326
A novel oral adjuvant for hepatitis B virus (HBV) vaccines
A plasma-derived HBV vaccine was administered to 86 healthy men ranging in age from 40-56 years. A relatively high rate of nonresponders was found among the men who received the HBV vaccine alone. There was a significantly hi&her HBs antibody response rate among the vaccinees who received an oral adjuvant (taurine) compared to those not receiving the adjuvant. In the vaccinees who received the oral adjuvant, mt in vitro palyclonal antibody response to taurine was detected in 17 (58.6%) of the 29 HBs responders, but in tmne of the HBs nonresponders. The development of oral adjwants other than aluminum may be a valuable approach to the study of HBV vaccinmion.
‘fbe availability
of HBV vaccine has prompted
exten-
sive trials for protection against HBV infection. In most studies on HBV vaccines (l-3). over 90% of the recipients acquired anti-HBs titres above 10 Uil after three vaccinations. However, a high frequency of nonresponsiveness to HBV vaccine in hemodialysis patients (4-6) and elderly persons has been reported (7,8). In mu previous experiments twine as an oral adjuvant was capable of restoring antibody responses to HBs antigens (Ag) in nonresponder BIO.M mice (9). These observations prompted us to explore whether the oral adjuvant could overcome hyporespansiveness m HEW vaccines in middle-aged merl.
ministered
only 20 fig of HB vaccine.
were measured Maleripls and Meth&
Group B consisted
of 43 men with a mean age of 48.1 years (range 40-56 years). The HBV vaccine (2O& was administered lntramuscularly to this group three times, along with 12 g taurine (Taisho Phamweuticals. Takvo. Jmmn) mallv ar day 0; the day prior to, and the da&f, &&tio,,:&. jects were immunized with three 20 pg doses of plasma HBV vaccine containing 50 pg of aluminium hydroxide (Green Cross, Osaka, Japan) via the deltoid muscle cm days 0.30 and 150. Anti-HBs levels WCICmeasured in the 86.subjec!s in both groups A and B for 6 months after the first vaccination, and for 12 months in 56 subjects. HBV serological markers (HBsAg, anti-H& and anti-HBc) by radioimntunoway
(Abbott
Iabomto-
ries, North Chicago, U.S.A.). HBs responders wae defined as vaccinees who had anti-HBs levels of greater than twice the cut-off value or 10 Lymphocyte culture. Lymphocytes were isolated from the peripheral blood of HBV vaccine reci~eots in the oral adjuvant group by antrlhtgation over Ficoll-Isopaque BP
wn.
Eighty-six healthy men were divided into two groups. All subjects were negative for HBV serologic markers, such as HBsAg, anti-HBs and anti-HBc, and had a sealanine aminatransfemse level of less than 35 IIJil before HBV vaccination. Group A, consisted of 43 males with a mean age of 47.5 years (range 40-55 years) who were ad-
described previously (10). The palpberdl Iymphocyta (2.106 celWml) were cultured with 10 mg/ml taurine in RPM1 1640 medium containing 10% heat-inactivated hu-
327 man AB serunt a, 37 T for 4 days.
Phque-forming cells.Theaumkrof polyclonal 1gG Dlaoue-fomtine cells i?FC) was derermined wine_~motein A plaque assays (11). In b;ief, 2.5~1 of cell suspension, 25 ~1 of a 1:s suspension of protein A-coupled sheep red blood cells @BBC), 25 81 of a 150 diluted solution of rabbit anti-human IgG (Dakopatts, Denmark) and 25 pl of
. .
_
guinea pig complement (diluted 15) were added to 0.5% agar (0.2 ml) containing 0.005% DEAE dextran. The mixture was placed on Petri dishes, covered with a coverglass, and incubatedat 37 ‘Cfor4 h. SmriPriEolanolvsiF. Tbe results listed in Table 1 and Fie. 2 were compared t&g the Fisher’s exact, twoail test and the ;i-analysis. Wilcoxon’s rank sum test was used to compare the anti-HBs levels in groups A and B.
