Skip to main content

Full text of "USPTO Patents Application 09848616"

See other formats


WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCT 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



Document A03 
Appl.No. 09/848,616 



(51) International Patent Classification 6 : 

A61K 39/00, 39/38, 39/29, C07K 7/00, 
15/00 



Al 



(11) International Publication Number: WO 99/40934 

(43) International Publication Date: 19 August 1999 (19.08.99) 



(21) International Application Number: PCT/US99/03055 

(22) International Filing Date: 1 1 February 1999 (11 .02.99) 



(30) Priority Data: 
60/074,537 



12 February 1998 (12.02.98) US 



(71) Applicant: IMMUNE COMPLEX, CORPORATION [US/US); 

3347 Industrial Court, San Diego, CA 92121 (US). 

(72) Inventor: BIRKETT, Ashley, J.; Apartment 3208, 878 Stevens 

Avenue, Solana Beach, CA 92075 (US). 

(74) Agents: GAMSON, Edward, P. et al.; Welsh & Katz, Ltd., 
22nd floor, 120 South Riverside Plaza, Chicago, IL 60606 
(US). 



(81) Designated States: AL, AU, BA, BB, BG, BR, BY, CA, CN, 
CU, CZ, EE, GE, HU, ID, IL, IS, JP ( KP, KR, LC, LK, LR, 
LT, LV, MG, MK, MN, MX, NO, NZ, PL, RO, SD, SG, 
SI, SK, SL, TR, TT, UA f UZ, VN, YU, ARIPO patent (GH, 
GM. KE, LS, MW, SD, SZ, UG, ZW), Eurasian patent (AM, 
AZ, BY, KG, KZ, MD, RU, TJ, TM), European patent (AT, 
BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, 
MC f NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, 
GA, GN, GW, ML, MR, NE, SN, TD, TG). 



Published 

With international search report. 



(54) Title: STRATEGICALLY MODIFIED HEPATITIS B CORE PROTEINS AND THEIR DERIVATIVES 



sm-HBc - 



0 O 
Na0 3 S.A o / ^ V 



pH 7.5 



sm-HBc- -N-C-^^CH ?r ^ 



II 



HS- 



hapten 



sm-HBc' 



pH7.0 



sm-HBc N" 



hapten 



(57) Abstract 



A strategically modified hepatitis B core protein is described, where an insert is provided, preferably in an immunodominant region 
of the nucleocapsid protein, containing a chemically reactive amino acid residue. The modified hepatitis B core protein or its aggregated 
nucleocapsid protein particles can be pendently linked to a hapten to form a modified nucleocapsid conjugate. Such a conjugate is useful 
in the preparation of vaccines or antibodies. TTie modified hepatitis B core protein can also be modified to include a T cell epitope. 



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\ 



AL 


Albania 


ES 


Spain 


LS 


Lesotho 


SI 


Slovenia 


AM 


Armenia 


Ft 


Finland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


FR 


Prance 


LU 


Luxembourg 


SN 


Senegal 


AU 


Australia 


GA 


Gabon 


LV 


Latvia 


sz 


Swaziland 


AZ 


Azerbaijan 


GB 


United Kingdom 


MC 


Monaco 


TD 


Chad 


BA 


Bosnia and Herzegovina 


GE 


Georgia 


MD 


Republic of Moldova 


TG 


Togo 


BB 


Barbados 


Gil 


Ghana 


MG 


Madagascar 


TJ 


Tajikistan 


BE 


Belgium 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


Turkmenistan 


BF 


Burkina Faso 


GR 


Greece 




Republic of Macedonia 


TR 


Turkey 


BG 


Bulgaria 


HU 


Hungary 


ML 


Mali 


TT 


Trinidad and Tobago 


BJ 


Benin 


IE 


Ireland 


MN 


Mongolia 


VA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


UG 


Uganda 


BY 


Belarus 


IS 


Iceland 


MW 


Malawi 


US 


United States of America 


CA 


Canada 


IT 


Italy 


MX 


Mexico 


UZ 


Uzbekistan 


CF 


Central African Republic 


JP 


Japan 


NE 


Niger 


VN 


Viet Nam 


CG 


Congo 


KB 


Kenya 


NL 


Netherlands 


YU 


Yugoslavia 


CH 


Switzerland 


KG 


Kyrgyzstan 


NO 


Norway 


ZVY 


Zimbabwe 


CI 


Cote d' I voire 


KP 


Democratic People's 


NZ 


New Zealand 






CM 


Cameroon 




Republic of Korea 


PL 


Poland 






CN 


China 


KR 


Republic of Korea 


PT 


Portugal 






cu 


Cuba 


KZ 


Kazakstan 


RO 


Romania 






cz 


Czech Republic 


LC 


Saint Lucia 


RU 


Russian Federation 






DE 


Germany 


U 


Liechtenstein 


SD 


Sudan 






DK 


Denmark 


LK 


Sri Lanka 


SE 


Sweden 






EE 


Estonia 


LR 


Liberia 


SG 


Singapore 







WO 99/40934 



PCT/US99/03055 



-1- 

STRATEGICALLY MODIFIED HEPATITIS B CORE 
PROTEINS AND THEIR DERIVATIVES 

Description 

5 

Technical Field 

The present invention relates to the 
intersection of the fields of immunology and protein 
engineering, and particularly to carrier proteins, 

10 and more particularly to a hepadnavirus nucleocapsid 
protein strategically modified with an inserted 
chemically-reactive amino acid residue, a pendently- 
1 inked hapten conjugate of that hepadnavirus 
nucleocapsid protein and to an immunogenic particle 

15 comprised of those conjugates. 

Background of the Invention 

It is known that antibodies can be raised 
to a small molecule by using a large immunogenic 

20 protein molecule as a carrier. The small molecule 
that derives enhanced immunogenicity by being 
conjugated to the carrier is called a hapten. The 
phenomenon of a relatively large molecule 
potentiating the immunogenicity of a small molecular 

25 entity to which it is attached is known in the art as 
the "carrier effect". 

The portion of an immunogen recognized by 
the helper T cell (Th cell) is the T cell determinant 
or epitope. The portion of an immunogen that is 

30 bound by antibody is the B cell determinant or 

epitope. Carrier effects can be defined as immunity 
to one determinant, the "helper" or T (T h ) cell 



WO 99/40934 



PCT/US99/03055 



-2- 

determinant, of a multideterminant immunogen 
enhancing the immune response to another determinant, 
the B cell determinant. 

It is now well established that most 
immunogens require T cell help to induce B cells to 
produce antibodies. Thus, T h cells, by recognizing 
helper determinants on the immunogen help B cells to 
make antibody against the immunogen. 

The antigenic determinants recognized by T 
helper cells (T h ) and B cells must be associated to 
form a single molecular entity, but they do not have 
to be covalently linked* See, Russel et al. f Nature, 
280:147 (1979), Lamb et al., J. Immunol., 129:1465 
(1982), Scherle et al., J. Exp. Med., 164:1114 (1986) 
and Lake et al. f Cold Spring Harbor Symp. Quant. 
Biol., 41:589 (1976). Some immunogens do not require 
T cell help to induce antibody formation, these are 
T - independent ant igens . 

A pathogen-related protein can be 
immunologically mimicked by the production of a 
synthetic polypeptide whose sequence corresponds to 
that of a determinant of the pathogen. Such 
polypeptide immunogens are reported by Sutcliffe et 
al., Nature, 287:801 (1980), and Lerner et al., Proc. 
Natl. Acad. Sci. USA, 78:3403 (1981). 

Gerin et al., Proc. Natl. Acad. Sci. USA, 
80:2365 (1983), showed limited protection of 
chimpanzees from hepatitis B virus upon immunization 
with carrier-bound synthetic polypeptides having 
amino acid residue sequences that correspond to the 
sequence of a determinant portion of HBsAg; in 



WO 99/40934 



PCT/US99/03055 



particular, residues 110-137 of the "S" (surface) 
region. However, the carrier protein used in those 
studies was keyhole limpet hemocyanin (KLH) , a 
T cell -dependent carrier that is not fit for use in 
5 medical applications to humans because it is a source 
of irritation that leads to severe inflammation. 

T cell -stimulating carrier proteins capable 
of enhancing the immunogenicity of haptens that do 
not produce unacceptable side effects in human 

10 subjects are often immunogenic natural proteins. For 
example, tetanus toxoid (TT) has been frequently used 
when a carrier suitable for human administration was 
needed. However, the use of tetanus toxoid as a 
carrier was restricted due to problems with dosage 

15 limitations and risk of sensitization to the toxoid 

itself. In addition, an epitope-specif ic suppression 
of response to the carried hapten can occur in 
individuals already immunized against tetanus. 

