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Appl.No. 09/848,616 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




per 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 5 : 
C07K 15/04, A61K 39/29 



Al 



(11) International Publication Number: 
(43) International Publication Date: 



WO 92/11291 

9 July 1992(09.07.92) 



(21) International Application Number: PCT/EP9 1/02422 

(22) International Filing Date: 16 December 1991 (16.12.91) 



(30) Priority data: 
9027623.9 
9105993.1 



20 December 1 990 (20. 12.90) GB 

21 March 1991 (21.03.91) GB 



(71) Applicant (for all designated States except US): SMITH- 

KLINE BEECHAM BIOLOGIC ALS (S.A.) [BE/BE]; 
Rue de 1' Inst hut 89, B-1330 Rixensart (BE). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only) : VAN WIJNENDALE, 
Frans [BE/BE1; Teckerstraat 12, B-3040 Ottenburg (BE). 
BAIJOT, Michelle [BE/BE]; Avenue de Broqueville 153, 
B-1200 Bruxelles (BE). PRIEELS, Jean-Paul [BE/BE]; 
Avenue de Fevrier 7, B-1200 Bruxelles (BE). 



(74) Agent: DALTON, Marcus, Jonathan, William; Smith- 
Kline Beecham, Corporate Patents, Great Burgh, Yew 
Tree Bottom Road, Epsom, Surrey KT18 5XQ (GB). 



(81) Designated States: AT (European patent), AU, BE (Euro- 
pean patent), CA, CH (European patent), DE (Euro- 
pean patent), DK (European patent), ES (European pa- 
tent), FR (European patent), GB (European patent), GR 
(European patent), IT (European patent), JP, KR, LU 
(European patent), MC (European patent), NL (Euro- 
pean patent), SE (European patent), US. 



Published 

With international search report. 



(54) Title: VACCINES BASED ON HEPATITIS B SURFACE ANTIGEN 



(57) Abstract 

Novel antigen are presented which are useful in vaccine formulations for the prophylactic treatment of a range of infec- 
tious diseases. The antigens comprise a hybrid polypeptide, one part being the S antigen of the hepatitis B virus, the other being a 
heterologous antigen, such as gD from HSV. The two antigens are linked by chemical spacers through a native sulphydryl group 
present on the surface of the S antigen. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify States party to the PCT on the front pages of pamphlets publishing international 
applications under the PCT. 



AT 


Austria 


AU 


Australia 


BB 


Barbados 


BE 


Belgium 


8F 


Burkina Fa*> 


BC 


Bulgaria 


BJ 


Benin 


BR 


Brazil 


CA 


Canada 


CF 


Central African Republic 


CC 


Congo 


CH 


Switzerland 


CI 


Cote (Tlvoiru 


CM 


Cameroon 


cs 


Czechoslovakia 


DE» 


Germany 


DK 


Denmark 



ES 


Spain 


PI 


Finlund 


PR 


France 


CA 


Gabon 


GB 


United Kingdom 


CN 


Guinea 


GR 


Greece 


HU 


Hungary 


IT 


Italy 


JP 


Japan 


KP 


Democratic People's Republic 




or Korea 


KR 


Republic or Korea 


LI 


Liechtenstein 


LK 


Sri Lanka 


LU 


Luxembourg 


MC 


Monaco 



MC 


Madagascar 


ML 


Mali 


MN 


Mongolia 


MR 


Mauritania 


MW 


Malawi 


NL 


Netherlands 


NO 


Norway 


PL 


Poland 


RO 


Romania 


SD 


Sudan 


SE 


Sweden 


SN 


Senegal 


SU+ 


Soviet Union 


TO 


Chad 


TC 


Togo 


US 


United State* of America 



+ Any designation of "SU" has effect in the Russian Federation. It is not yet known whether 
any such designation has effect in other States of the former Soviet Union. 



WO 92/11291 



PCT/EP91/02422 



Vaccines based on Hepatitis B surface antigen 

The present invention relates to improved immunogens which 
comprise an antigenic polypeptide chemically conjugated to 
5 hepatitis B surface antigen (HBsAg) . The invention further 
relates to novel vaccines and their use. 

Proteins or synthetic peptides comprising epitopes of 
different viruses represent potential immunogens for use in 
10 vaccines against the infectious diseases caused by the 
respective viruses. However, such polypeptides frequently 
require a combination of carriers and adjuvants to become 
sufficiently immunogenic for consideration as vaccines. 

15 It would appear that correct antigen presentation is the key 
requirement for an effective subunit vaccine and Valenzuala 
et al. ( Biotechnology , 1985, 3., 323-326) have concluded 
that a good immunogen should have the maximum number of its 
epitopes properly exposed* This requirement was stated by 

20 these authors to be difficult to achieve by random chemical 
coupling of antigens to a carrier molecule. Accordingly a 
new approach was tried in which HBsAg was used as a carrier 
and first antigen molecule and the gene encoding a second 
antigen was recombined with the gene for HBsAg so that the 

25 second antigen was assembled into and presented on the 
surface of the HBsAg particle. Using an N-terminal fusion 
with a truncated form of the HBsAg middle protein Valenzuala 
et al. (loc.cit.) observed particle assembly of a Herpes 
Simplex Glycoprotein D - HBsAg hybrid polypeptide. The 

30 glycoprotein D (gD) epitopes were found to be presented in a 
repetitive fashion at the at the surface of the particle, 
thereby greatly enhancing the immunogenicity of the gD 
component . 



SUBSTITUTE SHEET 



WO 92/11291 



PCT/EP91/02422 



-2- 

More recently it has been shown that the major repetitive 
epitope of P. falciparum circumsporozoite (CS) protein could 
be fused to HBsAg. Immunogenicity of the hybrid particles 
was found to be superior to that of an equivalent monomeric 
5 CS antigen (Rutgers et al., Biotechnology , 1988, 6, 1065) . 
Vaccines prepared from hybrid immunogenic particles 
comprising HBsAg protein are also described in European 
Patent Application Publication No, 0 278 940. In all cases 
the hybrid particles were obtained by gene fusion rather 
10 than chemical coupling techniques. 

Despite the emphasis on gene fusion as a means to designing 
polyvalent vaccines it has now been found, surprisingly, 
that effective immunogenic molecules can be prepared by 
15 chemical cross-linking of HBsAg protein (or a suitable 

fragment thereof) having at least one free sulphvdryl group 
on its surface with another antigen. 

Accordingly the present invention provides an immunogenic 
20 hybrid polypeptide comprising a first polypeptide component 
which is HBsAg or fragment thereof displaying the 
antigenicity of HBV surface antigen, covalently linked via a 
native sulphur atom in the first polypeptide component to a 
second polypeptide component. 

25 

An advantage of the invention is that an antigen may be 
coupled to the HBsAg particle with cross-linking agents 
without impairing the immunogenicity of the HBsAg or 
fragment thereof as defined hereinabove. Furthermore it is 

30 possible by the present invention to conjugate the second 
polypeptide antigen with a vector (the HBsAg particle) which 
is able to direct its processing via a non-endosomial route. 
In this way the second antigen can become associated with 
MHC I antigens and be recognised as such by cytotoxic 

35 lymphocytes. Finally chemical coupling allows a higher 
degree of freedom with regard to antigen (epitope) density 



SUBSTITUTE SHEET 



WO 92/H S29J FCT/EP91/02422 

-3- 

on the HBsAg particle and also the possibility of using 
non-immunogenic spacers whereby the distance from the 
attachment point to the particle can be varied at will. 