Two of the group A (HBs vaccine alane) and three of the group B subjects (HBV vaccioe plus tatwine) became anti-HBs positive 1 month after the second injection. Sixteea 137.2%) of the 43 vaccieinees in crow A and 29 (67.4&) of the 43 vaccinees in group i d&loped antiHBs at 1 month after the last vaccination. Anti-HBs was detected in 28.5% of group A (28 vaccinees) and 60.7% of group B (28 vaccinees) 12 months after the first vaccination (Table 1). We found no signifcant difference behvem the hvo groups in the anti-HBs levels of those positive for anti IlBs @ > 0.05 at each month). Two responders each fn group A and B becamz anti-HBs negative 12 mmttbs after the 6rs.t vaccinatian (Fig. 1). No subjects bad detectable HBsAg, anti-HBc or elevations of serum &mine amittotmnsferase levels. No side effec@ to HBV vaccines or the oral adjuvant found. Next, we investigated the responders and oonresponders to tati in group B. Peripheral blood lympbcqtes from group B were cultured for 4 days with taurbte to de-
(taurine) WCR
tennine the
tauine-induced polycIottal I@ antibody sesponses. Taurine respcmdets were detined as subjects whose cultured c&s induced an augmentatiott of polyclooat fgG antibody respmsm to taurine more than twice that seat with uttstimnlated cells. As &own in Fig. 2 polyclo”ai 1gG amibody w to taurine in 17 (58.6%) of the 29 HBs responders arere augmented, whereas lym phocytes from HBs nonresponders failed to respond with tatuine. Polyclonal IgG antibody resqonses induced by tatic are expressed as the mean ex&mental PFC per cmttml PFC x l@io Fig. 2.
The rate of HBs resqottse to the same vaccine used in the present trial has been reported to be 93.2% in male and %.7% in femalc recipients (12). Several studies btdicate that the rapomivenss to HBV vaccble dimbdabe.5 with advancing age (7.8). SimiMy, an&HBs amibodfes
s. ,SHlzAKA
et
at.
but not in taurine nonresponders. It is suggested, on the basis of these findings, that taurine may improve, at least in part, the nonresponsiveness to the vaccine in taurine responders. It is now known that many adjuvants, such as inorganic gels, lipophilic compounds, oil emulsions and components of bacteria, induce macropbage accumulation “t the site of injection and then stimulate the release of interkukin 1 (IL-l) and antigen-proeasing events by macropbages (13). Furthermore, a number of adjuvants also affect alterations in lymphocyte recirculation which lead to the augmentation of antibody responses and abrogation of tolerance (14). It is well established that taurine is one of the most abundant free amino acids present in mammalian tissues and that it acts as a neurotransmitter (15). a membrane stabilizer (Xi), a regulator of cardial contractility (17) and a detoxifier (l&19). The adjuvant effects of tatwine are likely to be caused by twine-induced IL-1 and pulyclonal antibody responses (9). In addition, tauriue, as a sulpbated amino acid, may be able to enhance HBs hyporespensiveness by altering lympbcqte recirctdatioo. since sulphated substances cause changes in lympboeyte recirculation (20-22). Taurine induced in vitro polyclonal antibody production in C57BU6 spleen cells (9) but not in DBA/2 spleen cells (unpublished data), indicating that the response to tatuine differs awoting to the mouse strain. These results are analogous to the individual dif-
were detected in only 37.2% of vaccinees (age 40-56 years) in this study (Table 1). Anti-HBs antibody levels were also found to be low in the middle-aged men (Fig. 1). Taurhte significantly increased the response rate to HBV vaccine in middle-aged men, but no statistically significant differences in anti-HBs antibody levels were observed between the oral adjuvam and vaccine mntml groups. This lack of significant difference
in the. HBs anti-
body levels between the two groups may be due to the fact that tautine exerts adjuvant effects in taurine responders
ferences oboerxd in humans. Tbe ability of Iwine to angment the anti-H& respoese is considered to be influenced by the genetic backgxxtnd 3f the individual. Although tuurine was unable to exert adjuvant effects on HBs responsiveness in the nonresponders to taurine (Fig. Z), it tney bc that twine contributes to the augmentation of HBs vaccination in HBs nonresponders who responded to teurhte in in vi:rocell culturns.
This study was suppaled
in part by a Grant-in-Aid
for
Scientific Research (No. 61480181) from the Ministty of Education, Science and Culture, Japan.