The hepatitis B surface protein has been 

20 proposed as a carrier for heterologous epitopes. 
Delpeyroux et al., Science, 233 : 472-475 (1986) , 
reported the use of the HBV surface protein (S 
protein) as a carrier for a poliovirus polypeptide 
hapten. Those investigators constructed a 

25 recombinant deoxyribonucleic acid (DNA) protein 

expression vehicle that produces a fusion protein, 
designated HBsPolioAg, capable of forming particles 
closely resembling authentic 22 -nanometer HBsAg 
particles. HBsPolioAg consists of HBV S protein 

30 having an 11 amino acid residue sequence insert 



WO 99/40934 



PCT7US99/03055 



corresponding to amino acids 93-103 of capsid protein 
VPI of poliovirus type 1 (Mahoney strain) . 

Hepadnavirus nucleocapsid proteins have 
been used as hapten carriers. Heterologous 
5 immunogenic peptide sequences inserted internally in 
the hepatitis B core, expressed as fusion particles, 
elicited very high immune responses in immunized 
animals in the absence of adjuvants. B.E. Clarke et 
al. Vaccines 91:313-318 (1991); F. Schodel et al . J m 

10 Virol. 66(1) :106-114 (1992). U.S. Patent Nos . 

4,818,527, 4,882,145, and 5,143,726, the disclosures 
of which are incorporated herein by reference, 
describe the use of the carrier effect with hepatitis 
B virus nucleocapsid protein to enhance the 

15 immunogenicity of an operatively linked polypeptide 
hapten. Those patents describe the linking of a 
polypeptide hapten to hepatitis B virus nucleocapsid 
protein through an amino acid residue side chain that 
occurs naturally in the hepatitis B nucleocapsid 

20 protein sequence. 

Hepadnavirus nucleocapsid proteins are 
fairly well studied. SEQ ID NOs:l and 2 are the DNA 
and amino acid sequences of the human hepatitis B 
core protein (HBc) , subtype ayw, as described in U.S. 

25 Patent Nos. 4,818,527, 4,882,145, and 5, 143 , 726 . 

Other hepadnavirus nucleocapsid protein sequences are 
also known in the art, see e.g. SEQ ID NOs: 3-13. 

There are reasons to select hepadnavirus 
nucleocapsid proteins as a carrier over other 

30 carriers used in the art, such as keyhole limpet 

hemocyanin (KLH) , BCG, tetanus toxoid and diphtheria 



WO 99/40934 



PCT/US99/03055 



-5- 

toxoid. KLH, BCG, tetanus toxoid and diphtheria 
toxoid are non-particulate, whereas hepadnavirus 
nucleocapsid proteins tend to aggregate into 
"particles". HBc particles tend to have a higher 
immunogenicity than hepatitis B surface antigen 
(HBsAg) particles. D.R. Milich et al . , Science, 
234:1398-1401 (1986). HBc is both a T cell- 
independent ar^d a T cell -dependent immunogen. Jd. 
HBc is one of the most immunogenic proteins known. 
Almost all hepatitis B-infected people develop a 
powerful immune response to core. J.H. Hoofnagle, 
Semin. Liver Dis., 1(1):7-14 (1981). HBc can provide 
universal responsiveness, irrespective of genetic 
background. Id. HBc directly activates T cells. 
HBc elicits strong T h cell responses. HBc is 
efficiently processed and presented by antigen- 
presenting cells. Due to the inherently high 
immunogenicity of HBc, complex adjuvants are 
typically not required, for example, the common and 
inexpensive alum is sufficient. 

The family hepadnaviridae is a family of 
enveloped animal viruses with a core of DNA that 
cause hepatitis B in humans. The hepadnaviridae are 
not responsible for human hepatitis A (a single- 
stranded RNA enterovirus) , human hepatitis C 
(Flaviridae family of single stranded RNA virus) , or 
human hepatitis D (a closed circular negative -sense 
RNA satellite virus, "delta virus", that requires 
hepatitis B virus for replication) . The 
hepadnaviridae family includes hepatitis viruses of 
other species, e.g. woodchuck, duck, ground squirrel, 



WO 99/40934 PCT/US99/03055 

-6- 

and heron, in addition to human and simian hepatitis 
B. Hepatitis B (HB) used hereinafter refers to the 
family hepadnaviridae , unless the discussion is 
referring to a specific example. 
5 Hepatitis B core protein monomers self- 

assemble into stable aggregates known as hepatitis B 
core protein particles (HBc particles). For example, 
human HBc particles are 27 nanometers (nm) in 
diameter. Conway et al., Nature, 386:91-94 (1997), 

10 describe the structure of human HBc particles at 9 

Angstrom resolution, as determined from cryo-electron 
micrographs. Bottcher et al., Nature, 386:88-91 
(1997) , describe the polypeptide fold for the human 
HBc monomers, and provide an approximate numbering 

15 scheme for the amino acid residues at which alpha 

helical regions and their linking loop regions form. 
Bottcher et al . propose a loop from about residues 78 
to 82 of the hepatitis B core protein. 

Using synthetic peptides and monoclonoal 

20 antibodies, the immunodominant loop region of HBc was 
mapped to about amino acid residues 75 to 83. G. 
Colucci et al., J. Immunol., 141:4376-4380 (1988). 
Two immunodominant linear epitopes were reported by 
other workers at amino acid residues 75 to 85 and 130 

25 to 140. Salfeld et al . J. Virol. 63:798 (1989). 

Insertion mutants of the hepatitis B core 
protein still are able to form core particles when 
foreign epitopes are cloned into the immunodominant 
loop region of HBc. P. Pumpens et al . , 

30 Intervirology, 38:63-74 (1995). The HBc fusion 



WO 99/40934 



PCT/US99/03055 



proteins form particles in prokaryotic and eukaryotic 
expression systems. Id. 

The ability to use a protein as a carrier 
for a pendent ly- linked* hapten depends upon several 
5 factors that have been studied with respect to HBc. 
Chemically-reactive amino acid side chains, such as 
lysine (K) , aspartic acid (D) , glutamic acid (E) , and 
reduced cysteine residues (C) , provide functional 
groups that can be useful for modifying polypeptides. 

10 The hepatitis B core protein sequence has 

several chemically-reactive amino acid side chains in 
the native sequence. Core has three primary amino 
groups, one at the amino terminus, and two lysine 
residues (K5 and K96) , along with four cysteine 

15 residues (C48, C61, C107 and C183) . There are 

several carboxylic acid groups, D (2, 4, 22, 29, 32, 
78, 83) and E (8, 14, 40, 43, 46, 64, 77, 113, 117, 
145, 179) and the carboxy terminus. 

However, the native, unmodified hepatitis B 

20 core protein particle does not exhibit appreciable 

chemical reactivity of the amino acid side chains in 
the native sequence. The chemical reactivity of an 
amino acid side chain in a protein depends upon the 
nature of the amino acid side chain, and its 

25 environment in the folded protein. 

As is discussed in detail hereinafter, it 
has now been found that the problem of low reactivity 
of the amino acid side chains in native hepatitis B 
nucleocapsid protein can be overcome by inserting a 

30 chemically-reactive amino acid side chain into the 
HBc protein sequence. A strategically modified 



WO 99/40934 



-8- 



PCT/US99/03055 



hepadnavirus core protein particle of the present 
invention exhibits substantially enhanced reactivity 
toward derivitization of HBc particles with 
chemically linked haptens, and provides enhanced 
immunogenicity to those linked hapten. 

These modified HBc proteins and their 
pendent ly- linked hapten conjugate derivatives are 
discussed in the disclosure that follows. 

Brief Summary of the Invention 

The present invention relates to a 
strategically modified hepatitis B core (HBc) protein 
that is linked to a pendent hapten through 
chemically- reactive amino acid residue inserted into 
the HBc sequence. The contemplated strategic 
modification of HBc is an insert mutation of the HBc 
protein. A contemplated insert is 1 to about 40 
amino acid residues in length, preferably 1 to 10 
amino acid residues in length, and includes a 
chemically-reactive amino acid residue. 

The insert is provided to the region 
corresponding to amino acid residues about 50 to 
about 100 of the hepatitis B core protein sequence 
shown in SEQ ID NO: 2. The preferred region of 
insertion corresponds to the hepatitis B core protein 
immunodominant loop region at about amino acid 
residue 70 to about 90, more preferably the loop tip 
region at about amino acid 78 to about 82 and most 
preferably at amino acid 78 to amino acid 80. 

Such an introduced chemically-reactive 
amino acid residue is characterized in that it has a 



WO 99/40934 



PCT/US99/03055 



-9- 

chemically- reactive side chain that provides a 
chemical group for pendent ly linking the 
strategically modified HBc to the hapten. Typically, 
the chemically-reactive amino acid residue is a 
lysine, cysteine, or histidine residue or a carboxyl- 
containing residue such as aspartic acid or glutamic 
acid, preferably lysine or a carboxyl -containing 
residue, and most preferably lysine . 

The hapten bonded to the chemically- 
reactive amino acid residue is any compound of 
interest for generating an immune response, and is 
typically a B cell determinant. Preferably, the 
hapten is a polypeptide hapten, a carbohydrate 
hapten, or a non-peptidal/non-saccharidal (chemical) 
hapten. In one embodiment of the invention, the 
hapten is a pathogen-related hapten, such as a B cell 
determinant of a pathogen. 

