Formaldehyde treatment of hepatitis B micelle vaccine

Journal of Virological Methods,

3 (198 1) 5 l-59

Elsevier/North-Holland

Biomedical

FORMALDEHYDE

TREATMENT

JACINTA

SKELLY,

Department

COLIN

51

Press

OF HEPATITIS

R. HOWARD

of Medical Microbiology,

B MICELLE VACCINE

and ARIE J. ZUCKERMAN

and WHO Collaborating Centre for Reference

Viral Hepatitis, London School of Hygiene and Tropical Medicine, London, (Accepted

18 March

Treatment method disruption

and Research on

U.K.

1981)

with formaldehyde,

for inactivation

during

of the 22 nm particles

under

conditions

the preparation with Triton

generally

of hepatitis X-100

used for viral inactivation,

B polypeptide

or after preparation

micelle

vaccine

is a suitable either before

of the micelles.

INTRODUCTION

In view of the repeated failure to grow and passage hepatitis B virus in tissue culture, it has not been possible to develop a conventional vaccine against this form of hepatitis. Attention has therefore been directed towards the use of other preparations for active immunisation. Since hepatitis B surface antigen, the essential protein coat of the virus, leads to the production of protective surface antibody as shown in experimental studies and seroepidemiological surveys, the use of purified and inactivated 22 nm spherical surface antigen particles is attractive. Available sources of the surface antigen include plasma from carriers,

the PLC/PRF/S

virus DNA, and mammalian 1981).

cell line, Escherichia

cells containing

The immunogenicity and protective efficacy of hepatitis B surface antigen particles which had been with formaldehyde was recently demonstrated by (Stevens et al., 1980; Maupas et al., 1981). These safe and essentially free of side-effects. Nevertheless,

many viral vaccines contain

coli

containing

cloned hepatitis

B

cloned viral DNA (reviewed by Zuckerman, vaccines prepared from purified from plasma and Szmuness et al. (1980) vaccines were found to

irrelevant

and sometimes

the 22 nm inactivated and others be entirely

reactogenic

com-

ponents derived from the host. The protection-inducing viral antigens in a highly purified form may offer a significant advantage over whole virus vaccines. In the case of influenza, for example, a vaccine consisting of haemagglutinin and neuraminidase subunits has been shown to be adequately immunogenic when given in two doses. and less reactogenic than whole inactivated virus. The prospect of a chemically well defined polypeptide subunit vaccine is particularly attractive in the case of hepatitis B, since many reports have indicated the presence in 22 nm surface antigen particles of contaminating host serum pro016660934/81/0000-0000/$02.50

@Elsevier/North-Holland

Biomedical

Press

52 teins which might depress the fevel and intensity

of the immune

response or induce un-

desirable immunological side-reactions. With this in mind, SkeIly et al. (1981) prepared water-soluble protein micelles from the polypeptides of the 22 nm hepatitis B surface antigen particles. The chemical purity, specific serological activity and immunogenicity the micelles,

taken together

their development Formaldehyde

with the ease of their preparation

of

on a large scale, favour

as an alternative ‘second generation’ hepatitis B vaccine. is widely used for the inactivation of viral vaccines, yet published

in-

formation on the effects of formaldehyde on hepatitis B surface antigen and its immunogenicity is lacking. We examined, therefore, the effects of formaldehyde treatment at two different stages of preparation of the vaccine micelles with regard to antigenicity and ~mmunogenicity, and the potential value of an inactivation safety of personnel during manufacture.

procedure

in terms of the

MATERIALSANDMETHODS ~i~~cati~n of the 22 nm hepatitis B surface antigen pm-ticks The purification of the 22 nm particles from the serum of a persistently infected chimpanzee has been previously described (Skelly et al., 1978, 1979). The preparations were radiolabelled with ‘25I using the Bolton and Hunter reagent. Is#~ati~~lof gp28/p23 c~rnp~e~es and fo~ation

of protein rn~ce~~es

The methods used to solubilise the 22 nm surface antigen particles, to isolate the gp28/p23 complex, and the formation of the protein micelles from this complex have been described in detail elsewhere (Skelly et al., 1978, 1979, 1981). ~-o~a~dehyde treatment Samples were incubated at 37°C for 72 h in the presence of formaldehyde at a concentration of 1 : 4000. Samples in the form of alum suspensions were washed by centrifugation and resuspension intact surface antigen, micelle formation.

in 0.01 M phosphate excess fo~aldehyde

buffer, pH 6.6, several times. In the case of was removed in the processes leading to

Polyacrylamide gel electrophoresis The conditions for sample disruption and electrophoresis in 10% discontinuous rical gels have been previously described (Skelly et al., 1978).

