678
BIOCHEMICAL SOCIETY TRANSACTIONS
Despite the inhibitory effect of (NH4)2WS4on the renal uptake of 64Cu, this compound increased the concentrations of non-radioactive copper in the kidneys. As with the plasma, this increase was greater in the rats receiving 28mg of copper/kg diet, and moreover could be correlated with the renal concentrations of tungsten. In this case there was always an equimolar increase in the concentrations of both metals. The (NH&WS4 also influenced the distribution of copper within the kidneys. Thus it decreased the amount of copper bound to metallothionein and increased that present in particulate and high-molecular-weight fractions. These observations are consistent with effects of (NH4)2WS4on the availability of copper to the rats both before and after its absorption from the intestine, as has been described for ammonium tetrathiomolybdate (Mills et al., 1978). Copper appears to accumulate in the blood and kidneys in forms where it cannot be utilized in a normal manner. The excellent stoicheiometric relationships between the concentrations of copper and tungsten in plasma and also in the kidneys may indicate that the metals are associated in some way with the same proteins, as was reported in molybdenumsupplemented sheep where copper and molybdenum were frequently found in the same chromatographic fractions from both plasma and kidneys (Bremner & Young, 1978). The effect of (NH&WS4 on copper metabolism in rats is therefore much greater than that of tungstate, which causes no change in plasma copper distribution, even when dietary concentrations of tungsten are 2g/kg (Cardin et al., 1976). It appears, therefore, that, as in the copper-molybdenum interaction, sulphur is an essential component of the copper-tungsten interaction. Brernner, I. & Young, B. W. (1978) Br. J. Nutr. 39,325-336 Cardin, C . J., Dennehy, A., Mason, J. & Roberts, C. (1976) Biochem. SOC.Trans. 4,873-877 Mills, C. F., Brernner, I., El-Gallad, T. T., Young, B. W. & Dalgarno, A. C. (1978) in Trace Element Metabolism in Man and Animals - 3 (Kirchgessner, M., ed.), pp. 150-157, Arbeitskreis fur TierernahrungsforschungWeihenstephan, Freising-Weihenstephan, Germany
Hepatitis-B-Surface-Antigen-ContainingLiposomes Enhance Humoral and Cell-Mediated Immunity to the Antigen EMAhXJEL K. MANESIS,* COLIN H. CAMERONt and GREGORY GREGORIADIS* *Division of Clinical Sciences, Clinical Research Centre, Watford Road, Harrow, Middx. HA1 3 UJ, U.K., and t Virology Laboratory, Middlesex Hospital Medical School, London W l P 7LD, U.K.
Accumulated evidence suggests that high titres of antibody against HB,Ag (hepatitis-B surface antigen), the outer coat of the putative virus, can protect man from natural hepatitis-B infection (Purcell et al,. 1978). The antigenicity of the HB,Ag in man is, however, weak, thus making necessary the use of an appropriate immunological adjuvant. Adjuvants applied in animals, although numerous, are by and large unacceptable to man because of side effects, and even those licensed for use in humans are far from ideal (World Health Organisation, 1976). More recently, attention has been drawn to the usefulness of liposomes as powerful and safe adjuvants potentially applicable in man (Gregoriadis, 1976a,b). In the present study we investigated the humoral and cellmediated immune responses of guinea pigs to free or liposome-associated HB,Ag and the possibility of a synergistic effect of liposomes and of Bordetella pertussis or saponin adjuvants. Purified HB,Ag (30-50,ug) was incorporated in liposomes composed of egg phosphatidylcholine (44,u~),cholesterol and dicetyl phosphate (7 :2: 1 molar ratio) (Manesis et al., 1978). Free or liposome-associated HB,Ag and its antibodies (anti-HB,) were measured by the AUSRIA I1 and AUSAB radioimmunoassay kits (Abbott G.m.b.H., Langen, Germany) respectively (Manesis et a[., 1978). Female outbred guinea pigs
1979
582nd MEETING, ST. ANDREWS
679
Table 1. Humoral conversion rate after immunization with HB,Ag Animals were treated on day 0, 18, 34 and 44 with free HB,Ag alone (I) or mixed with saponin (11) or B . pertussis (111) and with liposomal HB,Ag alone (IV) or mixed with saponin (V) or B. pertussis (VI). They were bled by cardiac puncture on days 18, 34,44 and 59. Four animals died during sampling. Conversion* Group I I1 I11 IV V VI
Day
...