In another embodiment of a strategically 
modified hepatitis B core protein conjugate, the 
strategically modified hepatitis B core protein also 
has a T cell stimulating amino acid residue sequence 
operatively linked to the carboxy terminus of the 
hepatitis B core amino acid sequence. Preferably, 
both the hapten and the T cell stimulating amino acid 
residue sequence are pathogen-related, most 
preferably, both are related to (from) the same 
pathogen. 

In the above embodiment of the invention, 
the response to a B cell epitope is boosted by also 
providing the T helper (T h ) cell determinant. In this 
preferred embodiment of the invention, such a T h cell 



WO 99/40934 



PCT/US99/03055 



-10- 

determinant is from the same pathogen as the B cell 
determinant hapten that is pendently linked to the 
strategically modified hepatitis B core protein in 
order to provide pathogen-specific T cell memory in 
5 addition to the hepatitis B core protein antigen- 
specific T cell memory. 

A strategically modified hepatitis B core 
protein conjugate contains a hapten that is pendently 
linked to a strategically modified hepatitis B core 

10 protein. Looked at differently, a before -described 
strategically modified hepatitis B core protein can 
be considered to have three peptide -linked domains, 
I, II and III (numbered consecutively from the amino 
terminus) . Domain I comprises a sequence that 

15 corresponds to residues numbered about 10 to 50 of 
the amino acid sequence of hepatitis B core protein 
of SEQ ID NO:2, and preferably corresponds to 
residues numbered 1 to 50 of that sequence. Domain . 
II is bonded to the carboxy terminal residue of 

20 Domain I. Domain II corresponds to residues numbered 
50 to 100 of the amino acid sequence of hepatitis B 
core protein of SEQ ID N0:2. Domain III comprises a 
sequence that is bonded to the carboxy terminal 
residue of Domain II. Domain III corresponds to 

25 residues numbered 100 to about 140 of the amino acid 
sequence of hepatitis B core protein, and preferably 
corresponds to residues numbered 100 to about 14 9 of 
that sequence . 

In an embodiment of the invention discussed 

30 before, a strategically modified hepatitis B core 

protein additionally has a Domain IV exogenous to HBc 



WO 99/40934 



PCT/US99/03055 



that is peptide -bonded to the carboxy terminal 
residue of Domain III to provide a fusion protein. 
Domain IV is a T cell epitope. 

A strategically modified hepatitis B core 
5 protein particle of the invention is made of 

assembled heptatitis B core protein where a plurality 
of the subunits are strategically modified hepatitis 
B core protein subunits. Also contemplated is a 
particle comprised of a mixture of strategically 

10 modified hepatitis B core protein subunits and other 
heptatits B core protein subunits. 

A contemplated strategically modified 
hepatitis B core protein particle conjugate is 
comprised of assembled hepatitis B core protein 

15 subunits where a plurality of the subunits are 
strategically modified hepatitis B core protein 
subunits. In this embodiment, a hapten is pendently 
linked to a hepatitis B core protein subunit. 
Preferably, the hapten is pathogen- related. As 

20 above, a T cell stimulating amino acid residue 
sequence can be peptide -bonded to the carboxy 
terminal residue of the sequence corresponding to 
hepatitis B core protein. Preferably that pathogen- 
related T cell determinant is related to the same 

25 pathogen as the pathogen-related hapten. 

A strategically modified hepatitis B core 
protein particle conjugate of the invention has 
pendently-1 inked hapten. In a contemplated 
embodiment of the particle conjugate, the particle is 

30 made up of a mixture of strategically modified 

hepatitis B core protein subunits having pendently- 



WO 99/40934 



PCT/US99/03055 



-12- 

linked haptens, and other hepatitis B core protein 
subunits. In one embodiment, about 0.1 to about 0.5 
of the strategically modified hepatitis B core 
protein subunits are pendently linked to a hapten. 
5 Also contemplated is a particle conjugate that is 
made, up of a mixture of strategically modified 
hepatitis B core protein subunits and other hepatitis 
B core protein subunits. 

A before-described strategically modified 

10 hepatitis B core protein of the invention includes a 
peptide insert containing a chemically-reactive amino 
acid residue. That insert can be, but is typically 
not itself a separate B cell antigenic determinant, 
although some B cell immunogenicity of the insert can 
. 15 be exhibited merely because of the placement of the 
insert into the HBc protein or particle. Such an 
insert can be and in some embodiments is a T cell 
epitope. Placement of an insert into the HBc loop 
region greatly dimishes the HBc immunogenicity and 

20 antigenicity of the resulting molecule. 

An inoculum of the invention comprises an 
immunogenic amount of the strategically modified 
hepatitis B core protein conjugate of the invention. 
When the pendently- linked hapten is a pathogen- 

25 related hapten, the inoculum can be used as a vaccine 
to protect a mammal treated with the inoculum from 
that pathogen. Thus, in one embodiment of the 
invention, a strategically modified hepatitis B core 
protein conjugate is used as a vaccine to provide 

30 protection against the pathogen from which the hapten 
is derived. More preferably, the inoculum is 



WO 99/40934 



PCT/US99/03055 



-13- 

comprised of strategically modified hepatitis B core 
protein particle conjugate as the immunogen. 

The present invention has several benefits 
and advantages . 
5 One benefit of a contemplated modified HBc 

protein is that the protein can be derivitized while 
in the aggregated form of HBc particles. 

An advantage of the invention is that the 
modified HBc protein displays appreciably enhanced 
10 chemical reactivity toward derivitization, as 

compared to use of the N-terminal primary amine, for 
example. 

Another benefit of a contemplated modified 
HBc protein is that the chemistry of derivitization 
15 of such side chains is well -studied, straightforward 
and relatively predictable. 

Another advantage of a contemplated 
modified HBc protein is that it enhances the 
immunologic response to the conjugated hapten with 
20 which it is derivitized. 

Yet another benefit of a contemplated 
modified HBc protein is that it is unlikely to 
produce undesirable immunologic side effects in 
humans . 

25 Still further benefits and advantages of 

the invention will be apparent to the skilled worker 
from the discussion that follows. 

Brief Description- of the Drawings 
30 In the figure forming a portion of this 

disclosure Scheme 1 illustrates a reaction sequence 



WO 99/40934 



PCT/US99/03055 



-14- 

for pendently linking a hapten to a strategically 
modified hepatitis B core protein (sm-HBc) particle 
using sulpho-succinimidyl 4- (N-maleimidomethyl) 
cyclohexane 1-carboxylate (sulpho-SMCC) . The sm-HBc 
particle is depicted as a box having (for purposes of 
clarity of the figure) a single pendent amino group. 

Detailed Description of thg Invention 

A. PQf initjQns 

The term "antibody" refers to a molecule 
that is a member of a family of glycosylated proteins 
called immunoglobulins, which can specifically 
combine with an antigen. 

The word "antigen 11 has been used 
historically to designate an entity that is bound by 
an antibody, and also to designate the entity that 
induces the production of the antibody. More current 
usage limits the meaning of antigen to that entity 
bound by an antibody, whereas the word "immunogen" is 
used for the entity that induces antibody production. 
Where an entity discussed herein is both immunogenic 
and antigenic, reference to it as either an immunogen 
or antigen will typically be made according to its 
intended utility. 

The word "hapten" is used to describe 
molecules that are capable of stimulating an immune 
response (e.g., production of antibody) when 
chemically coupled to a protein carrier. The word is 
often used for small nonantigenic molecules in the 



WO 99/40934 



PCT/US99/03055 



art, but herein, it merely refers to the molecule 
that is to be pendently linked to the carrier 
protein, even if it is antigenic or not small. 

"Antigenic determinant" refers to the 
5 actual structural portion of the antigen that is 

immunologically bound by an antibody combining site 
or T cell receptor. The term is also used 
interchangeably with "epitope". 

The noun "conjugate" as used herein refers 
10 to a molecule formed from a hapten pendently linked 
through an amino acid residue side chain to a 
carrier. 

The term "conservative substitution" as 
used herein denotes that one amino acid residue has 

15 been replaced by another, biologically similar 

residue. Examples of conservative substitutions 
include the substitution of one hydrophobic residue 
such as isoleucine, valine, leucine or methionine for 
another, or the substitution of one polar residue for 

20 another such as between arginine and lysine, between 
glutamic and aspartic acids or between glutamine and 
asparagine and the like. The term "conservative 
substitution" also includes the use of a substituted 
amino acid in place of an unsubstituted parent amino 

25 acid in place of an unsubstituted parent amino acid 

provided that antibodies raised to such a polypeptide 
also immunoreact with the corresponding polypeptide 
having the unsubstituted amino acid. 

The term "corresponds" in its various 

30 grammatical forms as used in relation to peptide 

sequences means the peptide sequence described plus 



WO 99/40934 



PCT/US99/03055 



-16- 

or minus up to three amino acid residues at either or 
both of the amino- and carboxy- termini and containing 
only conservative substitutions in particular amino 
acid residues along the polypeptide sequence. 
5 "Epitope" refers to that portion of a 

molecule that is specifically bound by a T cell 
antigen receptor or an antibody combining site. 
"Epitope" and "determinant" are used interchangeably. 