A novel oral adjuvant for hepatitis B virus (HBV) vaccines
A plasma-derived HBV vaccine was administered to 86 healthy men ranging in age from 40-56 years. A relatively high rate of nonresponders was found among the men who received the HBV vaccine alone. There was a significantly hi&her HBs antibody response rate among the vaccinees who received an oral adjuvant (taurine) compared to those not receiving the adjuvant. In the vaccinees who received the oral adjuvant, mt in vitro palyclonal antibody response to taurine was detected in 17 (58.6%) of the 29 HBs responders, but in tmne of the HBs nonresponders. The development of oral adjwants other than aluminum may be a valuable approach to the study of HBV vaccinmion.
‘fbe availability
of HBV vaccine has prompted
exten-
sive trials for protection against HBV infection. In most studies on HBV vaccines (l-3). over 90% of the recipients acquired anti-HBs titres above 10 Uil after three vaccinations. However, a high frequency of nonresponsiveness to HBV vaccine in hemodialysis patients (4-6) and elderly persons has been reported (7,8). In mu previous experiments twine as an oral adjuvant was capable of restoring antibody responses to HBs antigens (Ag) in nonresponder BIO.M mice (9). These observations prompted us to explore whether the oral adjuvant could overcome hyporespansiveness m HEW vaccines in middle-aged merl.
ministered
only 20 fig of HB vaccine.
were measured Maleripls and Meth&
Group B consisted
of 43 men with a mean age of 48.1 years (range 40-56 years). The HBV vaccine (2O& was administered lntramuscularly to this group three times, along with 12 g taurine (Taisho Phamweuticals. Takvo. Jmmn) mallv ar day 0; the day prior to, and the da&f, &&tio,,:&. jects were immunized with three 20 pg doses of plasma HBV vaccine containing 50 pg of aluminium hydroxide (Green Cross, Osaka, Japan) via the deltoid muscle cm days 0.30 and 150. Anti-HBs levels WCICmeasured in the 86.subjec!s in both groups A and B for 6 months after the first vaccination, and for 12 months in 56 subjects. HBV serological markers (HBsAg, anti-H& and anti-HBc) by radioimntunoway
(Abbott
Iabomto-
ries, North Chicago, U.S.A.). HBs responders wae defined as vaccinees who had anti-HBs levels of greater than twice the cut-off value or 10 Lymphocyte culture. Lymphocytes were isolated from the peripheral blood of HBV vaccine reci~eots in the oral adjuvant group by antrlhtgation over Ficoll-Isopaque BP
wn.
Eighty-six healthy men were divided into two groups. All subjects were negative for HBV serologic markers, such as HBsAg, anti-HBs and anti-HBc, and had a sealanine aminatransfemse level of less than 35 IIJil before HBV vaccination. Group A, consisted of 43 males with a mean age of 47.5 years (range 40-55 years) who were ad-
described previously (10). The palpberdl Iymphocyta (2.106 celWml) were cultured with 10 mg/ml taurine in RPM1 1640 medium containing 10% heat-inactivated hu-
327 man AB serunt a, 37 T for 4 days.
Phque-forming cells.Theaumkrof polyclonal 1gG Dlaoue-fomtine cells i?FC) was derermined wine_~motein A plaque assays (11). In b;ief, 2.5~1 of cell suspension, 25 ~1 of a 1:s suspension of protein A-coupled sheep red blood cells @BBC), 25 81 of a 150 diluted solution of rabbit anti-human IgG (Dakopatts, Denmark) and 25 pl of
. .
_
guinea pig complement (diluted 15) were added to 0.5% agar (0.2 ml) containing 0.005% DEAE dextran. The mixture was placed on Petri dishes, covered with a coverglass, and incubatedat 37 ‘Cfor4 h. SmriPriEolanolvsiF. Tbe results listed in Table 1 and Fie. 2 were compared t&g the Fisher’s exact, twoail test and the ;i-analysis. Wilcoxon’s rank sum test was used to compare the anti-HBs levels in groups A and B.