5 In one aspect, the hybrid polypeptide of the invention may 
be represented by formula (I) ; 

P^S-X-P 2 (I) 

10 in which the group P^—S- is HBsAg or fragment thereof 
displaying the antigenicity of HBV surface antigen bonded 
via a native sulphur atom; 

P A is a second antigenic polypeptide; and 

15 

X is either: 

a) a group -A-NH- wherein A is a spacer group and NH- is 

the residue of an amino group present in the side chain of 
20 an amino acid in P 2 ; 

or optionally if P 4 ^ comprises a cysteine residue and a 
hydrophobic anchor group: 

25 b) the sulphur atom of the said cysteine residue present 

in P 2 . 

Preferably P2 is selected from gD2t from HSV or is a peptide 
capable of binding to HIV neutralising antibodies and 
30 corresponds to the neutralising domain of the V-j loop from 
gpl20; and 

The V3 loop peptides utilised in the present invention are 
preferably between 10 to 21 amino acids in length and 
35 comprises a p turn flanked by at least one and preferably at 
least two amino acid on both sides of the P turn sequences. 



SUBSTITUTE SHEET 



WO 92/11291 



PCT/EP91/02422 



-4- 

Preferably the sequence corresponds to the sequence 310 to 
328 of the gpl60 protein. 

The following peptide is preferred. 

5 

TYR THR ARG LYS SER ILE ARG ILE GLN ARG GLY PRO GLY ARG 
ALA PHE VAL THR ILE GLY 

The C-terminal tyrosine is optional, as its primary function 
10 is to allow labelling with radioactive Iodine. 

An important advantage of the hybrid particles according to 
the present invention is their ability to induce cellular 
immunity mediated by T lymphocytes and in particular the 
15 ability to induce cytotoxic T lymphocytes (CTL) responses. 

It will be appreciated that formula (I) is a simplified 
representation since the second antigen polypeptide P 2 will 
not normally be bonded to P 1 solely through one sulphur 
20 atom. Furthermore, P 1 is preferably in particulate form as 
described below. 

It is essential to the invention that the HBsAg or fragment 
thereof used as the first polypeptide component has at least 
25 one free thiol on its surface (e.g. may be represented as 
pl_SH) and to this end it is important that the HBsAg or 
fragment thereof is obtained in the correct manner. 

Commercially available vaccines against HBV comprise 
30 Hepatitis B virus surface antigen (HBsAg) either in native 
or recombinant form. The authentic Hepatitis B virus 
surface antigen can be recovered from plasma of infected 
individuals as a particle of about 22nm comprised of two 



SUBSTITUTE SHEET 



WO 92/12291 



PCT/EP91/02422 



-5- 

proteins known as P24 and its glycosylated derivative GP28, 
both of which are encoded by the 226 amino acid coding 
sequence on the HBV genome known as the S-protein coding 
sequence or HBV S-gene; see Tiollais et al, Nature , 317 
5 (1985), page 489 and references therein. The complete amino 
acid sequence of, and nucleotide sequence encoding, HBsAg is 
given in Valenzuela et al , Nature , 280 (1979), page 815. 
The numbering system used by Tiollais et al. ( loc cit.) to 
define nucleotide and amino acid positions is used herein. 

10 

Insertion of HBV S-gene coding sequences under the control 
of yeast promoters on expression vectors to enable 
expression of HBsAg in .S. cerevisiae for vaccine production 
has been described by, for example, Harford et al in 

15 Develop. Biol. Standard . 54: page 125 (1983), Valenzuela et 
al. , Nature 298 , page 347 (1982) and Bitter et al., J. Med. 
Virol . 25, page 123 (1988) . Expression in Pichia pastoris 
has also been described by Gregg et al, Biotechnology , 5 
(1987), page 479 (see also European Patent Application 

20 Publication No. 0 226 846) as has expression in Hansenula 
polymorpha (see EP-A- 0 299 108) . 

Not all the above methods give HBsAg suitable for use in the 
present invention since recombinant HBsAg produced in 

25 mammalian cells or yeast by the method of Valenzuala and 
others does not have available free SH groups; it is 
believed that the cysteine residues of HBsAg are all 
involved in the formation of disulphide bonds (Wampler et 
al . ( Proc.Natl. Acid. Sci. U.S.A. 1985, 82, 6830-6834 and 

30 references therein) . 

EP-A-0 135435 (assigned to Merck and Co.) describes a method 
for efficiently converting the non-disulphide bonded HBsAg 
antigen into a fully intermolecular disulphide bonded 
35 particle, alleged to be ten times more immunogenic than the 



SUBSTSTUiESHEcT 



WO 92/11291 



PCT/EP91/02422 



-6- 

antigen which has not been so treated. 

However, recombinant HBsAg as produced in s. cerevisiae by 
SmithKline Beecham Biologicals for the preparation of the 
5 vaccine Engerix-B* (Harford et al. loc.cit. ) does have an 
average of four free cysteines per S monomer and does from 
particles and this has been found to be at least as 
immunogenic as the fully intermolecular disulphide bonded 
particle. It will be apparent that when HBsAg is in this 
10 form then free cysteines provide one or more native sulphur 
atoms which can be utilised for coupling the second 
polypeptide. It will also be appreciated that preferably 
the HBsAg forms a particle, typically a lipoprotein 
particle. 

15 

It is to be understood that the first polypeptide component 
in the hybrid according to the present invention may 
comprise all or part or parts of the HBsAg precursor protein 
encoded by the coding sequence which immediately precedes 
20 the HBV-S gene on the HBV genome referred to herein as the 
Pre-S coding sequence. 

The pre-S coding sequence normally codes for 163 amino acids 
(in the case the ay HBV sub type) and comprises a pre-Sl 
25 coding sequence and a Pre-S2 coding sequence. The latter 
codes for 55 amino acids and immediately precedes the 
S-protein coding sequence (see EP-A-0 278 940 for further 
details) . 

30 In one preferred aspect the first polypeptide component P 1 
is the HBsAg S-protein having one or more, preferably up to 
4, sulfhydryl groups on its surface. 

* Engerix-B is a Trade Mark 

35 



SUBSTITUTE SHEET 



WO 92/1 1291 



PCT/EP9H/02422 



-7- 

From the foregoing it will be appreciated that the first 
polypeptide component is preferably prepared by recombinant 
DNA techniques, for example by expression in S. cerevisiae 
as described by Harford et al ( loc . cit . ) . i.e. corresponds 
5 to or comprises the HBsAg antigen present in the commercial 
vaccine Engerix-B*. 

In another aspect the first polypeptide component may 
comprise a fragment or truncate of the HBsAg S-protein 
10 provided the said fragment has at least one free sulphydryl 
group on its surface available for coupling to the group X, 
and provided that particle assembly is not adversely 
affected. 

15 In yet another aspect the first polypeptide component may be 
part of a composite particle comprising at least two 
polypeptides corresponding to part or all of a protein 
having the biological activity of one of the hepatitis B 
surface antigens wherein the particle presents at least two 

20 antigenic determinants provided by the S-protein, 

pre-S2-protein or pre-Sl-protein, said particle optionally 
containing host specific lipids, as described in copending 
European Patent Application No. 0414 374. 

25 An example of such a composite particle may be represented 
by (L,S) where L is the large protein of HBsAg (including 
the pre-Sl, pre-S2 and S coding sequences as hereinabove 
defined) and S is the HBsAg S-protein. 