As used herein, the term "fusion protein" 

10 designates at least two amino acid residue sequences 
not normally found linked together in nature 
operatively linked together end-to-end (head-to-tail) 
by a peptide bond between their respective terminal 
amino acid residues. 

15 The phrase "hepatitis B" as used here 

refers in its broadest context to any member of the 
family hepadnaviridae t a family of enveloped DNA- 
containing animal viruses that can cause hepatitis B 
in human. 

20 The phrase "HBc" as used here refers to T 

cell stimulating proteins having an amino acid 
residue sequence that corresponds to an amino acid 
residue sequence encoded by the hepatitis B virus 
(HBV) nucleocapsid protein gene. An exemplary well- 

25 known naturally occurring protein encoded by the 

human HBV nucleocapsid gene is the "core" protein, 
subtype ayw, having the biological sequences of SEQ 
ID NOs: 1 and 2. Galibert, et al . , Nature 281:646 
(1979) . HBeAg protein, the precursor to HBc, 

30 includes the sequence of the hepatitis B core protein 
and a "pre-core" sequence at the amino terminus 



WO 99/40934 



PCT/US99/03055 



-17- 

t hereof, as shown in SEQ ID NOs: 8 and 9 in the case 
. of a ground squirrel hepatitis B nucleocapsid gene. 
The core protein sequence begins at amino acid 
position 31 therein, thus corresponding to amino acid 
5 residue number 1 of SEQ ID NO:2. The sequences for 
other hepatitis B core proteins are known in the art. 
Human hepatitis B virus core protein subtype adr is 
provided in SEQ ID NOs: 3 and 4, and subtype adw is 
provided in SEQ ID NOs : 5 and 6 . Ono et al w Nucl . 

10 Acids Res. 11:1747 (1983). Sequences are also 

provided for woodchuck hepatitis B core protein at 
SEQ ID NO: 7 [Schodel et al . , Adv. Viral Oncol. 8:73- 
102 (1989)], ground squirrel at SEQ ID NOs : 8 and 9, 
heron at SEQ ID NOs : 10 and 11, and duck at SEQ ID 

15 NOs: 12 and 13. For clarity, the amino acid numbering 
system shown in SEQ ID NOs : 1 and 2 with respect to 
human hepatitis B core protein subtype ayw is used as 
a benchmark herein. Other HBc sequences can be 
aligned with that sequence using standard biological 

20 sequence alignment programs and protocols to 

determine the amino acid residues that "correspond to 
the hepatitis B core protein sequence of SEQ ID 
N0:2 H , see e.g. Schodel et al., Adv. Viral Oncol. 
8:73-102 (1989) . 

25 If reference is made to a polypeptide 

portion of any of the above described naturally 
occurring HBV nucleocapsid gene encoded proteins, 
that reference is explicit, either by stating, for 
example, that a T cell stimulating portion of the 

30 particular protein is referred to or by explicitly 
designating the particular portion of the sequence, 



WO 99/40934 



PCT/US99/03055 



-18- 



as by indication of the included amino acid residue 
positions . 

The term "immunoreact " in its various forms 
means specific binding between an antigen as a ligand 
5 and a molecule containing an antibody combining site 
such as a Fab portion of a whole antibody. 

The phrase "operatively linked" as used 
herein means that the linkage does not interfere with 
the ability of either of the linked groups to 
10 function as described; e.g., to function as a T or B 
cell determinant. 

The phrase "pendently linked" refers to a 
single linkage, either direct or via a bridge, from a 
HBc protein to another molecule at other than the 
15 amino or carboxy termini. The phrase is used herein 
to describe the linkage between a hapten and a 
chemically- reactive amino acid side chain of a 
strategically modified hepatitis B core protein. 

"Macromolecular assembly 11 refers to a non- 
20 covalently bonded aggregate of protein subunits. 

Typically in this invention, the protein subunit is a 
strategically modified hepatitis B core protein 
monomer. As described in more detail hereinafter, 
those core protein monomers usually self -assemble 
25 into spherical "core particles" having either 90 or 
120 core protein dimers (a total of 180 or 240 core 
protein subunits) . A spherical core particle is an 
example of a macromolecular assembly. 

The phrase "pathogen-related" as used 
30 herein designates a B cell or T cell immunogen that 
is capable of inducing the production of antibodies 



WO 99/40934 



PCT/US99/03055 



that immunoreact with a pathogen in native form. 
Exemplary pathogen- related B cell and T cell 
immunogens are illustrated hereinafter. 

The words "polypeptide" and "peptide" as 
5 used interchangeably throughout the specification and 
designate a linear series of amino acid residues 
connected one to the other by peptide bonds between 
the alpha -amino and carboxy groups of adjacent amino 
acids. Polypeptides can be variety of lengths, 

10 either in their neutral (uncharged) forms or in forms 
which are salts, and either free of modifications 
such as glycosylation, side chain oxidation, or 
phosphorylation or containing these modifications. 
It is well understood in the art that amino acid 

15 residue sequences contain acidic and basic groups, 

and that the particular ionization state exhibited by 
the peptide is dependent on the pH of the surrounding 
medium when the protein is in solution, or that of 
the medium from which it was obtained if the protein 

20 is in solid form. Also included in the definition 
are proteins modified by additional substituents 
attached to the amino acid side chains, such as 
glycosyl units, lipids, or inorganic ions such as 
phosphates, as well as modifications relating to 

25 chemical conversions of the chains, such as oxidation 
of sulfhydryl groups. Thus, "polypeptide" or its 
equivalent terms is intended to include the 
appropriate amino acid residue sequence referenced, 
subject to those of the foregoing modifications which 

30 do not destroy its functionality. A peptide or 

polypeptide used as a hapten typically contains fewer 



WO 99/40934 



PCT7US99/03055 



-20- 

than 70 amino acid residues, and more typically 
contains a linear chain of about 5 to about 4 0 amino 
acid residues, and more preferably about 10 to about 
25 residues. It is noted that a contemplated 
polypeptide hapten can be longer than 70 residues, 
but such a polypeptide is shorter than the naturally 
occurring protein that shares its sequence. 

The word "protein" designates a polypeptide 
having about 70 or more amino acid residues, and is a 
naturally occurring entity. 

The words "secrete" and "produce" are often 
used interchangeably in the art as to cells from 
which antibody molecules are obtained. Cells that 
produce antibodies may, however, not secrete those 
molecules into their environment. Herein, the 
antibody molecules are secreted and are obtained from 
the blood stream (humoral antibody) . Nevertheless, 
antibodies are generally referred to as being 
"produced" in keeping with the phrase utilized in the 
art . 

All amino acid residues identified herein 
are in the natural or L- configuration. In keeping 
with standard polypeptide nomenclature, [J. Biol. 
Chem., 243, 3557-59 (1969)], abbreviations for amino 
acid residues are as shown in the following Table of 
Correspondence, Table 1. 



WO 99/40934 



PCT/US99/03055 



Table 1. TABLE OF CORRESPONDENCE 
SYMBOL 

1 -Letter 3 -Letter AMINO ACID 

' ) 



Y 


Tyr 


L-tyrosine 


G 


Gly 


glycine 


F 


Phe 


L-phenylalanine 


M 


Met 


L-methionine 


A 


Ala 


L-alanine 


S 


Ser 


L-serine 


I 


He 


L-isoleucine 


L 


Leu 


L- leucine 


T 


Thr 


L- threonine 


V 


Val 


L-valine 


P 


Pro 


L-proline 


K 


Lys 


L- lysine 


H 


His 


L-histidine 


Q 


Gin 


L-glutamine 


E 


Glu 


L-glutamic acid 


z 


Glx 


L-glutamic acid 






or 






L-glutamine 


w 


Trp 


L- tryptophan 


R 


Arg 


L-arginine 


D 


Asp 


L-aspartic acid 


N 


Asn 


L-asparagine 


B 


Asx 


L-aspartic acid 






or 






L-asparagine 


C 


Cys 


L- cysteine 



5 B. Strategically Modified Hepatitis B Core Protein 
The present invention contemplates a 
strategically modified hepadnaviridae core ("HBc") 
protein that has an inserted chemically reactive 
amino acid residue for pendently linking with haptens 
10 such as polypeptides and carbohydrates. The 

strategic modification of the invention is the 



WO 99/40934 



PCT/US99/03O55 



-22- 

insertion of 1 to about 4 0 amino acid residues 
including a chemically- reactive amino acid residue 
into the hepatitis B core protein sequence in the 
region corresponding to amino acid residues 50 to 100 
5 of the HBc sequence of SEQ ID NO: 2. Such an 

introduced chemically-reactive amino acid residue has 
a side -chain that provides a functional group for 
pendently linking a hapten to the strategically 
modified carrier. 

