Journal of Virologikal Methods, 4 (1982) 177-185

Elsevier Biomedical Press

177

PREPARATION OF HEPATITIS B POLYPEPTIDE MICELLES FROM HUMAN

CARRIER PLASMA

PAUL YOUNG, MARK VAUDIN, JILL DIXON and ARIE J. ZUCKERMAN

Department of Medical Microbiology, and WHO Collaborating Centre for Reference and Research on

Viral Hepatitis, London School of Hygiene and Tropical Medicine, London WCIE 7HT, U.K.

(Accepted 14 December 1981)

Water-soluble protein micelles consisting of the 28,000 (gp28) and 23,000 (~23) molecular weight

polypeptide complex of hepatitis B surface antigen can be readily prepared from human plasma

containing the surface antigen and other markers of infection with hepatitis B virus. The antigenic

activity of the polypeptides was preserved throughout the process of solubilisation and reassociation

into micelles. Such preparations are therefore eminently suitable as ‘second generation’ hepatitis B

vaccines.

hepatitis B polypeptides micelles hepatitis B vaccine

INTRODUCTION

The formation of micelles composed of the 28,000 molecular weight glycoprotein

(gp28) and 23,000 molecular weight protein (~23) extracted from purified 22 nm hepa-

titis B surface antigen particles from the serum of a persistently infected chimpanzee

has been previously described (Skelly et al., 1981). Potency testing in SWR/J mice has

shown the micelles to be highly immunogenic. Indeed, consistently higher levels of

hepatitis B surface antibody were induced in mice immunised with the micelles than in

those receiving equivalent weight doses of intact 22 nm hepatitis B surface antigen

particles. The difference in antibody response was observed at each dose level tested.

In addition, the chemical purity, specific serological activity and ease of large-scale

preparation has favoured the development of micelle preparations as an alternative

‘second generation’ hepatitis B vaccine. We now report the relative efficiency of micelle

formation from surface antigen particles purified from plasma pooled from asymptomatic

human carriers.

MATERIALS AND METHODS

Plasma

A plasma pool containing a high titre of hepatitis B surface antigen and other markers

of hepatitis B virus: the complete virion, e antigen, DNA polymerase, and core antibody,

0166-0934/82/0000-0000/$02.75 @ 1982 Elsevier Biomedical Press

178

was obtained for base-line studies before commencing antiviral therapy from a persistent-

ly infected chimpanzee (Zuckerman et al., 1978). Plasma from asymptomatic carriers of

hepatitis B was kindly supplied by Dr. I. Murray-Lyon and Dr. M. Anderson of Charing

Cross Hospital, London. The titre of the surface antigen in the pool was 252 X lo3

British standard units/ml, and other markers of hepatitis B virus were present including