30 In yet a further aspect the first polypeptide component may 
be a modified L protein of HBsAg as described in copending 
European Patent Application No. 0414 374 wherein the 
modified L protein comprises a modified hepatitis B virus 
large surface protein comprising an amino acid sequence 

35 

* Engerix-B is a Trade Mark 



SUBSTITUTE SHEET 



WO 92/11291 



PCT/EP91/02422 



-8- 

encoding the L protein characterized by a modification in at 
least one of the following sequences: a sequence sensitive 
to protease digestion, a sequence necessary for. 
myristylation, a sequence necessary for N-linked 
5 glycosylation, a sequence necessary for O-linked 
glycosylation, and a sequence necessary for binding of human 
serum albumin. 

In a preferred aspect the modified L protein may be 
10 represented by L* wherein L* has an amino acid sequence 
comprising residues 12-52 followed by residues 133-145 
followed by residues 175-400 of the L protein. 

In another preferred aspect the first polypeptide in the 
15 compound of the present invention may be part of a composite 
particle (I** f S) wherein L* and S are as hereinabove defined. 
Such composite particles may be prepared as described in 
copending European Patent Application No. 0414 374. 

20 The second polypeptide P 2 is an antigen useful in the 
preparation of a polyvalent vaccine and may be of any 
suitable structure. 

Specific antigens for P 2 include the recombinant DNA 
25 envelope protein gD of Herpes Simplex Virus (HSV) , 

particularly the truncated form of gD known as gD 2 t from 
HSV2. Other antigens which may be mentioned are malaria 
antigens, particularly those derived from the 
circumsporozoite protein, or antigens derived from HIV 
30 envelope protein. 

The human immunodeficency virus (HIV) has been identified as 
the causative pathogen of acquired immunodeficency syndrome 
(AIDS) . Like other members of the retroviral family, the 
35 genes encoding the major structural proteins of the virus 

* Engerix-B is a Trade Mark 



-Ql IRCTfTi rrc: oljetct 



WO 92/1 1291 PCT/EP91/02422 

-9- 

,are definded within the genome by env (viral envelope 
glycoprotein) and gag (core proteins) genes . 

The envelope, glycoprotein is known as gpl20. After 
5 infection with the AIDS virus, human beings develop 
antibodies against this glycoprotein. In many patients 
neutralising antibodies are produced although, it is also 
known that different HIV isolates exhibit a diverse array of 
sequence variation, particularly in the envelope gene. 

10 

Peptides from the major neutralising domain located in the 
envelope protein gp!20 in a region known as the V 3 loop are 
known to bind to neutralising antibodies generated in vivo . 
Nonetheless in order for those peptides to generate 
15 neutralising antibodies, correct presentation of the peptide 
is required. The present invention achieves this, by 
presenting such peptides on the surface of HBsAg in the 
manner described herein. 

20 In certain circumstances, the second polypeptide may 

comprise a hydrophobic anchor group (a hydrophobic 'foot') 
which may be naturally or synthetically attached to its 
amino terminal . 

25 Suitable hydrophobic anchor groups include fatty acid 
residues such as myristoyl, palmitoyl and lauryl. 

The advantage of having such a hydrophobic anchor group in 
the second polypeptide component is that it can, via 
30 hydrophobic interaction, become embedded in the lipidic 
membrane associated with the first polypeptide component. 

If the second polypeptide component also comprises an 
accessible cysteine residue the thiol group of the said 
35 cysteine can then, by spontaneous oxidation, form an 

intermolecular disulphide bond with a native thiol group in 
the first polypeptide component, thereby contributing to the 



SUBSTITUTE SHEET 



WO 92/11291 PCT/EP91/02422 

-In- 
stability of the formed complex. In such a case it is 
unnecessary to use a chemical cross linking agent to form 
the hybrid according to the invention (i.e. the group X as 
hereinabove defined may represent a sulphur atom which is 
5 native to P 2 as hereinabove defined) . 

The linker group A as hereinabove defined represents a 
linear spacer group bonded at one end to a native sulphur 
atom of the first polypeptide and at the other end to the 
10 second polypeptide P 2 via an amino acid containing side 

2 

chain m P . 

Examples of the group A are substituted C 2 -C 10 alkanes or 
linear polymers such as polyethylene glycol. 

15 

Particular groups A include: 

o 
I 



20 O 



and 

25 



— % - Oi 2 OA^ CO - 



The present invention further provides a process for the 
preparation of a hybrid polypeptide of formula (I) : 

P 1 -S-X-P 2 <D 

30 

wherein P 1 , P 2 and X are as hereinabove defined which 
process comprises the steps of 



SUBSTITUTE SHEET 



W0 92/11291 



PCT/EP91/02422 



-11- 

a) when X is an a group -ANH-: 

i) reacting the second polypeptide P 2 with a compound of 
formula (II) : 

5 

Y-A-B (II) 

wherein Y is a group capable of reacting with a native thiol 
group in the first polypeptide; B is a group which is 
10 specific for one or more amino acid side chains on the 
second polypeptide P and A is as hereinabove defined; and 
thereafter 

ii) reacting the product with the first polypeptide 
15 P 1 -SH; or 

b) when X is S and F 2 comprises a hydrophobic anchor group 
and a cysteine residue: 

20 i) mixing the first polypeptide and second polypeptide 

in aqueous solution; and 

ii) allowing an intermolecular disulphide bond between P 1 
and P^ to form by spontaneous oxidation. 

25 

Heterobifunctional reagents of formula (II) are known in the 
art and include N-succinimidyl 3- (2-pyridylthio) propionate 
[SPDP; (III)], succinimidyl 6-maleimidyl hexanoate [EMCS 
(IV) ], and N-succinimidyl 4- (iodoacetyl) amino benzoate [SIAB 
30 (V) ] . 



SUBSTITUTE SHEET 



WO 92/11291 



PCT/EP91/02422 



-12- 




o 

o * 




5 



-S-S-CH,-CH,-C-CMv| 



(III) 



10 



0 n 0 

< », > > 
NO-C(CH2}-N 

O 0 




(IV) 



o / v o \ 

II H /^\ II / 
I-CH,-C-N-( ( ) )-c-o-n 




15 



In method a) according to the above process steps i) and ii) 
my be carried out standard conditions as known in the art 
for cross-linking proteins, and the final product may be 
20 purified by, for example, preparative high pressure liquid 
chromatography . 

Particular hybrids which are the subject of the present 
invention include: 



HBsAg S protein crosslinked to gD 2 t of herpes simplex virus 
2 using reagent (V) ; 

HBsAg S protein linked to LCF6 as hereinbelow defined via an 
30 intermolecular disulphide bond, and HBsAg S-protein linked 



25 



to V 3 peptides from HIV gp!20; 



SUBSTITUTE SHEET 



WO 92/11291 



FCI7EF91/02422 



-13- 

Typically the ratio of the second polypeptide to the first 
polypeptide will be in the range 0.1 to 1.0 molecules per 
monomer. Although in the case of V 3 peptides this may be in 
the range of 1.0 to 4 molecules per monomer. 

5 

In a further embodiment of the present invention, the 
polypeptide P 1 may be mixed with a mixture of V 3 peptides. 
The resulting fusion with then have different v 3 peptide 
attached to the surface of P 1 . 

10 

In the present case the efficiency of the reaction is such 
that approximately forty peptides are linked to each 
particle it being understood that the hybrids according to 
the invention are preferably in particulate form. 