10 Hepadnaviridae have a nucleocapsid, or 

core, surrounded by a lipid envelope containing 
surface proteins. The nucleocapsid is a generally 
spherical aggregate of core proteins ("core antigen", 
HBcAg) dimers. In vitro, the hepatitis B core 

15 protein self -assembles into "particles", spherical 
shells of icosahedral symmetry made up of 90 or 120 
hepatitis B core protein dimers, thus 180 or 240 
protein subunits. The particles are about 280 or 310 
Angstroms in diameter, respectively. B. Bottcher et 

20 al., Nature, 386:88-91 (1997); J. P. Conway et al . 
Nature 386:91-94 (1997). 

A contemplated strategically modified 
hepatitis B core protein also forms a macromolecular 
assembly. Such a particle can be present in the form 

25 of 180 or 240 protein subunits, although it does not 
have to be such a 90 or 120 dimer. 

Hybrid core proteins with exogenous amino 
acid residues inserted in the region near amino acid 
residue 80 are reported to assemble into regular 

30 shells, even with inserts as large as 4 6 amino acids 
in length. A.I. Brown et al., Vaccine, 9:595-601 



WO 99/40934 



PCIYUS99/03055 



-23- 

. (1991) ; F. Schodel et al . , J. Virol., 66:106-114 
(1992) . 

The hepadnaviridae core protein sequence 
used as a benchmark sequence herein is that of the 
5 human hepatitis B core protein, subtype ayw, shown in 
SEQ ID NOs : 1 and 2 . Other subtypes of the human 
hepatitis B virus are known. SEQ ID Nos: 3 and 4 are 
human HBc, subtype adr, and SEQ ID NOs: 5 and 6 are 
HBc subtype adw. The sequences of various animal 

10 hepatitis core proteins are also published. The 

biological sequence of duck hepatitis core protein is 
disclosed herein as SEQ ID NO: 12 and 13; a portion of 
the ground squirrel hepatitis nucleocapsid gene is at 
SEQ ID NO: 8 and 9; woodchuck hepatitis core is at SEQ 

15 ID NO: 7 and heron hepatitis core at SEQ ID NOs : 10 and 
11 . Exemplary animal hepatitis B core proteins are 
aligned with human hepatitis B core protein by F. 
Schodel et al . , Adv. Viral Oncology 8:73-102 (1989). 

20 i. gtratogiq MocUfjc^tiQn of the Cere Prptein 

The present invention contemplates a 
strategically modified hepatitis B core protein 
conjugate that comprises a hapten that is pendently 
linked to a strategically modified hepatitis B core 

25 protein (HBc) . The strategically modified hepatitis 
B core protein itself comprises an amino acid 
sequence corresponding to the hepatitis B core 
protein amino acid sequence of SEQ ID NO: 2 including 
the amino acid residues numbered about 10 to about 

30 14 0 of that sequence. That HBc amino acid residue 
sequence additionally contains an exogenous amino 



WO 99/40934 



PCT/US99/03055 



-24- 

acid residue insert in the region corresponding to 
amino acid residues numbered about 50 to about 100 
from the HBc amino terminus, wherein the exogenous 
insert (i) is 1 to about 40 amino acid residues in 
5 length, and (ii) contains a chemically-reactive amino 
acid residue. The hapten is pendently linked to the 
strategically modified HBc protein by means of a 
chemically-reactive amino acid residue present in the 
insert . 

10 It is preferred that residues 1 through 10 

of SEQ ID NO: 2 be present in the strategically 
modified HBc protein molecule. It is further 
preferred when any residue is absent or deleted from 
position 1 to 10 that those residues be deleted in 

15 sequence and that the remaining residues be present 
in sequence. Thus, if a five residue deletion were 
contemplated, the deleted residues would be numbered 
1-5, leaving residues 6 through the desired HBc - 
carboxy terminus present, plus the insert. 

20 It is similarly preferred that residues 

numbered about position 140 through 149 of SEQ ID 
NO: 2 be present in a strategically modified HBc 
protein molecule. As noted elsewhere herein, the 
region of HBc numbered 150 through the carboxy 

25 terminus contains a plurality of arginine residues. 

Those residues bind nucleic acids on purification of 
HBc particles after expression, and the sequence 
containing those residues is preferably omitted from 
a strategically modified HBc protein molecule. As 

30 was the case with the residues of positions 1 through 
10, it is preferred that residues between about 



WO 99/40934 



PCT/US99/03055 



-25- 

position 140 and 149 be present and correspondingly 
absent in a sequential manner. Thus, where the 
carboxy terminal residue corresponds to the residue 
of position 146, the residues of positions 141-145 
5 are also present and those of 147-149 are absent. 

Most preferably, a contemplated HBc sequence is that 
shown in SEQ ID NO: 2 from position 1 through position 
149, plus the sequence of the insert. 

The insert can be placed within the HBc 

10 sequence in the region of positions numbered about 50 
through about 100, as already noted. Preferably, the 
insert is present in the region corresponding to 
amino acid residues numbered about 70 to about 90. 
More preferably, the insert is present in the region 

15 corresponding to amino acid residues numbered 78 to 
82. Most preferably, the insert is located in the 
region corresponding to residues numbered 78 through 
80. 

A strategically modified hepatitis B core 
20 protein of the invention has from 1 to about 4 0 amino 
acid residues inserted. Preferably, the insert is 1 
to 10 amino acid residues in length. The insert 
contains a chemically-reactive amino acid residue. 
The insertion of more than one chemically-reactive 
25 amino acid residue is also contemplated. 

It is contemplated that restriction 
endonuclease sites be provided in the gene construct 
for the strategically modified hepatitis B protein 
near the desired insert region. The nucleotides of 
30 the restriction endonuclease site will be translated 
into amino acids upon expression, and that effect has 



WO 99/40934 



PCT/US99/03055 



-26- 

some bearing on the choice of endonuclease . Several 
restriction endonucleases are commercially available 
(e.g. from Promega Corp., Madison, Wisconsin) and 
their recognition site sequences and cleavage sites 
5 well known in the art. Example 1 describes such a 
construct for a strategically modified hepatitis B 
core protein. 

In one preferred embodiment, the insert is 
a single residue that is added as the chemically- 

10 reactive residue. In other preferred embodiments, 

the use of restriction enzymes and their recognition 
sequences causes about three to about five residues 
to be inserted, including the desired chemically 
reactive residue. 

15 An insert containing a chemically-reactive 

amino acid residue is inserted into the native 
hepatitis B core protein at a position corresponding 
to an amino acid residue position from about 50 to 
about 100. The preferred region of insertion into 

20 the hepatitis B core protein is in the immunodominant 
loop region (about amino acid residue 70 to about 
90) , more preferably in the loop region that 
corresponds to the native hepatitis B core protein 
position from about amino acid 78 to about 82 . Most 

25 preferably, the insert is placed at residues numbered 
78 to 80 Of SEQ ID N0:2. 

As used herein when it is said that the 
insert is "at a position 1 ' it is meant that the amino 
terminus of the insert is peptide bonded to the 

30 carboxy terminus of the corresponding amino acid 
residue of the hepatitis B core protein sequence 



WO 99/40934 



PCT/US99/03055 



having that amino acid residue number. In other 
words, the insert immediately follows the residue at 
that stated position. 

Insertion can be effected by generally 
5 utilized methods in the art. Genetic manipulation, 
by a PCR-based method is illustrated in Examples 1 
and 5. In addition, oligonucleotide -mediated site 
directed mutagenesis as discussed in J.Sambrook et 
al. Molecular Cloning: a laboratory manual . 2 nd ed., 

10 Cold Spring Harbor Laboratory Press, 15.51-ff. (1989) 
can be used to add one codon by hybridizing a desired 
DNA sequence with a template that adds a codon for 
single residue, followed by filling in the remaining 
nucleic acid sequence. Dawson et al . , Science 

15 266:776-779 (1994), describe a method of linking 

polypeptide chains at their peptide backbone, so that 
a fusion could be built up of peptide fragments. 

The chemically-reactive amino acid residue 
can be at any position within the insert. 

20 Preferably, the chemically- reactive amino acid 

residue is in a position that corresponds to amino 
acid residue numbered 70 to 90 of the native (wild 
type) core, and most preferably at residue position 
78 to 82. For example, when a 10 amino acid residue 

25 long insert is inserted at a position corresponding 
to native core protein residue 73, then the 
chemically-reactive amino acid residue is preferably 
at position 5 to 9 of the insert. When a 30 amino 
acid residue long insert is inserted a position 

30 corresponding to native core protein residue 58, then 



WO 99/40934 



PCIYUS99/03055 



-28- 

the chemically-reactive amino acid residue is 
preferably at position 22 to 24 of the insert. 

An introduced chemically-reactive amino 
acid residue has a chemically-reactive side chain 
5 that provides a functional group for derivitizing the 
strategically modified HBc (i.e. conjugating a hapten 
to the modified HBc) . Useful side chain functional 
groups include epsilon-amino groups, beta-or gamma - 
carboxyl groups, thiol (-SH) groups and aromatic 

10 rings (e.g. tyrosine and histidine) . The chemically- 
reactive amino acid residue is typically a lysine, 
cysteine, or histidine residue or a carboxyl - 
containing residue such as aspartic acid or glutamic 
acid. Lysine is a particularly preferred chemically- 

15 reactive amino acid residue. 