34 115 115 016 515 516 616
18 015 016 016 316 116 416
44
. 59
115 315 416 515 616 616
214 415 516 515 616 616
Delayed-type hypersensitivityt 80
014 115 016 3I5 016 515
* Responders from total in' the group. t Animals showing delayed-typehypersensitivityfrom total in the group.
t
0
t 20
t
40
t
60
Time (days)after immunization Fig. 1. Increase in anti-HB, titres in guinea pigs immunized with HB,Ag Animals were treated (arrows) as described in Table 1. Each dose (0.6ml) given subcutaneously in both hind-limbs contained 1.4pg of free or liposornal (0.8mg of lipid) HB,Ag and, when appropriate, 1 . 2 10'' ~ bacteria or 50pg of saponin. 0 , Group 1; A , Group 11; 0, Group 111; 0 , Group IV; A , Group V ; W, Group VI. VOl. 7
680
BIOCHEMICAL SOCIETY TRANSACTIONS
(Hartley strain) were divided into groups of six and injected with liposomal o r free antigen alone or mixed with B. pertussis or saponin (see the legend to Table 1). Delayedtype hypersensitivity, as a measure of cell-mediated immunity, was determined (Herbert, 1978) by the intradermal injection of 2.0,ug HB,Ag in 0.1 ml of phosphate-buffered saline (0.01 M-sodium phosphate buffer, pH 7.2, containing 1 % NaCI). Local induration was compared 48 h later with that produced by the injection of 0.1 ml of phosphatebuffered saline. Table I shows that humoral conversion rates varied among groups, with some of them responding earlier than others. A chi-square analysis of the time to first response showed a highly significant difference between pooled groups of animals treated with the liposomal (Groups IV, V and V1) and the free antigen (Groups I, I1 and 111) (x: = 22.10, P
BIOCHEMICAL SOCIETY TRANSACTIONS
Despite the inhibitory effect of (NH4)2WS4on the renal uptake of 64Cu, this compound increased the concentrations of non-radioactive copper in the kidneys. As with the plasma, this increase was greater in the rats receiving 28mg of copper/kg diet, and moreover could be correlated with the renal concentrations of tungsten. In this case there was always an equimolar increase in the concentrations of both metals. The (NH&WS4 also influenced the distribution of copper within the kidneys. Thus it decreased the amount of copper bound to metallothionein and increased that present in particulate and high-molecular-weight fractions. These observations are consistent with effects of (NH4)2WS4on the availability of copper to the rats both before and after its absorption from the intestine, as has been described for ammonium tetrathiomolybdate (Mills et al., 1978). Copper appears to accumulate in the blood and kidneys in forms where it cannot be utilized in a normal manner. The excellent stoicheiometric relationships between the concentrations of copper and tungsten in plasma and also in the kidneys may indicate that the metals are associated in some way with the same proteins, as was reported in molybdenumsupplemented sheep where copper and molybdenum were frequently found in the same chromatographic fractions from both plasma and kidneys (Bremner & Young, 1978). The effect of (NH&WS4 on copper metabolism in rats is therefore much greater than that of tungstate, which causes no change in plasma copper distribution, even when dietary concentrations of tungsten are 2g/kg (Cardin et al., 1976). It appears, therefore, that, as in the copper-molybdenum interaction, sulphur is an essential component of the copper-tungsten interaction. Brernner, I. & Young, B. W. (1978) Br. J. Nutr. 39,325-336 Cardin, C . J., Dennehy, A., Mason, J. & Roberts, C. (1976) Biochem. SOC.Trans. 4,873-877 Mills, C. F., Brernner, I., El-Gallad, T. T., Young, B. W. & Dalgarno, A. C. (1978) in Trace Element Metabolism in Man and Animals - 3 (Kirchgessner, M., ed.), pp. 150-157, Arbeitskreis fur TierernahrungsforschungWeihenstephan, Freising-Weihenstephan, Germany
Hepatitis-B-Surface-Antigen-ContainingLiposomes Enhance Humoral and Cell-Mediated Immunity to the Antigen EMAhXJEL K. MANESIS,* COLIN H. CAMERONt and GREGORY GREGORIADIS* *Division of Clinical Sciences, Clinical Research Centre, Watford Road, Harrow, Middx. HA1 3 UJ, U.K., and t Virology Laboratory, Middlesex Hospital Medical School, London W l P 7LD, U.K.