15 

In a further aspect the invention provides a vaccine 
composition comprising an immunoprotective amount of a 
hybrid polypeptide according to the invention together with 
a conventional carrier or adjuvant. 

20 

A preferred adjuvant, according to the present invention is 
de - 3 - 0 - acylated monophosphorylated lipid A (3D-MPL) in 
a suitable carrier. This adjuvant system provides high 
neutralising antibody titres. 

25 

3D-MPL may be obtained by the methods described in U.K. 
patent No. 2,211502 (RIBI) . 

In the case of utilising HIV peptides the present inventors 
30 have found that exceptional results may be achieved by first 
adsorbing the HBsAg-V 3 peptide conjugate of the present 
invention on to alum and then admixing with 3D-MPL. 



SUBSTITUTE SHEET 

I 



WO 92/11291 



PCT/EF91/02422 



-14- 

Additionally, oil in water emulsions containing 3D-MPL 
provide excellent results. The oil in water emulsion 
formulation provided by the present invention most 
5 preferably comprises, 3D-MPL, squalane, pluronic L -121 and 
phosphate buffered saline. 

The emulsion is preferably passed through a microfluidizer 
to provide submicron particles in the emulsion. This 
10 enhances the activity of the formulation. 

Alternatively vaccines containing the hybrid polypeptide 
according to the invention are prepared by conventional 
techniques and will contain an immunoprotective amount of 

15 the hybrid preferably in buffered physiological saline and 
admixed or adsorbed with any of the various known adjuvants 
including aluminium hydroxide and aluminium phosphate. By 
' 'immunoprotective" is meant that enough of the hybrid is 
administered to elicit a sufficient protective antibody or 

20 cell mediated immune response to confer protection against 
an infectious agent without serious side effects. The 
amount of hybrid to be administered will depend on whether 
the vaccine is adjuvanted and will generally comprise 
between 1 to 1000 meg of total protein, for example 1 to 200 

25 meg total protein, more preferably 5 to 40 meg total 

protein. The amount and number of doses to be administered 
can be determined in standard dose range studies involving 
observation of antibody titres and other responses in 
subjects. 

30 

The hybrid polypeptide according to the invention may also 
be mixed with other antigens such as composite HBsAg 
particles containing all or part or parts of the PreSl or 
PreS2 polypeptides for vaccine formulation. It may also be 
35 mixed with fused or other chemically synthesised hybrid 



SUBSTITUTE SHEET 



WO 92/11291 



PCT/EP91/02422 



-15- 

HBsAg particles carrying epitopes from proteins from other 
organisms and with other immunogens to form multivalent 
vaccines. Vaccine preparation is generally described in 
"Vaccines", edited by Voller et at, University Park Press, 
5 Baltimore, MD, U.S.A., 1978. 

Accordingly in a further aspect, the invention provides a 
method of immunising a subject against viral infection which 
comprises administering to a subject in need of such 
10 immunisation an effective amount of a vaccine composition 
according to the invention. 

The following examples illustrate the invention. 
15 Examples 

A. Determination of free sulfhvdrvls on the recombinant 

HBsAg particle 

20 A.l. Material and Methods 

a) BNP Method 

A method for identifying cysteine-containing peptides in 
25 proteins was applied using 2-bromoacetamido-4-nitrophenol 
(BNP) to introduce an easily detectable probe. The 
formation of a covalent bond between the protein sulfhydryl 
group and the acetamido moiety of BNP introduces a 
chromophore with an absorbance maximum at 410 mm. The 
30 modified protein can then be cleaved with appropriate 
proteases and the resulting peptides separated by 
chromatographic methods. Monitoring the effluent at a 
single wavelength (405 nm) provides a rapid and simple 
method for detecting and isolating only those peptides which 
35 contain cysteine residue (s) . 



SUBSTITUTE SHEET 



W0 92/1129H 



PCT/EP91/02422 



-16- 

Materials 

HBsAg was from SmithKline Biologicals. NH 4 HC0 3 , 
2-bromoacetamido-4-nitrophenol (BNP) and chymotrypsin were 
5 from Sigma. Trifluoroacetic acid (TFA) and acetonitrile 
were from Baker (HPLC grade) . N,N-dimethylformamide (DMF) 
was from Janssen Pharmaceutica . 

Protein labelling 

10 

Protein (± 1 mg) was dialyzed against labelling buffer (0.1 
M Tris-HCl, EDTA 2 mM, Urea 8M, pH 8.6). 2,5 mg of BNP 
(dissolved in DMF) per mg of protein was added to the 
denatured protein. After 90 minutes of incubation at room 
15 temperature, unreacted BNP was removed by extensive dialysis 
against NH 4 HC0 3 lOOmM, pH 8.5. 

Digestion of modified protein 

20 Duplicate addition of chymotrypsin (t Q and t Q + 4 hours) (2% 
W/W) was made. Incubation was then carried out at 37°C 
overnight. The digested protein was stored at 4°C. 

HPLC separation of the peptides was accomplished on a Waters 
25 600 HPLC system fitted with a 250 mm x 4.6 mm Vydac C 4 

reverse-phase column. The column was equilibrated with HPLC 
buffer (NH 4 HC0 3 100 mM, pH 8.5) prior to injection of the 
sample (± 1 mg) . Peptides were eluted with a linear 
acetonitrile gradient from 0 to 50% in 50 minutes. The 
30 elution was monitored at 405 nm and at 224 nm on a Waters 
490 Multiwave Detector. 



SUBSTITUTE SHEET 



WO 92/1 1291 

-17- 

Secruence analysis of the BNP peptides 



PCT/EP91/02422 



Dried sample was redissolved in 60 \il TFA 6%, and applied on 
the glass fiber membrane of the sequenator. The membrane 
5 was preferably treated with Biobrene. Edman automatic 
sequential degradation was performed with a liquid phase 
sequenator (Applied Biosystem 477A) coupled with an amino 
acid analyser (Applied Biosystem 120A) . 

10 The program used is analogous to that of Hewick et al. J. 
Biol. Cheitu , 1981, 256 , 7990-7997 . Phenylthiohyantoin 
derivatives of cleaved amino acids were identified by RPLC, 
following the gradient system described by Hunkapiller and 
Hood, (Methods Enzymol . 1983, 91, 486-493) . 

15 

b) PDS Method 

The method of Grasetti et al ( Arch . Biochem . Biophvs . , 1967, 
119 , 44-4 9) was followed. Upon reaction of protein thiols 
20 with 2, 2' -dithiodipyridine (thiol disulfide exchange 

reaction) 2-thiopyridine is liberated and measured at 343 nm 

< £ 343 nm =8 ' 08x 1q3m > • 
Materials 

25 

HBsAg was from SmithKline Biologicals. Dithiothreitol (DTT) 
and 2, 2' -dithiodipyridine (PDS) were from Serva. PD 10 gel 
filtration columns were from Pharmacia. 

30 Assay procedure 

Protein (± 750 jig/ml) was dialyzed against buffer (Urea 8M, 
EDTA 2 mM # 0.1 M Tris-HCl, pH 8,5). PDS (25 M in excess, 
dissolved in ethanol) was added to the denatured protein. 
35 After one hour of incubation at room temperature, excess of 



SUBSTITUTE SHEET 



WO 92/11291 



PCT/EP91/02422 



-18- 

PDS was removed by gel filtration (PD 10 column) . 
Absorbance of the modified protein was measured at 280 nm. 
Addition of DTT (10 mM final concentration) was made to 
measure the absorbance of the free thiones at 343 nm. 