It is noted that the amino acid residue 
sequence of the hepatitis B core protein encoded by 
and shown in SEQ ID NOs:l and 2, respectively, has 
two consecutive endogenous carboxyl -containing 

20 residues, existing glutamic (glu, E) and aspartic 

(asp, D) acids, at positions 77 and 78. However, the 
present invention contemplates the introduction of at 
least one additional, exogenous chemically- reactive 
amino acid residue. European Patent No. 385610 

25 reports that unsatisfactory results were achieved in 
attempts to chemically couple polypeptide haptens to 
HBc particles. It is noted that those coupling 
attempts were directed toward amino groups and not 
carboxyl groups of the amino acid side chains. 

3 0 In addition of the use of an individual 

chemically-reactive amino acid residue in the insert 



WO 99/40934 



PCT/US99/03055 



-29- 

such.as aspartic acid or lysine, substantially any 
sequence of the desired length that contains a 
chemically- reactive amino acid residue can be used. 
Exemplary inserts of greater than a single residue 
5 include the B cell HRV-2 VP2 epitope discussed in 
B.E. Clarke et al . Vaccines 91:313-318 (1991), the 
HBsAg Pre-S (1) 27-53 sequence discussed in F. Schodel 
et al. J. Virol. 66 (1) : 106-114 (1992) and the HBsAg 
Pre-S (2) 133-143 sequence discussed in F. Schodel et 
10 al. Vaccines 90:193-198 (1990). An appropriate T 
cell epitope discussed hereinafter as a hapten can 
also be used as an insert. Exemplary sequences 
include those of SEQ ID NOs: 28, 32, 33, 34, 47, 48, 
49, 50 and 55. 

15 

ii. Additional Modification of the Core Protein 
It is also contemplated that a hepatitis B 
core protein strategically modified as described 
above has other modifications. The contemplated 

20 modifications of the strategically modified core 
include the nature of the insert containing the 
chemically-reactive amino acid residue, truncation of 
the amino terminus, truncation of the carboxy 
terminus, fusion at the carboxy terminus, pendent 

25 linking to the carboxy- terminal region. 

The insert containing the chemically- 
reactive amino acid residue to which the hapten is 
conjugated can have a use in addition to providing 
the chemically-reactive amino acid residue. It is 

30 contemplated that an insert containing a chemically- 
reactive amino acid residue is a T cell stimulating 



WO 99/40934 PCT/US99/03055 

-30- 

amino acid sequence. Such a T cell stimulating amino 
acid sequence is preferably a T cell epitope from the 
same source as the B cell epitope that will be the 
conjugated antigen, e.g. both from Mycobacterium 
5 tuberculosis. Exemplary epitopes are discussed 
hereinafter. 

An insert containing a chemically- reactive 
amino acid residue can also be chosen in order to 
confer additional desirable properties, such as 

10 stability-enhancing or solubility-enhancing 
properties . 

A strategically modified hepatitis B core 
protein can be chemically modified by methods well 
known in the art. Numerous such techniques are 

15 disclosed in Roger L. Lundblad, Techniques in Protein 
Modification , CRC Press (Ann Arbor, Michigan: 1994) . 
Such chemical modifications are made to enhance or 
diminish properties, for example, a lysine amino 
group can be blocked to change the isoelectric point 

20 of the protein, causing it to separate differently on 
a chromatographic ion exchange resin. 

It is also contemplated that the carboxy 
terminus of the core protein sequence be truncated, 
preferably down to about amino acid residue position 

25 140. The arginine-rich sequence present beginning at 
residue 150 of SEQ ID NO: 2 binds to nucleic acids and 
can hinder the purification and handling of the 
expressed core protein. In SEQ ID N0:2, the 
arginine-rich stretches begin at position 150. A 

30 preferred strategically modified HBc protein has a 
carboxy terminal valine (V) residue of residue 149. 



WO 99/40934 



PCT/US99/03055 



-31- 

iii. Making Strategically-Modified Core Protein 
The strategic modification of the hepatitis 
B core protein is typically made by known processes 
5 in the art on the DNA level, for example by inserting 
the codons corresponding to the amino acids to be 
inserted. The engineered gene is then expressed in a 
convenient system known in the art, for example in a 
viral culture in infected immortalized cells. 

10 Methods for producing HBcAg proteins in 

general and the pre-core, core and HBeAg proteins in 
particular, are well known in the art. The same 
methods readily adapted to the isolation of the 
modified core protein particles of the invention. In 

15 addition, HBcAg and HBeAg can be produced by a 

variety of well known recombinant DNA techniques. 
See, for example, U.S. Pat. No. 4,356,270 to Itakura 
and 4,563,423 to Murray et al., respectively. Those 
recombinant DNA techniques can be easily adapted to 

20 produce modified core particles of the invention. 
The modified core proteins can be conjugated with 
hapten before or after particle formation, preferably 
after core particle formation and purification. 



25 C. Modified Hepatitis B Core Protein Conjugate 
Any hapten against which antibody 
production is desired can be linked to a 
strategically modified hepatitis B core protein to 
form an immunogenic strategically modified hepatitis 

3 0 B core protein conjugate of this invention. The 

hapten of interest typically is a B cell determinant. 



WO 99/40934 



PCT/US99/03055 



-32- 

The hapten can be a polypeptide, a carbohydrate 
(saccharide) , or a non-polypeptide, non- carbohydrate 
chemical such as 2 , 4-dinitrobenzene . 

Methods for operatively linking individual 
5 haptens to a protein or polypeptide through an amino 
acid residue side chain of the protein or polypeptide 
to form a pendent ly- linked immunogenic conjugate, 
e.g., a branched- chain polypeptide polymer, are well 
known in the art. Those methods include linking 

10 through one or more types of functional groups on 

various side chains and result in the carrier protein 
polypeptide backbone being pendently linked- - 
covalently linked (coupled) to the hapten but 
separated by at least one side chain. 

15 Methods for linking carrier proteins to 

haptens using each of the above functional groups are 
described in Erlanger, Method of Enzymology, 70:85 
(1980), Aurameas, et al . , Scand. J. Immunol . , Vol. 8, 
Suppl. 7, 7-23 (1978) and U.S. Pat. No. 4,493,795 to 

20 Nestor et al. in addition, a site-directed coupling 
reaction, as described in Rodwell et al., Biotech. , 
3, 889-894 (1985) can be carried out so that the 
biological activity of the polypeptides is not 
substantially diminished. 

25 Furthermore, as is well known in the art, 

both the HBcAg protein and a polypeptide hapten can 
be used in their native form or their functional 
group content can be modified by succinylation of 
lysine residues or reaction with cysteine- 

30 thiolactone. A sulfhydryl group can also be 

incorporated into either carrier protein or conjugate 



WO 99/40934 



PCT/US99/03055 



-33- 

by reaction of amino functions with 2-iminothiolane 
or the N-hydroxysuccinimide ester of 3- (3- 
dithiopyridyl ) propionate . 

The HBc protein or hapten can also be 
modified to incorporate a spacer arm, such as 
hexamethylene diamine or other bi functional molecules 
of similar size, to facilitate the pendent linking. 

Polypeptide hapten. Methods for covalent 
bonding of a polypeptide hapten are extremely varied 
and are well known by workers skilled in the 
immunological arts. For example, following U.S. 
Patent No. 4,818,527, m-maleimidobenzoyl-N- 
hydroxysuccinimde ester (ICN Biochemicals, Inc.) or 
succinimidyl 4- (N-maleimidomethyl) cyclohexane-1- 
carboxylate (SMCC, Pierce) is reacted with a 
strategically modified hepatitis B core protein to 
form an activated carrier. That activated carrier is 
then reacted with a polypeptide that either contains 
a terminal cysteine or to which an additional amino- 
or carboxy- terminal cysteine residue has been added 
to form a covalently bonded strategically modified 
hepatitis B core protein conjugate. As an 
alternative example, the amino group of a polypeptide 
hapten can be first reacted with N-succinimidyl 3- (2- 
pyridylthio) propionate (SPDP, Pharmacia), and that 
thiol -containing polypeptide can be reacted with the 
activated carrier after reduction. Of course, the 
sulfur-containing moiety and double bond- containing 
Michael acceptor can be reversed. These reactions 
are described in the supplier's literature, and also 
in Kitagawa, et a!., J. Biochem. , 79:233 (1976) and 



WO 99/40934 PCT/US99/03055 

-34- 

in Lachmann et al . , in 3,99$ Synthetic Peptides 3g 
Antigens , (Ciba Foundation Symposium 119), pp. 25-40 
(Wiley, Chichester: 1986) . 

U.S. Patent No. 4,767,842 teaches several 
5 modes of covalent attachment between a carrier and 
polypeptide that are useful here. In one method, 
tolylene diisocyanate is reacted with the carrier in 
a dioxane-buf f er solvent at zero degrees C to form an 
activated carrier. A polypeptide hapten is 

10 thereafter admixed and reacted with the activated 

carrier to form the covalently bonded strategically 
modified hepatitis B core protein conjugate. 