cylind-

53

Samples were tested for HBsAg by reverse passive haemagglutination the Hepatest reagents (Burroughs Wellcome, Beckenham,

Micelle samples were adsorbed

to aluminium

(RPHA) using

England).

hydroxide

(AI(OH

The concentra-

tions of samples were adjusted so that each dose contained 10 @g of protein in a 100 ~1 volume of 0.4% Al(OH& , Male SWR/J mice (6-8 weeks) were inoculated three times intrape~tonealIy at weekly intervals. Seven to 10 days after the final inoculation the mice were bled, and the levels of serum hepatitis B surface antibody were determined using a protein A radioimmunoassay

(Skelly et al., 1981).

RESULTS Two fo~aldehyde treatment procedures were examined: 1) purified, intact 22 nm surface antigen particles were treated prior to extraction of the gp28/p23 complex and formation of micelles, and 2) pre-formed micelles were treated after adsorption to the alum adjuvant, as a final ‘fail-safe’ inactivation procedure. Effect afire-treff~ent

of intact 22 nm surface antigen porticoes with fo~u~de~yde

In order to determine whether formaldehyde treatment interfered with the micelle preparation procedure and/or reduced the serological activity or immunogenicity of the final product, a pre-treated sample of 22 nm sample at each stage of the micelle preparation three stages: 1) disruption of the particles with p23 complex by affinity chromatography; and

particles was compared with a control procedure. This procedure consisted of Triton X-100; 2) extraction of the gp28/ 3) the formation of micelles by sucrose

density gradient centrifugation (Skelly et al., 1981). Table 1 shows the titres of hepatitis B surface antigen obtained by RPHA assay at each stage. Formaldehyde treatment had no apparent effect on the titre of the surface antigen before or after disruption with Triton X-100. When both control and treated samples were fractionated on a column of concanavalin A-Sepharose these behaved identically (Fig. 1). The distribution of radioactivity between the fraction which did not bind to concanavalin A-Sepharose and the fraction eluted by o-methyl mannoside was the same in both cases. The titre of surface antigen in the a-methyl mannoside eluates, which contain the gp281p23 complex, indicated that the serological activity of the fo~aldehyde-treated material appeared to be better preserved than that of the untreated material (Table 1). Equal amounts of material from the a-methyl mannoside eluate were centrifuged onto 20-50% w/v sucrose gradients in order to form protein micelles from the gp28/p23 complex. The distribution of radiolabel in the gradients is shown in Fig. 2. The peak

Fiy. I. Fractionation of disrupted hepatitis B surface antigen an a column of cnncanavalin A-Sephas rose. W, Control; O--O, formaldehyde-treated antigen.

of fo~aldehyde-treated

material was more diffuse and irregular than that of the control material, When the peak fractions of each gradient were pooled and adjusted to the same protein concentration, the titre of the surface antigen activity was again higher in the

formaldehyde-treated sample (Table I). Polyacrylamide gel electrophoresis of the micelles derived from fo~aldeh~de-treated surface antigen showed the presence of gp28 and ~23 (Fig. 3). However, there was an amaunt of material at the top of the gel, not observed in control preparations which

might indicate

that cross-~ink~g

of the two polypeptides

plexes of higher molecular weight. In order to determine the immunogenicity trol preparations,

each was adsorbed

had occurred

to produce com-

of micelles derived from treated and con-

to an alum adjuvant

and inoculated

into SWR/J

TABLE 1 Titres of controi and formaldehyde-treated

hepatitis B surface antigen during preparation of micelles

Stage of preparation

Control

Formaldehyde-treated

Intact 22 nm particles Triton X- 1O~d~rupted antigen Con-A ~ Sepharose eluate Micelies from sucrose gradient

32,000 14,000 102,400 16

32,000 16,000 409,600 64

Titres are expressed as the reciprocal dilution in a reverse passive haemagglutination assay (Hepatest, Burroughs Wellcome). It should be noted that the large difference in surface area of micelles compared with intact 22 nm particles implies that direct comparisons of such titres are not a quantitative measure of their relative antigenicity. For comparisons of relative anti~eni~ity by competitive radioimm~lnoprecipitation, refer to Skelly et al. (1981)

2

4

6

8

10

12

FRACTION

14

16

18

20

I

1

22

24

NUMBER

Fig. 2. Sucrose density-gradient centrifugation formaldehyde-treated (O--O) antigen.

of gp28/p23 complex derived from control (M)

or

56

~23

0

0.5 Relative mlgratlon

0

Fig. 3. SDS-PAGE of micelles derived from formaldehyde-treated

antigen.

mice. The levels of hepatitis B surface antibody in the mouse sera were determined. Fig. 4 shows the average serial dilution titres obtained in the protein A-radioimmunoassay for each inoculum. Although the pre-treated preparation elicited higher titres of surface antibody than the control, the difference was not significant.