5 

c) Carboxvmethvl-cvsteine determination 

Materials 

10 Iodoacetamide was from Merck. HBsAg was used from 
SmithKline Biologicals. 

Carboxymethvlation 

15 Protein (1 mg/ml) was dialyzed against buffer (Tris 100 mM, 
EDTA 2mM, pH 8.0) with or without Urea 8M. The 
S-carboxymethylation of the thiol groups was performed by 
addition of iodoacetamide (100 moles per mole of sulfhydryl 
groups) for 20 minutes, in the dark at room temperature. 

20 

Amino acid analysis 

Hydrolysis: Aliquots of protein (± 100 |ig) were dried in 
conic hydrolysis tubes, in a Speed Vac Concentrator. 
25 Hydrolysis was performed by addition of HC1 6N (500 

containing 0.5% phenol, at 110°C during 24 hours. Samples 
were done in triplicate. 

After cooling, hydrolysate was evaporated to dryness, washed 
30 in 500 p.1 water, and dried again. Having been dissolved in 
200 |il of 0.2 N pH 2.2 citrate buffer and filtered on a 45 
Jim membrane, 50 \il of each sample is injected on the 
analyser column. 



SUBSTITUTE SHEET 



WO 92/11291 



PCT/EP91/02422 



-19- 



Amino acid analysis ; was performed with an Automatic 
Analyser (Alpha Plus-LKB 4151) on a polystyrene sulfonated 
column. A three buffer elution system was used to separate 
the different amino acids: 



10 Amino acid detection was made by post-column reaction with 
ninhydrin and by colorimetric measurement at 440 and 570 nm. 
For quantification, optical densities at 440 and 570 nm are 
added together and chromatographic data integration were 
performed on a Shimadzu CR-A3 integrator. 

15 

A. 2 . Results 

a) BNP Method 

20 After modification of the free thiols by BNP and 

chymotryptic digestion, peptides containing the BNP label 
were separated by RPLC and identified by sequencing, 
following the procedure of Gardner et al . Anal. Biochem . , 
1987, 677, 140-144. 

25 

HPLC analysis of 5 different HBsAg chymotryptic digestions 
yielded 5 well separated major peaks indicating a 
reproducible oxidation state for all batches. The sequences 
of the BNP labelled peptides allow the localization of 4 
30 thiol groups (cys 48, 65, 121 and 124) . 

The identical analysis was performed on HBsAg produced in 
mammalian cells. No BNP labelled peak could be detected 
indicating that no free cysteines were available. 



5 



2. 



3. 



1. 



citrate buffer 0.2 N pH 3.2 at 54°C 
citrate buffer 0.2 N pH 4.25 at 56°C 
borate buffer 0.2 N pH 10.0 at 90°C 



35 



SUBSTITUTE SHEET 



WO 92/11291 



PCT/EP91/02422 



-20- 

b) PDS Method 

Four thiol groups per monomer of HBsAg were detected after 
application of the PDS method on 3 different batches of 
5 HBsAg particles from SmithKline Biologicals (Batches 1 to 3 
in Table 1 below) No SH free per monomer could be detected 
in a batch of HBsAg expressed in Chinese hamster ovary (CHO) 
cells (Batch 4) . 

10 Table 1 ; 



PDS Method: FREE THIOL GROUPS DETERMINATION 



15 


BATCH 


SH FREE PER 




NUMBER 


MONOMER 




1 (yeast) 


4.38 




2 (yeast) 


3.18 


20 


3 (yeast) 


4.2 




4 (CHO cells) 


0 



25 c) Carboxvmethyl - cvsteine determination 

Cysteines in HBsAg particles were carboxymethylated with or 
without denaturing agent (Urea 8M) . 

30 Subsequent amino acid analysis showed the presence of 3 
carboxymethylcysteine per monomer of HBsAg in both cases. 



Ol IRQTITI !TF= SHEET 



PCT/EP91/02422 

-21- 

Examples of coupling antigens to the HBsAa particle 

EXAMPLE 1; Covalent coupling of the glycoprotein D 
of Herpe s Simplex 2 Virus to a particulate carrier 

5 

B.l.l. Introduction 

The glycoprotein D of HSV 2 (gD 2 t) expressed in CHO cells 
(Lasky and Dowbenko DNA, 1984, 3(1), 23-29) is covalently 
10 coupled to a recombinant HBsAg particle containing free SH 
groups . 

B.1.2. Materials and Methods 
15 a. Agents 

5,5'Dithiobis 2-nitrobenzoic acid (Ellman's reagent 
or DTNB) and N-succinimidyl (4-iodoacetyl) - aminobenzoate 
(SIAB) were purchased from PIERCE, 

20 

2, 4, 6-Trinitrobenzenesulfonic acid (TNBS) was 
obtained from SERVA. 

Recombinant gD 2 t was expressed in CHO cells and 
25 purified by SmithKline Biologicals. 

gD 2 t was iodinated by the enzymobeads method of 

PIERCE. 

30 . HBsAg particles were produced by SmithKline 

Biologicals . 

b. Methods 



1291 

B. 



SUBSTITUTE SHEET 



WO 92/11291 



PCT/EP91/02422 



-22- 

b . 1 . Characterization of gDot 

b.1.1 Quantitative determination of sulfhydryl groups: 

5 To 150 ^1 of gD 2 t (23 HM in Na 2 HP0 4 0.02 M pH 7) 1 ml of 
DTNB (3.28 mM in Na 2 HP0 4 0.04 M pH 8) is added. 

After 5 min, the optical density at 412 nm is determined 
against a blank lacking protein. An extinction coefficient 
10 of 1.3 x lO^M- 1 cm- 1 is used to calculate the concentration 
of sulfhydryl groups reacting (Ellman, Arch. Biochem, 
Biophvs . , 1959, 82/ 7 0) . 

b.1.2 Quantitative determination of lysine residues: 

15 

50 \il of TNBS/H 2 0 24.5 mM are added to 50 \il of gD 2 t (58 \XM 
in Na 2 HP0 4 0.02 M pH 7) diluted in 200 Jil of borate buffer 
(0.05 M Na 2 B 4 0 7 adjusted to pH 9.5 with 0.05 M NaOH) . 

20 After 3 hours in the dark at room temperature the change in 
absorbance at 367 nm is followed against a blank without 
protein . 

The extent of trinitrophenylation is calculated on the basis 
25 of an e 367 nm = 1.1 x 10 4 M- 1 cm- 1 (Plapp et al . , J. Biol. 
Chem . 1971, 246 (4), 939-945). 

b.2. Activation of gD o t with SIAB 

30 100 of gD 2 t (23 \m in Na 2 HP0 4 0.02 M pH 7) + 100 Jll gD 2 t 
I 125 are incubated for 30 min at 37°C with 2 jil SIAB (25 mM 
in DMSO) which corresponds to a molar ratio 
succinimide/lysine of 2. The excess of cross-linker is 
eliminated by dialysis (2 hours against Na 2 HP0 4 0.02 M pH 8) 

35 and the reaction mixture is concentrated to 100 p.1 by 



SUBSTITUTE SHEET 



WO 92/11291 PCT/EF91/02422 
ultrafiltration on an YM 10 centricon. 

b.3. Coupling to HBsAq particle 



5 gD 2 t (100 , concentrated (1 mg/ml) and SIAB-activated, is 
incubated with 53 ill of HBsAg particles (1 mg/ml in Na 2 HP0 4 
10 mM pH 7.2, NaCl 150 mM) for various times at 37C°C. 