Particularly useful are a large number of 
heterobi functional agents that form a disulfide link 

15 at one functional group end and a peptide link at the 
other, including N-succidimidyl-3- (2-pyridyldithio) 
propionate (SPDP) . This reagent creates a disulfide 
linkage between itself and a thiol in either the 
strategically modified hepatitis B core protein or 

20 the hapten, for example a cysteine residue in a 
polypeptide hapten, and an amide linkage on the 
coupling partner, for example the amino on a lysine 
or other free amino group in the carrier protein. A 
variety of such disulf ide/amide forming agents are 

25 known. See for example Immun. Rev. (1982) 62:185. 

Other bifunctional coupling agents form a thioether 
rather than a disulfide linkage. Many of these 
thioether- forming agents are commercially available 
and include reactive esters of 6-maleimidocaproic 

30 acid, 2-bromoacetic acid, 2-iodoacetic acid, 4-(N- 
maleimido-methyl) cyclohexane- 1-carboxylic acid and 



WO 99/40934 



PCT/US99/03055 



-35- 

the like. The carboxyl groups can be activated by 
combining them with succinimide or 1 -hydroxy- 2 -nitro- 
4-sulfonic acid, sodium salt. The particularly 
preferred coupling agent for the method of this 
invention is succinimidyl 4- (N-maleimidomethyl) 
cyclohexane-l-carboxylate (SMCC) obtained from Pierce 
Company, Rockford, 111. The foregoing list is not 
meant to be exhaustive, and modifications of the 
named compounds can clearly be used, e.g., the 
sulpho-SMCC depicted in the figure. 

A polypeptide hapten can be obtained in a 
number of ways well known in the art. Usual peptide 
synthesis techniques can be readily utilized. For 
example, recombinant and PCR-based techniques to 
produce longer peptides are useful . Because the 
desired sequences are usually relatively short, solid 
phase chemical synthesis is useful. 

As discussed below, DNA sequences that 
encode a variety of polypeptide haptens are known in 
the art. The coding sequence for peptides of the 
length contemplated herein can easily be synthesized 
by chemical techniques, for example, the 
phosphotriester method of Matteucci et al., J. Am. 
Chem. Soc. 103:3185 (1981). Of course, by chemically 
synthesizing the coding sequence, any desired 
modification can be made simply by substituting the 
appropriate bases for those encoding the native 
peptide sequence. The coding sequence can then be 
provided with appropriate linkers and ligated into 
expression vectors now commonly available in the art, 



WO 99/40934 



PCT/US99/03055 



-36- 

and the regulating vectors used to transform suitable 
hosts to produce the desired protein. 

A number of such vectors and suitable host 
systems are now available. For example promoter 
5 sequences compatible with bacterial hosts are 
provided in plasmids containing convenient 
restriction sites for insertion of the desired coding 
sequence. Typical of such vector plasmids are, for 
example, pUC8, and pUC13 available from J. Messing, 

10 at the University of Minnesota (see, e.g., Messing et 
al., Nucleic Acids Res. 9:309 (1981)) or pBR322, 
available from New England Biolabs. Suitable 
promoters include, for example, the beta- lactamase 
(penicillinase) and lactose (lac) promoter systems 

15 (Chang, et al., Nature 198:1056 (1977) and the 

tryptophan (trp) promoter system (Goeddel et al . , 
Nucleic Acids Res. 8:4057 (1980) ) . The resulting 
expression vectors are transformed into suitable 
bacterial hosts using the calcium chloride method 

20 described by Cohen, et al., Proc. Natl. Acad. Sci. 

U.S.A. 69:2110 (1972). Successful transf ormants may 
produce the desired polypeptide fragments at higher 
levels than those found in strains normally producing 
the intact pili. Of course, yeast or mammalian cell 

25 hosts can also be used, employing suitable vectors 
and control sequences. 