Two of the group A (HBs vaccine alane) and three of the group B subjects (HBV vaccioe plus tatwine) became anti-HBs positive 1 month after the second injection. Sixteea 137.2%) of the 43 vaccieinees in crow A and 29 (67.4&) of the 43 vaccinees in group i d&loped antiHBs at 1 month after the last vaccination. Anti-HBs was detected in 28.5% of group A (28 vaccinees) and 60.7% of group B (28 vaccinees) 12 months after the first vaccination (Table 1). We found no signifcant difference behvem the hvo groups in the anti-HBs levels of those positive for anti IlBs @ > 0.05 at each month). Two responders each fn group A and B becamz anti-HBs negative 12 mmttbs after the 6rs.t vaccinatian (Fig. 1). No subjects bad detectable HBsAg, anti-HBc or elevations of serum &mine amittotmnsferase levels. No side effec@ to HBV vaccines or the oral adjuvant found. Next, we investigated the responders and oonresponders to tati in group B. Peripheral blood lympbcqtes from group B were cultured for 4 days with taurbte to de-
(taurine) WCR
tennine the
tauine-induced polycIottal I@ antibody sesponses. Taurine respcmdets were detined as subjects whose cultured c&s induced an augmentatiott of polyclooat fgG antibody respmsm to taurine more than twice that seat with uttstimnlated cells. As &own in Fig. 2 polyclo”ai 1gG amibody w to taurine in 17 (58.6%) of the 29 HBs responders arere augmented, whereas lym phocytes from HBs nonresponders failed to respond with tatuine. Polyclonal IgG antibody resqonses induced by tatic are expressed as the mean ex&mental PFC per cmttml PFC x l@io Fig. 2.
The rate of HBs resqottse to the same vaccine used in the present trial has been reported to be 93.2% in male and %.7% in femalc recipients (12). Several studies btdicate that the rapomivenss to HBV vaccble dimbdabe.5 with advancing age (7.8). SimiMy, an&HBs amibodfes
s. ,SHlzAKA
et
at.
but not in taurine nonresponders. It is suggested, on the basis of these findings, that taurine may improve, at least in part, the nonresponsiveness to the vaccine in taurine responders. It is now known that many adjuvants, such as inorganic gels, lipophilic compounds, oil emulsions and components of bacteria, induce macropbage accumulation “t the site of injection and then stimulate the release of interkukin 1 (IL-l) and antigen-proeasing events by macropbages (13). Furthermore, a number of adjuvants also affect alterations in lymphocyte recirculation which lead to the augmentation of antibody responses and abrogation of tolerance (14). It is well established that taurine is one of the most abundant free amino acids present in mammalian tissues and that it acts as a neurotransmitter (15). a membrane stabilizer (Xi), a regulator of cardial contractility (17) and a detoxifier (l&19). The adjuvant effects of tatwine are likely to be caused by twine-induced IL-1 and pulyclonal antibody responses (9). In addition, tauriue, as a sulpbated amino acid, may be able to enhance HBs hyporespensiveness by altering lympbcqte recirctdatioo. since sulphated substances cause changes in lympboeyte recirculation (20-22). Taurine induced in vitro polyclonal antibody production in C57BU6 spleen cells (9) but not in DBA/2 spleen cells (unpublished data), indicating that the response to tatuine differs awoting to the mouse strain. These results are analogous to the individual dif-
were detected in only 37.2% of vaccinees (age 40-56 years) in this study (Table 1). Anti-HBs antibody levels were also found to be low in the middle-aged men (Fig. 1). Taurhte significantly increased the response rate to HBV vaccine in middle-aged men, but no statistically significant differences in anti-HBs antibody levels were observed between the oral adjuvam and vaccine mntml groups. This lack of significant difference
in the. HBs anti-
body levels between the two groups may be due to the fact that tautine exerts adjuvant effects in taurine responders
ferences oboerxd in humans. Tbe ability of Iwine to angment the anti-H& respoese is considered to be influenced by the genetic backgxxtnd 3f the individual. Although tuurine was unable to exert adjuvant effects on HBs responsiveness in the nonresponders to taurine (Fig. Z), it tney bc that twine contributes to the augmentation of HBs vaccination in HBs nonresponders who responded to teurhte in in vi:rocell culturns.
This study was suppaled
in part by a Grant-in-Aid
for
Scientific Research (No. 61480181) from the Ministty of Education, Science and Culture, Japan.