Accumulated evidence suggests that high titres of antibody against HB,Ag (hepatitis-B surface antigen), the outer coat of the putative virus, can protect man from natural hepatitis-B infection (Purcell et al,. 1978). The antigenicity of the HB,Ag in man is, however, weak, thus making necessary the use of an appropriate immunological adjuvant. Adjuvants applied in animals, although numerous, are by and large unacceptable to man because of side effects, and even those licensed for use in humans are far from ideal (World Health Organisation, 1976). More recently, attention has been drawn to the usefulness of liposomes as powerful and safe adjuvants potentially applicable in man (Gregoriadis, 1976a,b). In the present study we investigated the humoral and cellmediated immune responses of guinea pigs to free or liposome-associated HB,Ag and the possibility of a synergistic effect of liposomes and of Bordetella pertussis or saponin adjuvants. Purified HB,Ag (30-50,ug) was incorporated in liposomes composed of egg phosphatidylcholine (44,u~),cholesterol and dicetyl phosphate (7 :2: 1 molar ratio) (Manesis et al., 1978). Free or liposome-associated HB,Ag and its antibodies (anti-HB,) were measured by the AUSRIA I1 and AUSAB radioimmunoassay kits (Abbott G.m.b.H., Langen, Germany) respectively (Manesis et a[., 1978). Female outbred guinea pigs
1979
582nd MEETING, ST. ANDREWS
679
Table 1. Humoral conversion rate after immunization with HB,Ag Animals were treated on day 0, 18, 34 and 44 with free HB,Ag alone (I) or mixed with saponin (11) or B . pertussis (111) and with liposomal HB,Ag alone (IV) or mixed with saponin (V) or B. pertussis (VI). They were bled by cardiac puncture on days 18, 34,44 and 59. Four animals died during sampling. Conversion* Group I I1 I11 IV V VI
Day
...
34 115 115 016 515 516 616
18 015 016 016 316 116 416
44
. 59
115 315 416 515 616 616
214 415 516 515 616 616
Delayed-type hypersensitivityt 80
014 115 016 3I5 016 515
* Responders from total in' the group. t Animals showing delayed-typehypersensitivityfrom total in the group.
t
0
t 20
t
40
t
60
Time (days)after immunization Fig. 1. Increase in anti-HB, titres in guinea pigs immunized with HB,Ag Animals were treated (arrows) as described in Table 1. Each dose (0.6ml) given subcutaneously in both hind-limbs contained 1.4pg of free or liposornal (0.8mg of lipid) HB,Ag and, when appropriate, 1 . 2 10'' ~ bacteria or 50pg of saponin. 0 , Group 1; A , Group 11; 0, Group 111; 0 , Group IV; A , Group V ; W, Group VI. VOl. 7
680
BIOCHEMICAL SOCIETY TRANSACTIONS
(Hartley strain) were divided into groups of six and injected with liposomal o r free antigen alone or mixed with B. pertussis or saponin (see the legend to Table 1). Delayedtype hypersensitivity, as a measure of cell-mediated immunity, was determined (Herbert, 1978) by the intradermal injection of 2.0,ug HB,Ag in 0.1 ml of phosphate-buffered saline (0.01 M-sodium phosphate buffer, pH 7.2, containing 1 % NaCI). Local induration was compared 48 h later with that produced by the injection of 0.1 ml of phosphatebuffered saline. Table I shows that humoral conversion rates varied among groups, with some of them responding earlier than others. A chi-square analysis of the time to first response showed a highly significant difference between pooled groups of animals treated with the liposomal (Groups IV, V and V1) and the free antigen (Groups I, I1 and 111) (x: = 22.10, P