e antigen and core antibody.

~~~cati~~ of hepatitis E surface antigen Puri~cation of the 22 nm hepatitis B surface antigen particles from the plasma of a

persistently infected chimpanzee and from asymptomatic human carriers was carried

out as described previously (Skelly et al., 1978). Electron microscopy showed that both purified preparations were composed entirely of the 22 nm spherical particles.

Preparation of protein micelles from the gp28/p23 polypeptide complex The preparation of the micellar form of the gp28/p23 complexes purified from the

surface antigen particles was carried out as described previously (Skelly et al., 1979,

1981) with some modifications. Purified surface antigen in 0.01 M Tris-HCl, pH 7.3,

was disrupted by overnight incubation at 37°C in the presence of 2% Triton X-100.

The disrupted antigen was added to 1 ml of concanavalin A (Con-A)-Sepharose 4B

~ha~acia Fine Chemicals, Uppsala, Sweden) which had been equ~ibrated with 0.01 M

Tris-HCl buffer at pH 7.3, containing 2% Triton X-100, 0.5 M NaCI, 1 mM CaC12 and

1 mM MnCl, . The suspension was gently mixed on a rotator for 60 min and then packed

in a 1 X 10 cm column and washed with buffer to remove unbound material. Bound

material was eluted with 0.01 M Tris-HCI, pH 7.3, containing 2% Triton X-100, 0.5 M

NaCl and 5% cr-methyl-D-mannoside. Peak fractions were then centrifuged into 20-60%

w/v linear sucrose gradients in 0.01 M Tris-HCl buffer, pH 7.3, at 120,000 X g for 18 h

at 15°C in a Beckman SW40 rotor.

Electron microscopy Samples in either buffer or sucrose were applied directly to carbon formvar-coated

copper grids and allowed to adsorb for 5 min. The grids were washed by inversion over a

drop of distilled water and then stained with 1% uranyl acetate at pH 4.4 and containing

0.1% bacitracin. After staining, the grids were examined in a JEOL CX 100 electron

microscope operated at 80 kV.

Polyacrylamide gel electrophoresis (PAGE) Samples were solubilised for 2 min at 100°C in 0.1 M Tris-phosphate buffer, pH 6.7,

containing 2% sodium dodecyl sulphate (SDS), 1% 2-mercaptoethanol and 10% glycerol.

After addition of bromophenol blue as a tracking dye, the samples were applied to the

wells of a polyac~lamide slab gel composed of a 7.5-15% gradient resolving gel in

0.375 M Tris-HCl buffer, pH 8.9, and a 3% stacking gel in 0.08 M Tris-phosphate buffer,

pH 6.7. Electrophoresis was carried out at a constant 50 volts for 18 h. Gels were fixed

and then stained with Coomassie brilliant blue.

179

Radioimmunoassay Samples were tested for hepatitis B surface antigen activity by radioimmunoassay

(Ausria II, Abbott Laboratories, North Chicago, IL). A standard preparation of surface

antigen of known concentration (British standard preparation containing 2 units/ml) was

titrated in parallel.

RESULTS

Formation of micelles from the polypeptides of hepatitis B surface antigen

The 28,000 molecular weight glycoprotein (gp28) and the 23,000 molecular weight

protein (~23) can be readily isolated from intact surface antigen particles by solubilisation

with 2% Triton X-100 and affinity chromatography on a column of Con A-Sepharose

4B (Skelly et al., 1981). The binding efficiency of this column for the gp28/p23 complex

of either chimpanzee or human origin is 100% as determined by reverse passive haemag-

glutination (Hepatest, Burroughs Wellcome, Beckenham, Kent, U.K), and recovery

following elution with a-methyl-D-mannoside is greater than 90%. If this complex is

then centrifuged into a detergent-free sucrose gradient, protein micelles are formed which

retain specific antigenicity for eliciting an antibody response to hepatitis B surface

antigen (Almeida et al., 1981; Skelly et al., 1981; Tabor et al., 1982). The micelles

prepared in this study from purified 22 nm hepatitis B surface antigen particles of human

and chimpanzee carrier origin have buoyant densities in sucrose of 1.24 g-ml-’ and

1.22 g-ml-’ respectively (Fig. 1).

The SDS-PAGE profiles of both the human and chimpanzee micelles and the 22 nm

hepatitis B surface antigen particles from which they were prepared are shown in Fig. 2.

Apart from variations in some of the minor proteins which were resolved, reflecting the

incorporation of host antigens (Popper and MacKay, 1972; Zuckerman and Howard,

1973; Neurath et al., 1974; Burrell, 1975) the polypeptides of the 22 nm surface antigen

particles from both human and chimpanzee origin were found to be identical. The profiles

of both micelle preparations show them to be composed of the gp28 and p23 poly-

peptides, in the same proportions found in the original surface antigen particle.

Electron microscopy

Examination of both preparations showed the micelles to be spheroidal particles of

variable diameter in the range of 140-250 nm (Figs. 3 and 4). Measurement of over

200 particles of each preparation indicates that the human micelles are slightly larger,

with a mean diameter of 200 nm, compared with 180 nm for the micelles prepared from

chimpanzee surface antigen. Morphologically, however, both preparations are identical

(Fig. 4) the surface of the particles being composed of discrete globular and stranded

units.

The recovery of specific hepatitis B surface antigen activity was manitared by either reverse passive haemagglutination or radioimmunoassay after extraction of the gp2&/p23 complex from intact particles and after their formation into micelles, No Ioss of sero- logical activity was detected after disruption with 2% Triton X-f 00 or fo~o~~~g &&ion from the Con A-Sepharose coh~mn. fn fact, an increase in specific activity was recorded, which was probably a result of the rernaval of non-~mm~noreact~ve co~t~tuenta present in the originai particle (Helenius and Simons, 1975; Skelly et al., 1981). The intact surface antigen particles and the protein micelles reacted independently of suurce when titrated by radio~mu~oa~~y (Fig. 51, with both mice&? preparations showing a similar ~rjat~on in the slope of’ the dilution curve when compared with the intact 22 nm surface antigen particles.

P

182

Fig. 4. High magnification electron micrographs of a) human hepatitis B micelles; b) chimpanzee

hepatitis B micelles with added purified 22 nm intact hepatitis B surface antigen particles as a size

marker. The scale bars represent 100 nm.

DISCUSSION

The gp28/p23 complex was isolated and prepared in a micellar form from intact