Treatment of pre-formed hepatitis B polypeptide micelles with formaldehyde Pre-formed micelles adsorbed to an alum adjuvant were compared with an untreated control by the same mouse potency method. The results are shown in Fig. 5. Again, no significant difference was observed between treated and control preparations. DISCUSSION

Treatment with formaldehyde under conditions generally used for viral inactivation had little effect on the antigenicity or immunogenicity of hepatitis B surface antigen, whether in intact 22 nm particulate form or as protein micelles. Pre-treatment with formaldehyde had no effect on the ability of Triton X-100 to disrupt the particles, nor did it alter the behaviour of the disrupted material in affinity chromatography. There was a difference, however, in the distribution of radioactivity in the sucrose gradients during micelle formation between treated and control preparations. The formaldehyde-treated material was more broadly distributed throughout the gradient. The reason for this alteration is not known, but it is clear that it results in a lower total yield of micelles. Polyacrylamide gel electrophoresis suggested a certain amount of intra-complex cross-linking between gp28 and p23 as shown by the amount of protein which remained at the top of the gel. The immunogenicity of the pretreated preparation was well maintained, and it elicited a slightly, but not significantly greater surface antibody response than the control preparation. Pre-treatment of the 22 nm spherical particles is therefore

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RECIPROCAL

Fig.

4.

Average

derived

from

serial two-fold control

assay for surface vals, and bled described

(u)

antibody.

11 days

in Methods.

after

128 256

512 1024 2048 4096

DILUTION

dilution

curves

obtained

or formaldehyde-treated Five SWR/J

\Ij

with sera from

mice inoculated

(M)

in a protein

mice were inoculated

the final inoculation.

The protein

antigen with three

with micelles

A radioimmuno-

10 r.rg doses at weekly

A radioimmunoassay

was carried

interout as

58

i L

\

I

I

2

4

I1

b

8

16

32

I1

RECIPROCAL

Fig. 5. Average

serial

control

(M)

portion

was treated

two-fold with

for Fig. 4.

formaldehyde

SWR/J

128 256

I1

I

I

516 1024 2048 4096

DILUTION

dilution

or formaldehyde-treated

were used to inoculate

1

64

curves (O--O)

obtained

with

micelles.

as described

sera from

Micelles

in the Methods.

mice and the levels of surface

SWR/J

mice inoculated

were adsorbed

antibody

The treated induced

to Al(OH), and control

measured

with and a samples

as described

59

a feasible and suitable inactivation

procedure,

with the only drawback that yields may be

somewhat reduced (Fig. 2). However, micelles inactivated at the final stage of preparation, in the adjuvant form, showed no significant loss of immunogenicity. This procedure may perhaps be preferable to pre-treatment

in that yield is not affected.

In conclusion, the results show that formaldehyde treatment is a suitable method of inactivation in the preparation of a hepatitis B micelle vaccine. There is little alteration in antigenicity or immunogenicity of the micelles following formaldehyde treatment, regardless of the stage of the preparation at which it is introduced. Either of the procedures examined may therefore be appropriate for the production of the hepatitis B polypeptide micelle vaccine for use in man. REFERE:NCES

Maupas,

P., J.-P.

1981, Lancet

Chiron,

F. Barin,

P. Coursaget,

A. Goudeau,

J. Perrin,

Skelly, J., C.R. Howard

and A.J. Zuckerman,

1978, J. Gen. Virol. 41, 477.

, C.R. Howard

and A.J. Zuckerman,

1979, J. Gen. Viral. 44, 679.

J., C.R. Howard

and A.J. Zuckerman,

Skelly, J Skelly, Stevens,

C.E., W. Szmuness,

Szmuness,

W., C.E. Stevens,

Morrison Zuckerman, cerpta

and A. Kellner, A.J.,

Medica,

F. Denis and I. Diop-Mar,

1, 289.

1981,

AI.

Goodman,

E.J. Harley,

290,51.

E.A.

Zang,

W.R. Olesko,

1980, Lancet D.C. William,

2, 1211. R. Sadovsky,

J.M.

1980, N. Engl. J. Med. 303,833.

in: Liver Annual,

Amsterdam)

1981, Nature

S.A. Wesely and M. Fotino,

p. 8 1.

Vol. 1, eds. I.M. Arias,

M. Frenkel

and J.H.P.

Wilson

(Ex-