The initial molar ratio gD 2 /S monomer is 1/1. 
10 The particulate gD 2 t is purified by a 1,5 M CsCl gradient 
(45 hours, 65000 rpm in a 70.1 Ti rotor) . 



b.4 . Quantification of qD2t coupled per particle 

15 50 \il of water are added to a vial of enzymobeads . After 
one hour, 50 \il of Na 2 HP0 4 0.2 M pH 7.2, 25 jil gD 2 t (1 
mg/ml), 0.5 mCi Nal 125 (Amersham) and 25 ill 1% p-D-Glucose 
are added. 



20 After 20 min at room temperature, the reaction is completed 
and the iodinated protein is separated from free iodine by 
chromatography on DOWEX Ag 1 x 8 resin saturated by BSA 1%. 

The specific activity of the gD 2 t involved in the coupling 
25 may be determined by the radioactivity detected in the 
mixture of labelled and non-labelled gD 2 t. The amount of 
gD 2 t coupled to particles may be determined by this specific 
activity. 



SUBSTITUTE SHEET 



WO 92/11291 



PCT/EP91/02422 



-24- 

B.1.3. Results 

a. Characterization of gD2t 

5 a.l. Quantitative determination of sulfhvdrvl groups No 
free thiol is detected on the gD 2 t by DTNB. This result 
fits the aminoacid sequence of the protein . The truncated 
gD 2 t molecule used (283 aa) contains 6 cysteine residues, 
each involved in disulfide bridges that constitute 
10 discontinuous epitopes. Therefore, gD 2 t is an ideal 

molecule for the activation step with a heterobifunctional 
cross-linker without risk of homopolymerisation. 

a. 2. Quantitative determination of lysine residues 

15 

The number of free amino groups are detected by TNBS either 
on the native or on the SIAB activated gD 2 t. The number of 
free lysines decreases as a function of the excess of SIAB. 

20 With a molar ratio SIAB/lysine of 2, four residues are 
activated. 

The number of detected lysines on the native protein (10) is 
close to the number determined in the amino acid sequence 
25 (11) . 

b. Activation of gD2t by SIAB 

After an activation of 30 min at 37°C with a molar ratio of 
30 SIAB/lysine of 2, the absence of homopolymers of gD 2 t is 
checked by gel filtration. 

On a TSK 3000 column, the homopolymers elute in the void 
volume (8 min) and the monomeric gD 2 t has a retention time 
35 of 15 min. 



ftl INSTITUTE SHEET 



WO 92/11291 



PCT/EP91/02422 



-25- 

Despite the absence of cysteine residues in the protein, 
formation of homopolymers is observed when gD 2 t is activated 
at a concentration of 2.5 mg/ml. An aspecific reactivity of 
the halogen in SIAB for lysine, methionine or histidine 
5 residues may explain this phenomenon (see Means and Feeney, 
Chemical Modifications of Proteins; Holden Day publ., 1971, 
page 107) . 

Initial protein concentration is a decisive factor in 
10 homopolymerisation events. If gD 2 t is activated at a 
concentration of 0.5 mg/ml, the formation of homopolymers 
decreases from 50 to 10%. 

c. Coupling to HBsAq particle 

15 

The HBsAg-gD 2 t conjugate obtained after a 30 min, 2 hrs or 
over night incubation at 37°C is purified by CsCl gradient. 
The gD 2 t homopolymers have a different density to the 
carrier and do not contaminate the conjugate. The yield of 
20 coupling increases with time as shown in Table 2. 0.2 gD 2 t 
molecules are coupled per S monomer (twenty per particle) as 
calculated by radioactivity detected in the particle's 
density area. 

25 Table 2 : Influence of incubation on the yield of coupling 



Time 


gD 2 t/S monomer 


30 min 


0.08 


2 hrs 


0.14 


15 hrs 


0.20 



SUBSTITUTE SHEET 



WO 92/11291 PCT/EP91/02422 

-26- 

B.2. Example 2 

B.2.1. Incorporatio n T.f!F6 rLaurovl-Cy_S-Tjn:-Gly_-Glv.- 
(NPNA161 into HBsAg pa rticles 

5 

10 Hi of LCF6 solution (1 mg/ml in phosphate 10 mM pH 7, 
NaCl 150 mM) are incubated with 10 Hi of a solution of yeast 
expressed HBsAg particle (ex SmithKline Biologicals) at 1 
mg/ml in phosphate 10 mM P H 7, NaCl 150 mM overnight at 
10 37°C. 

B.2-2. Assay 

Quantitation of the HBsAg-LCF6 hybrid is performed by a 
15 sandwich ELISA test using as coating antibody a polyclonal 
IgG against HBsAg at a concentration of 5 ul/ml and as 
detecting antibody a biotinylated monoclonal IgM directed 
against the repeat sequence of LCF6. 

20 An antigen R16 HBsAg solution (a recombinant repeat 

Malaria-HBsAg particle) of known concentration is used as 
standard. This ELISA presents the advantage of monitoring 
the hybrid without prior separation of the free peptides. 
The results are expressed in ug/ml equivalent R16 HBsAg. 

25 

B.2.3. implication of a cov* lent bond in the LCF6 
incorporation into HB sAa particle 

To evaluate the relative important of the disulfide bridge 
30 and of the hydrophobic foot, the incorporation of the 
peptide without cysteine and of the peptide without the 
lauroyl group is compared. The ELISA shows that the 
lipopeptide lacking cysteine (LF6) fails to incorporate into 
the HBsAg particle. The peptide without the lauroyl group 
35 (CF6) is nearly ineffective. Therefore the synergy between 



SUBSTITUTE SHEET 



WD 92/11291 PCT/EP9S/02422 

-27- 

hydrophobic and covalent interactions is critical for the 
peptide's incorporation into HBsAg particle. 

The results from LCF6 incorporation into different HBsAg 
5 particles with variable oxidation state were compared. 

The lipopeptide is incubated with classical HBsAg particle 
(± 4 free SH groups/S monomer), with more oxidized particles 
(1 free SH group/S monomer), or with totally oxidized 
10 particles (no free SH group; particles synthesized in CHO 
cells) . 

These results indicated that a disulfide bridge between LCF6 
and S monomer stabilizes the peptide incorporation. The 
15 ELISA for anti-HBsAg-LCF6 show decreasing coupling as a 
function of an increasing oxidation state of HBsAg 
particles. 

The existence of covalent linkage between LCF6 and HBsAg 
20 particle is also demonstrated by analysis on SDS-PAGE. 
Bands at 26 and 50 Kd appear in non-reducing conditions. 
These bands correspond to the LCF6 peptide coupled to the S 
monomer and dimer . 

25 In contrast, in reducing conditions, these bands disappear. 

B3 . Example 3 : Inco rporation of V 3 peptides into HBsAg 
particles 

30 3.1. V3 loop structure 

The peptide used represents the sequence from amino acid 310 
to 328 (G. Larosa, Science 1990, 24 9-932) comprised in a 
disulfide bridged loop in the third variable region of the 
35 external protein gp!20. Its conservation is over 80% in 9 



SUBSTITUTE SHEET 



WO 92/11291 



PCT/EP91/02422 



-28- 

out of 14 positions in the central portion and its predicted 
structural motif is of the type p strand-type Hp turn-p 
strand-a helix. 