22 nm surface antigen particles of both chimpanzee and human origin. The morphology

of both micelle preparations was identical. The micelles are composed of discrete globular

or stranded units randomly arranged on the surface and are spheroidal in shape and

variable in size, with only a slight difference in mean diameter between the chimpanzee

and human preparations. There was, however, a significant difference in the buoyant

density of the two micelle preparations, although intact human surface antigen and

chimpanzee surface antigen particles both have a density of 1 .16 g-ml-’ in sucrose. The

183

Reciprocal dilution

Fig. 5. Serial two-fold dilution curves obtained by radioimmunoassay as described in Materials and Methods of a) surface antigen particles; b) hepatitis B protein miceties. M, Chimpanzee; o--O, human.

reason for this variation is not clear, but it may reflect minor alterations in the structure

and confo~ation of the gp28/p23 complexes and hence the way in which they interact.

These conformational alterations could arise through variations in glycosylation, which

is host-mediated, and also through interactions with the proteins that are host-derived.

The SDS-PAGE profiles of the two micelle preparations show that the two component

polypeptides were present in the same stoichiometric ratio as in the original intact surface

antigen particles, indicating that neither polypeptide was preferentially packaged. This

can also be inferred from the evident linkage between the two proteins. which is demons-

trated by the binding ability of the non-glycosylated 23,000 molecular weight poly-

peptide to the Con A-Sepharose column. The recognition of a disulphide linkage be-

tween gp2.8 and p23 followed an SDS-PAGE analysis of both intact surface antigen particles and micelles in non-reducing conditions (data not shown). Samples were sol-

ubilised in the absence of 2-mercaptoethanol at 100°C for 2 min in 2% SDS prior to electrophoresis. It was found that approximately 2% of the total complement of gp28

and p23 existed as a dimer of molecular weight 50,000 in the intact surface antigen

particle, an observation which is in agreement with that reported by Mishiro et al. (1980).

This dimer was not detected in the micelle preparations. Approximately 5% was present

as individual polypeptides, but more than 90% of these two proteins found in the intact

particles and in the micelles remained at the top of the gel. Similarly, Mishiro et al.

(1980) found that following SDS solubilisation, 90% of the serological activity of the

surface antigen was collected as large aggregates in the void volume of a Sephadex G-200

184

column. These observations suggest that the gp28/p23 complex exists in its native form

as an extensive, disulphide-linked oligomer. This is further supported by electron

microscopy of preparations following solubilisation with either Triton X-100 or SDS

which shows large protein aggregates and frequently spherical particles with a ‘hazy’

surface and a diameter little less than that of the intact surface antigen particle. Large

aggregates of gp28/p23, free of other proteins, are also found in the fractions eluted

from the Con A-Sepharose column and may explain the formation of such large micellar

forms of the surface antigen. Previous reports of micelles prepared from a variety of

other membrane polypeptides describe homogeneous structures of 20-30 nm in dia-

meter (Simons et al., 1978) compared with a mean diameter of 200 nm for the micelles

of hepatitis B surface antigen.

The antigenic activity of the surface antigen polypeptides, as determined by radio-

immunoassay, was preserved throughout solubilisation and reassociation. In addition, the

radioimmunoassay titrations show that the micelles react independently of the source of

the surface antigen. The titration of the micelle preparations yielded dilution curves with

shallower slopes compared with those for intact surface antigen particles and suggests an

altered affinity for antibody for the polypeptides in micelle form. Such differences are

expected for particles which vary so dramatically in size, composition and in the distri-

bution and interaction of their antigenic sites.

No direct comparison of the titres, as presented, will allow a quantitative estimate of

the relative antigenicity of intact 22 nm particles and the much larger micelles. However,

it is estimated that at least 60-70s of the surface antigen activity was recovered in

micellar form when correction is made for the difference in surface area of the micelles

and the original 22 nm surface antigen particles.

In conclusion, the results show that protein micelles of the gp28/p23 complex of

hepatitis B surface antigen can be readily prepared from either a chimpanzee or human

source. Antigenicity is retained with a recovery approaching 70%. Studies on the im-

munogenicity of the micelles of human origin are in progress.

ACKNOWLEDGEMENTS

We are most grateful to Dr. I. Murray-Lyon and Dr. M. Anderson of Charing Cross

Hospital, London, for the supply of human plasma, to Dr. J. Skelly for helpful discussion

and to Mr. M. Bowerman for his technical help. The vaccine development programme is

supported by generous grants from the British Technology Group (previously the National

Research Development Corporation) and the Department of Health and Social Security.

REFERENCES

Almeida, J.D., J. Skelly, C.R. Howard and A.J. Zuckerman, 1981, J. Virol. Methods 2, 169.

Burrell, C.J., 1975, J. Gen.Virol. 27, 117.

Helenius, A. and K. Simons, 1975, Biochim. Biophys. Acta 415,29.

Mishiro, S., M. Imai, K. Takahashi, A. Machida, T. Gotanda, Y. Miyakawa and M. Mayumi, 1980,

J. Immunol. 124.1589.

185

Neurath, A.R., A.M. Prince and A. Lippin, 1974, Proc. Nat]. Acad. Sci. U.S.A. 71, 2663.

Popper, H. and I. MacKay, 1972, Lancet 1, 1161. Simons, K., A. Helenius, K. Leonard, M. Sarvas and M.J. Gethmg, 1978, Proc. Natl. Acad. Sci. U.S.A.

7595306. Skelly, J., CR. Howard and A.J. Zuckerman, 1978, J. Gen. Virol. 41,477. SkelJy, J., CR. Howard and A.J. Zuckerman, 1979, J. Gen. Virol. 44,679. SkeJly, J., CR. Howard and A.J. Zuckerman, 1981, Nature 290,51. Tabor, E., CR. Howard, J. Skelly, P. Snoy, A. Goudeau, A.J. Zuckerman and R.J. Gerety, 1982,

J. Med. Virol. (in press). Zuckerman, A.J. and C.R. Howard, 1973, Nature 246,445. Zuckerman, A.J., A. Thornton, CR. Howard, K.N. Tsiquaye, D.M. Jones and MR. Brambeii, 1978,

Lancet 2,652.