaccination.
Guinea pigs were injected intradermally at three sites with 0.1 ml of antigen on their backs; hair was removed by chemical depilatory. HBsAg (1 µg) and gE : gI (1 µg) were used for the dose-finding experiment and HBsAg (2 µg) and gE : gI (3.2 µg) were used for the other experiments. The erythematous area was measured at 8, 24 and 48 h and the size (area) of the cutaneous reaction was determined at three sites at 24 h (Kamiyama et al., 2000; Sato et al., 1998
; Shiraki et al., 2001
).
Sera were tested for antibody titres to gE : gI and HBsAg by ELISA (Kamiyama et al., 2000; Sato, 1998; Shiraki et al., 2001
). Sera were diluted 1 : 80 with PBS containing 2 % skimmed milk and applied to each well treated with 1 µg HBsAg and 0.5 µg gE : gI for antibody titres to HBsAg and gE : gI, respectively. Then the anti-guinea pig IgG, goat IgG antibody conjugated with peroxidase (whole molecule, Cappel) was distributed to the wells and the reaction was visualized by HAT-EIA (Denka Seiken). Epitope analysis of HBsAg was performed using 16 species of its constituent peptides, as reported by Kamiyama et al. (2000)
. The statistical significances of the data were assessed using an unpaired Student's t-test.
Fig. 1 shows the dose response effects of HBsAg in the Oka varicella vaccine mixture on the antibody response and DTH to gE : gI and HBsAg. There was no significant difference in the magnitude of antibody responses to gE : gI among the various immunization groups. Antibody responses to gE : gI were not affected by the amount of HBsAg. Antibody responses to HBsAg were significantly lower in the heat-inactivated vaccine groups than those in the live vaccine groups (P<0.05). Weak responses to gE : gI remained in the heat-inactivated vaccine groups (with HBsAg at 20 and 40 µg) until 24 h; these were thought to be Arthus reactions from the time-course of reaction. Significantly strong DTH was induced in the live vaccine groups, especially at doses of 20 and 40 µg (P<0.05). The antibody response and the magnitude of DTH to HBsAg increased similarly depending on the amount of HBsAg in the live vaccine mixture and the response was almost saturated when 20 µg HBsAg was in the mixture. Thus, live Oka varicella vaccine showed significant adjuvant activity towards HBsAg by inducing DTH and enhancing antibody production and these responses were almost saturated when 20 µg HBsAg was in the mixture.
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Antibody decline during the year after immunization with combined vaccination was compared with HBsAg or PBS immunization alone (Fig. 3). Antibody titres to gE : gI declined gradually. Antibody titres to HBsAg were significantly higher in the combined vaccine group than in the other groups during this 1 year period. Antibody titres in the HBsAg alone group at the first month were equivalent to those of the combined vaccine group at 9 months (P<0.05) and the enhanced level of antibody titres by Oka varicella vaccine corresponded to the antibody decline seen at the 9 month period. DTH to HBsAg was observed in the combined vaccination group but not in the other immunization groups. The adjuvant effect of Oka varicella vaccine on cell-mediated and humoral immunity to HBsAg continued for at least 1 year.
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Patients with chronic hepatitis B who are treated with lamivudine might need cell-mediated immunity to HBV to recover from their carrier state; otherwise, they need prolonged treatment until complete elimination of HBV (Lau et al., 2000). A Th1-dominant immune response is important for virus clearance and recovery from chronic hepatitis B (Rossol et al., 1997
). The results with this combined vaccine suggested the possibility of improving the immune status in patients with chronic hepatitis B.
This is the first report that demonstrates the adjuvant activity of VZV using Oka varicella vaccine for inducing a cell-mediated immune response by simply mixing the candidate antigen with Oka varicella vaccine. Therefore, the utilization of Oka varicella vaccine as a novel adjuvant vector may contribute to a new vaccine development for the infectious diseases in which a Th1-dominant immune response may play a beneficial role.
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ACKNOWLEDGEMENTS |
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Received 5 July 2002;
accepted 1 October 2002.