5 The primary structure of the peptide can be represented by 
the following 

TYR THR ARG LYS SER ILE ARG ILE GLN ARG GLY PRO GLY ARG 
ALA PHE VAL THR ILE GLY 

10 

3.2. Synthesis 

The peptide is synthesized according to the Merrifield solid 
phase method, giving, after purification by reverse phase 
15 hplc, a peptide of 97% purity. The peptide is homogeneous 
by SDS-PAGE and gel filtration on TSK 2000 column. Its 
molecular weight is 2800. 



20 3.3. Coupling strategy 

EMCS, (succinimidyl 6-maleimidyl hexanoate an 
heterofunctional crosslinker containing a primary amino 
reactive group N-hydroxysuccinimide (NHS) and a thiol group 
25 (maleimide) , was chosen. 

3.3.1. Activation 

The V3 peptide contains one Lysine group which was activated 
30 with EMCS for 30 min at 37°C, pH 7, in a 1:1 to 4:1 ratio. 

3.3.2. Coupling to HBs 

After elimination of the excess of crosslinker on a G 10 
35 column, HBsAg particles were added in a 1:1 ratio with the 



SUBSTITUTE SHEET 



WO 92/11291 



PCT/EP91/02422 



-29- 

V 3 peptide. The reaction took place overnight at 37°C, pH 
6.5 - 7.5. 

The conjugate obtained is purified from the unconjugated 
5 peptide by gel filtration on HR 200 column. 

3.3.3. Coupling efficiency 

As assayed by radioimmunoassay and ELISA, the coupling 
10 efficiency was of 0.4 peptide coupled per HBs monomer, which 
is equivalent to 40 V3 peptides per HBs particle or to 1 mg 
V 3 per 25 mg HBsAg. 

15 Example C 

Antigen-Adjuvant preparations 

1. V3 HBsAg particles in oil water emulsion 

20 

The vehicle is prepared as follows. To phosphate-buffered 
saline (PBS) containing 0.4% (v/v) Tween 80 is added 5% 
(v/v) Pluronic L121 and 10% squalane. This mixture is then 
microf luidized. For microf luidization, the emulsion is 

25 cycled ten times through a microf luidizer (Model MHO 
Microf luidics Corp., Newton, Mass.). After five passes 
through the Microfluidizer, the resulting emulsion comprises 
only submicron particles.. 50 \ig 3D-MPL is then added to 
this emulsion. One volume of this emulsion containing 3D-MPL 

30 is mixed with an equal volume of twice concentrated V3 HBsAg 
and vortex briefly to ensure complete mixing of the 
components. The final preparation consists of 0.2% Tween 
80, 2.5% Pluronic L121, 5% squalane, 50 ng 3D-MPL and 1 ^g 
equivalent V3 peptide (corresponding to 25 ^g HBsAg) in a 

35 250 \il injection dose. 



SUBSTITUTE SHEET 



WO 92/11291 



PCT/EP91/02422 



-30- 

1.2 V 3 HBsAq/Aluminium Hvdroxvde plus 3D-MPL 

1 \ig equivalent V 3 is adsorbed overnight at 4°C on alum 
corresponding to 0.5 mg equivalents Al 3+ in 0.25 ml of 150 
5 mM NaCI, 10 mM phosphate buffer pH 6.8. After overnight 
incubation, the adjuvant preparation is centrifuged and its 
supernatant removed. An equal volume of adsorption buffer 
containing 50 ^g 3D-MPL is then added to the alum-bound 
HBsAg - V 3 peptide. 



SUBSTITUTE SHEET 



WO 92/11291 



PCT/EP91/02422 



-31- 
VACCINES 

Claims 

5 1. An immunogenic hybrid polypeptide comprising a first 
polypeptide component (P 1 ) which is HBsAg or a fragment 
thereof displaying the antigenicity of HBV surface antigen, 
covalently linked via a native sulphur atom in the first 
polypeptide component to a second polypeptide component 
10 (P 2 ) . 

2. A hybrid polypeptide of the formula 

P 1 -S-X-P 2 

15 

in which P 1 -^- is HBsAg or fragment thereof displaying the 
antigenicity of HBV surface antigen bonded via a native 
sulphur atom; 

20 

o . 

P is a second antigenic polypeptide; and 
X is either: 

25 a) a group -A-NH- wherein A is a spacer group and NH- is 

the residue of an amino group present in the side chain of 
an amino acid in P 2 ; 

or optionally if P 2 comprises a cysteine residue and a 
30 hydrophobic anchor group: 

b) the sulphur atom of the cysteine residue in P 2 . 



SUBSTITUTE SHEET 



WO 92/12291 



PCT/EP91/02422 



-32- 

3. A hybrid polypeptide as claimed in claim 1 or 2, 
wherein P 2 is selected from r gD of Herpes Simplex Virus or a 
derivative thereof, gpl20 from HIV or a derivative thereof, 
the circumsporozoite antigen or derivative thereof* 

5 

2 

4. . A hybrid polypeptide as claimed in claim 3 wherexn 
is gD2t. 

5. A hybrid polypeptide as claimed in claim 3 wherein P 2 
10 is a peptide corresponding to the V 3 loop of HIV gp!20. 

6. A hybrid polypeptide as claimed in any of claims 1 to 5 
wherein P 1 comprises all or part or parts of the HBs Ag 
precursor protein. 

15 

7. A hybrid polypeptide as claimed in any of claims 1 to 6 
wherein p 1 is monomer of the particle. 

8. A hybrid protein as claimed herein, wherein before 

20 fusion P 1 is HBsAg-S-protein having one or more sulphydryl 
groups on its surface. 

9. A hybrid protein as claimed herein wherein P 1 is a part 
of a composite particle comprising at least two polypeptides 

25 corresponding to part or all of a protein having the 
biological activity of the hepatitis B surface antigen 
wherein the particle presents at least two antigenic 
determinants provided by the S-protein, pre S2-protein or 
pre Sl-protein, said particle optionally containing host 

30 specific lipids. 

10. A hybrid protein as claimed in claim 9 wherein the 
composite particle maybe represented by L, S, wherein L is 
the large protein of HBsAg, including pre SI, pre S2 and S, 

35 and S is HBsAg S-protein. 



SUBSTITUTE SHEET 



WO 92/11291 



PCT/EP91/02422 



-33- 

11. A hybrid protein as claimed in claim 10, characterised 
is that the L protein comprises one or more of the following 
modifications in the following sequences: 

5 a sequence sensitive to protease digestion, 
a sequence necessary for myristylation, 
a sequence necessary for N-linked glycosylation, 
a sequence necessary for O-linked glycosylation, and 
a sequence necessary for binding of human serum albumin. 

10 

12. A hybrid protein as claimed in claim 10 or 11, wherein 
L is modified and has an amino acid sequence comprising 
residues 12-52, followed by 133-145, followed by residues 
175-400 of the L-protein. 

15 

13. A hybrid protein as claimed in any of claims 2 to 12 
wherein A is a substituted C 2 -C 10 alkane or a linear polymer 
such as polyethylene glycol or a compound 

o 

25 ^ ^^^H-Q^co. 

and S-CH 2 CH 2 CO- 

14. A vaccine formulation comprising a hybrid protein as 
30 claimed in any of claims 1 to 13 in conjunction with a 

pharmaceutically acceptable excipient. 

15. A vaccine formulation as claimed in claim 14 further 
comprising de - 3 - 0 - acylated monophosphorylated lipid A 

35 (3D-MPL) in a suitable carrier. 



SUBSTITUTE SHEET 



WO 92/11291 



PCT/EP91/02422 



-34- 

16.. A vaccine formulation as claimed herein comprising 3D- 
MPL and alum. 

17. A vaccine formulation as claimed herein wherein the 
5 carrier is an oil in water emulsion. 

18. A vaccine formulation as claimed in claim 18 wherein 
the oil in water emulsion comprising 3D-MPL, squalane, 
pluronic L - 121 and phosphate buffered saline. 

10 

19. A process for the preparation of a hybrid polypeptide 
of formula (I) : 

P 1 -S-X-P 2 (I) 

15 

wherein P 1 , P 2 and X are as defined in claim 2 which process 
comprises the steps of 

a) when X is an a group -ANH-: 

20 

i) reacting the second polypeptide P 2 with a compound of 
formula (II) : 

Y-A-B (II) 

25 

wherein Y is a group capable of reacting with a native thiol 
group in the first polypeptide; B is a group which is 
specific for one or more amino acid side chains on the 
second polypeptide P 2 and A is as hereinabove defined; and 
30 thereafter 

ii) reacting the product with the first polypeptide 
P 1 -SH; or 



SUBSTITUTE SHEET 



WO 92/11291 



PCT/EP91/02422 



-35- 

b) when X is S and comprises a hydrophobic anchor group 
and a cysteine residue: 

i) mixing the first polypeptide and second polypeptide 
5 in aqueous solution; and 

ii) allowing an intermolecular disulphide bond between P 1 
and P* to form by spontaneous oxidation. 



SUBSTITUTE SHEET 



INTERNATIONAL SEARCH REPORT 

International Application No 

L CLASSIFICATION OF SUBJECT MATTER Qf wwril classification symbols tpply, Indicate all)* 

Aceonling to International Patent Classification (IPC) or to both National aerification and IPC 

Int. CI. 5 CQ7K15/04; A61K39/29 



PCT/EP 91/02422 



O. FIELDS SEARCHED 



Minimum Documentation Searched? 



Classification System 



Classification Symbols 



Int. CI. 5 



C07K ; 



A61K 



Documentation Searched other than Minimum Documentation 
to the Extent that such Documents are included Is the Fields Searched 1 



Ed. DOCUMENTS CONSIDERED TO BE RELEVANT* 



Category 0 



Citation of Document," with indication, where appropriate, of the relevant passages** 



Relevant to Claim No" 



3, 


line 10 - 


line 


30 


6, 


line 55 - 


page 


7, 


7, 


line 48 - 


line 


64 


11, 


line 9 - 


line 


21 



EP,A,0 271 302 (SCRIPPS CLINIC AND RESEARCH 



line 33 



see page 12, line 54 - page 13, Hne 25 

EP,A,0 326 109 (NEW YORK BLOOD CENTER) 2 August 
1989 

see page 2, Hne 28 - page 3, Hne 37 
see page 5, Hne 35 - page 8, Hne 15 

EP,A,0 385 610 (THE WELLCOME FOUNDATION) 5 
September 1990 

see page 2, Hne 38 - page 3, Hne 20 



1-19 



1-19 



1-19 



° S pedal categories of cited documents : 10 

*A' document defining the general rtatt of the art which Is not 

considered to be of particular relevance 
*E* earlier document but published on or after the International 

filing date 

*L' document which may throw doubts on priority dalm(s) or 
which Is died to establish the publication date of another 
dtation or other special reason (as specified) 

"O* document referring to an oral disclosure, use, exhibition or 
other means 

"P* document published prior to the international filing date but 
later than the priority date dalmed 



"T* later document published after the international filing date 
or priority date and not In conflict with the application but 
dted to understand the principle or theory underlying the 
invention 

~X" document of particular relevance; the daimed invention 
cannot be considered novel or cannot be considered to 
involve an inventive step 

*Y" document of particular relevance; tha claimed invention 
cannot be considered to Involve an Inventive step when the 
document Is combined with one or more other such does* 
meats, such combination being obvious to a person skilled 
In the art 

"A* document member of the same patent family 



IV. CERTIFICATION 



Date of the Actual Completion of the International Search 

16 MARCH 1992 



Date of Mailing of this international Search Report 

1 9. 03. 92 




International Searching Authority 

EUR PEAN PATENT OFFICE 



Signature of Authorized Officer 

K0RSNER S.E. 



Fans PCT/ISA/ZIO (Km* ik«clHJ«ay HeS) 



International AppDcatfon No 



PCT/EP 91/02422 



~ nereis raNSIDEBED TO BE RELEV ANT (CONTINUE D FROM THE SECOND SHEET) 



Clarion of Pocamtnt, with Indication. wt>«> »ppwpri««, of th« tdwint pnags 



PROCEEDINGS OF THE AMERICAN NATIONAL ACADEMY OF 
SCIENCE 

vol. 85, 1988, WASHINGTON 
pages 1932 - 1936; 

PALKER ET AL: 'Type-sped f 1c neutralization of 
the human Immunodeficiency virus with antibodies 
to ENV-coded synthetic peptides 

* P. 1932 ( Abstract ) * 

* P. 1933 ( Col. 1, top ) * 

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE 

Sol! A 85, 1988, WASHINGTON 
pages 4478 - 4482; 

GOUDSMIT ET AL: 'Human Immunodeficiency virus 
type I neutralization epitope with conserved 
architecture elicits early type-specific 
antibodies 1n experimentally Infected 
chimpanzees' 

* P. 4478 ( Abstract ) * 

JOURNAL OF PROTEIN CHEMISTRY 
vol. 2, no. 3, 1983, NEW YORK 
pages 263 - 277; 

PARTIS ET AL: 'Cross-Unking of protein by 
W-male1m1do alkanoyl N-hydroxysucc1n1m1d1 
esters' 

* P. 267-270 * 
BIOTECHNOLOGY 

vol. 3, no. 4, 1985, LONDON 
pages 323 - 326; 

VALENZUELA ET AL: 'Antigen engineering 1n yeast: 
Synthesis and assembly of hybrid hepatitis B 
surface antigen-herpes simplex 1 gD particles 

* P. 323 * 



1-19 



1-19 



1-19 



1-19 



Fam PCT/XSA/2LO (exxn thtxt ) <J«**ary IMS) 



ANNEX TO THE INTERNATIONAL SEARCH REPORT 
ON INTERNATIONAL PATENT APPLICATION NO. EP 91°Z* ZZ 



SA 



54130 



Tha annex fats the patent family members rentinl to the patent documents cited in the above-mentioned international search report. 

The members are at raooancd in toe European Patent Office EDP Ele on /rio/QO 

The European Patent Office is in no way babte for tbeae particulars which are merer* erven for the purpose of raformahoo. lb/US/ 



Patent docanent 


Phiblkation 


Patent rvnOj 


PubKcafioD 


cited in search report 


date 


nxnibcr(a) 





EP-A-0271302 



15-06-88 



EP-A-0326109 



02-08-89 



EP-A-0385610 



05-09-90 



US-A- 


4882145 


21-11-89 


US-A- 


4818527 


04-04-89 


AU-A- 


8223187 


09-06-88 


JP-A- 


1025800 


27-01-89 


US-A- 


5039522 


13-08-91 


AU-A- 


4975590 


18-10-90 


JP-A- 


3027400 


05-02-91 



For more details about this annex : see Official Journal of the European Patent Office, No. 12/82