WO 99/40934 



PCT/US99/03055 



-37- 

Table 2 
Polypeptide haptens 



Organism 


Antigen 


T or B 
cell 


Sequence 


SEQ 


Streptococcus 
pneumoniae 


PspA 


B 


KLEBLSDKIDELDAE 


25 


Streptococcus 
pneumoniae 


PspA 


B 


SQKKYDEDQKKTEEKAALEKA 
ASEEMDKAVAAVQQA 


26 


Cryptosporidium 
parvum 


P23 


B 


QDKPADAPAAEAPAAEPAAQQ 
DKPADA 


27 


HIV 


P24 


T 


GPKEPFRDYVDRFYKC 


28 


HIV 


GP120 


B 


RKRIHIGPGRAFYITKN 


29 


Foot and Mouth 
Disease Virus 


VP1 


B 


YNGECRYNRNAVPNLRGDLQV 
LAQKVARTLP 


30 


Corynebacterium 
diphtheriae 


toxin 


T 


FQWHNSYNRPAYSPGC 


31 


Borrelia 
burgdorferi 


OspA 


T 


VE I KEGTVTLKRE IDKNGKVT 
VSLC 


32 


Borrelia 
burgdorferi 


OspA 


T 


TLSKNISKSGEVSVELNDC 


33 


Influenza A8/PR8 


HA 


T 


SSVSSFERFEC 


34 


Influenza A8/PR8 


HA 


B 


YRNLLWLTEK 


35 


Yersinia pestis 


V Ag 


B 


DILKVIVDSMNHHGDARSKLR 
EELAELTAELKIYSVIQAEIN 
KHLSSSGTINIHDKSINLMDK 
NLYGYTDEE I FKASAEYKI LE 

FLGSENKRTGALGNLKNSYSY 
NKDNNELSHFATTCSD 


36 


Haemophilus 
influenzae 


pBOMP 


B 


CSSSNNDAAGNGAAQFGGY 


37 


Haemophilus 
influenzae 


pBOMP 


B 


NKLGTVSYGEE 


38 


Haemophilus 
influenzae 


pBOMP 


B 


NDEAAYSKNRRAVLAY 


39 


Moraxella 
catarrhalis 


COpB 


B 


LDIEKDKKKRTDEQLQAELDD 
KYAGKGY 


40 


Moraxella 
catarrhalis 


copB 


B 


LD I EKNKKKRTEAELQAELDD 
KYAGKGY 


41 


Moraxella 
catarrhalis 


copB 


B 


IDIEKKGKIRTEAELLAELNK 
DYPGQGY 


42 



WO 99/40934 



PCT/US99/03055 



-38- 



Porphyromonas 
gingivalis 


HA 


8 


GVS PKVCKD VTVEG SNE FAP V 
QNLT 


43 


Porphyromonas 
gingivalis 


HA 


B 


RIQSTWRQKTVDLPAGTKYV 


44 


Trypanosoma 
cruzi 




T 


SHNFTLVAS VI I EEAPSGNTC 


45 


Trypanosoma 
cruzi 




B 


KAAIAPAKAAAAPAKAATAPA 


46 


Plasmodium 
falciparum 


MSP1 


T 


SVOIPKVPYPNGIVYC 


47 


pi a Qinofl i nm 

falciparum 


MSP1 


T 


D PNW YYTT . KTflT iF ADC 


48 


sobrinus 


Aa T / T T 


B & T 


V DP t> T V T? L. KT . A OW HTTP 


A Q 


oLieptococcus 
sobrinus 


a rr T /TT 
n^j 1/ 11 


U JC T 


A If ATYVT?AITT J^f"W'E , TfTYT' 1 /"'• 


O V 


lyrnphocy t i c 
Choriomeningitis 
vi rus 


IMP 


X 


KFyAbtj V i wcjInLi i Aye 




Shigella 
t icxneri 


Invasin 


B 


KDRTLIEQK 


52 


respiratory 

e> y lit* L. icl J. VIXUib 


G 


B 


CSICSNNPTCWAICK 


53 




CS 


g 






Clostridium 
tetani 


tox 


T 


QYIKANSKFIGITEIiC 


55 


Entamoeba 
histolytica 


lectin 


B 


VECASTVCQNDNSCP I IADVE 
KCNQ 


56 


Schistosoma 
j aponicum 


para 


B 


DLQSEISLSLENGELIRRAKS 
AESLAS EIiQRR VD 


57 


Schistosoma 
mansoni 


para 


B 


DLQSEISLSLENSEL IRRAKA 
AESLASDLQRRVD 


58 


Plasmodium vivax 




B 


DRAAGQPAGDRADGQPAG 


83 


Influenza virus 


Infl 


B 


CNNPHRIL 


84 


Influenza virus 


Infl 


T 


CPKYVKQNTLKLATGMRNVPE 
KQTR 


85 


Influenza virus 


Infl 


B 


SIMRSDAPIGTCSSECITPNG 
SIPNDKPFQNVNKITY 


14 


Influenza virus 


Infl 


B 


RGIFGAIAGFIENGWEGMIDG 
WYGFRHQN 


15 


Influenza virus 


Infl 


B 


EKQTRGIFGA 


16 


Mycobacterium 
tuberculosis 




T 


AVLEDPYILLVSSKV 


86 


Mycobacterium 
tuberculosis 




T 


LLVSSKVSTVKDLLP 


87 



WO 99/40934 



PCT/US99/03055 



-39- 



Mycobacterium 
tuberculosis 




T 


LLPLLEKVIGAGKPL 


88 


Mycobacterium 
tuberculosis 




T 


AILTGGQVISEEVGL 


89 


Mycobacterium 
tuberculosis 




T 


IAFNSGLEPGWAEK 


90 


Mycobacterium 
tuberculosis 




T 


ARRGLERGLNALADAVKV 


91 


Mycobacterium 
tuberculosis 




T 


EKIGAELVKEVAKK 


92 


Mycobacterium 

w UvCi <w U -L s/O X O 




T 


GLKRGIEKAVEKVTETL 


93 


Mycobacterium 

L. U.LJC- X UUlUDlO 




T 


IEDAVRNAKAAVEEG 


94 


Feline leukemia 
virus 


FeLV 


B 


CDI IGNTWNPSDQEPFPGYG 


95 


Feline leukemia 
virus 


FeLV 


B 


C I GTVPKTHQ ALCNETQQGHT 


96 


Feline leukemia 
virus 


FeLV 


B 


GNYSNQTNPPPSC 


97 


Feline leukemia 
virus 


FeLV 


B 


TD I QALEES I S ALEKSLTS LS 


98 


Feline leukemia 
virus 


FeLV 




AKLRERLKQRQQLF 


99 


Feline leukemia 
virus 


FeLV 




DSQQGWFEGWFNKSPWFTTLI 
cc 


100 


Feline leukemia 
virus 


FeLV 




OVMTITPPOAMG PNLVLP 


101 


Feline leukemia 
virus 


FeLV 




DQKPPSRQSQI ESRVTP 


102 


Feline leukemia 
virus 


FeLV 




RRGLDILFLQEGGLC 


103 


Feline leukemia 
virus 


FeLV 




QEGGLCAALEECQIGGLCAAL 
KEEC 


104 


Plasmodium 
falciparum 




B 


NANPNANPNANP 


105 


Circumsporozoite 






QAQGDGANAGQP 


113 



Chemical Hapten. Related chemistry is used 
to couple chemical compounds to carrier proteins. 
5 Typically, an appropriate functional group for 

coupling is designed into the chemical compound. 

Antibodies to the compound 6-0- 
phosphocholine hydroxyhexanoate protect against 
Streptococcus pneumoniae. Randy T. Fischer et al . J. 
10 Ijnmunol., 154:3373-3382 (1995). 



WO 99/40934 



PCT/US99/03055 



-40- 



• Table 3 
Chemical Haptens 



Chemical Hapten 


Citation 


piperidine N-oxide 


U.S. Patent No. 5,304,252 


phospholactone or 
lactamide 


U.S. Patent No. 5,248,611 


metal ion complexes 


U.S. Patent No. 5,236,825 


[2.2.1] or [7.2.2] 
bicyclic ring 
compounds 


U.S. Patent No. 5,208,152 


ionically charged 
hydroxyl -containing 
compounds 


U.S. Patent No. 5,187,086 


phosphonate analogs 
of carboxylate 
esters 


U.S. Patent No. 5,126,258 


cocaine analogs 


Carrera et al., Nature 
378:725 (1995) 



5 Carbohydrate Hapten. There are many 

methods known in the art to couple carrier proteins 
to polysaccharides. Aldehyde groups can be generated 
on either the reducing end [Anderson, Infect. Immun., 
39:233-238 (1983); Jennings, et al., J. Zmmunol . , 
10 127:1011-1018 (1981); Poren et al. t Mol . Immunol . , 
22:907-919 (1985)] or the terminal end [Anderson et 
al., J. Immunol., 137:1181-1186 (1986); Beuvery et 
al., Dev. Bio. Scand., 65:197-204 (1986)] of an 
oligosaccharide or relatively small polysaccharide, 



WO 99/40934 



PCT/US99/03055 



which can be linked to the carrier protein via 
reductive amination. 

Large polysaccharides can be conjugated by 
either terminal activation [Anderson et al., J. 
5 Immunol., 137:1181-1186 (1986)] or by random 

activation of several functional groups along the 
polysaccharide chain [Chu et al., Jnfect. Immun. , 
40:245-256 (1983); Gordon, U.S. Patent 4,619,828 
(1986); Marburg, U.S. Patent 4,882,317 (1989)]. 

10 Random activation of several functional groups along 
the polysaccharide chain can lead to a conjugate that 
is highly cross -linked due to random linkages along 
the polysaccharide chain. The optimal ratio of 
polysaccharide to carrier protein depend on the 

15 particular polysaccharide, the carrier protein, and 
the conjugate used. 

See Dick et al., in Contributions to 
Microbiology and Immunology . Vol. 10, Cruse et al . , 
eds., (S. Karger: 1989), pp. 48-114; Jennings et 

20 al., in NeoglyqQCQiQiuqates ; Preparation and 

Applications. Lee et al., eds., (Academic Press: 
1994), pp. 325-371; Aplin et al . , CRC Crit. Rev. 
Biochem., 10:259-306 (1981); and Stowell et al., Adv. 
Carbohydr. Chem. Biochem., 37:225-281 (1980) for 

25 detailed reviews of methods of conjugation of 
saccharide to carrier proteins. 

The carbohydrate itself can be synthesized 
by methods known in the art, for example by enzymatic 
glycoprotein synthesis as described by Witte et al., 

30 J. Am. Chem. Soc, 119:2114-2118 (1997). 



WO 99/40934 



PCT/US99/03055 



-42- 



10 



15 



20 



Several oligosaccharides, synthetic and 
semi -synthetic, and natural, are discussed in the 
following paragraphs as examples of oligosaccharides 
that are contemplated haptens to be used in making a 
strategically modified core protein conjugate of the 
present invention. 

An oligosaccharide hapten suitable for 
preparing vaccines for the treatment of Haemophilus 
Influenza type b (Hib) is made up of from 2 to 20 
repeats of D-ribose-D-ribitol -phosphate (I, below) , 
D-ribitol-phosphate-D-ribose (II, below) , or 
phosphate-D-ribose-D-ribitol (III, below) . Eduard C. 
Beuvery et al . , EP-0 276 516-B1. 



.OH 




O — I 



™/\*0 OH 



— OH 
— OH 



■OH O 
II 

-o— p°-N^ 
I 

OH 



OH 
OH 



-o— p- 

I 



■O OH 



OH 




I — OH 

-oh in 

I — OH 

U.S. Patent 4,220,717 also discloses a 
polyribosyl ribitol phosphate (PRP) hapten for 
Haemophilus influenzae type b. 

Ellena M. Peterson et al . , Infection and 
Immunity, 66 (8) : 3848-3855 (1998), disclose a 
tri saccharide hapten, aKdo(2 8)aKdo(2 4)aKdo, that 



II 



WO 99/40934 



PCT/US99/03055 



-43- 

provddes protection, from Chlamydia pneumoniae. 
Chlamydia pneumoniae is a cause of human respiratory 
infections ranging from pharyngitis to fatal 
pneumonia. Kdo is 3-deoxy-D-/nanno-oct-2-ulosonic 
5 acid. 

Bengt Andersson et al., EP-0 126 043-A1, 
disclose saccharides that can be used in the 
treatment, prophylaxis or diagnosis of bacterial 
infections caused by Streptococci pneumoniae. One 

10 class of useful saccharides are derived from the 

disaccharide GlcNAcPl 3Gal . Andersson et ai . also 
found neolactotetraosylceramide to be useful, which 
is Galpl 4GlcNAcpl 3Gaipi 4Glc-Cer. 

European Patent No. 0 157 899 -Bl, the 

15 disclosures of which are incorporated herein by 

reference, discloses the isolation of pneumococcal 
polysaccharides that are useful in the present 
invention. The following table lists the 
pneumococcal culture types that produce capsular 

2 0 polysaccharides useful as haptens in the present 
invention. 



Table 4 

Polysaccharide Hapten Sources 



Danish Type 
Nomenclature 


U.S. 
Nomenclature 


1978 ATCC Catalogue 
Number 


1 


1 


6301 


2 


2 


6302 


3 


3 


6303 


4 


4 


6304 


5 


5 





WO 99/40934 



PCTAJS99/03055 



-44- 



6A 


6 


6306 


6B 


26 


6326 


7F 


51 


10351 


8 


8 


6308 


9N 


9 


6309 


9V 


68 




10A 


34 




11A 


43 




12F 


12 


6312 


14 


14 


6314 


15B . 


54 




17F 


17 




18C 


56 


10356 


19A 


57 




19F 


19 


6319 


20 


20 


6320 


22F 


22 




23F 


23 


6323 


25 


25 


6325 


33F 


70 





Moraxella (Branhamella) catarrhalis is a 
known cause of otitis media and sinusitis in children 
and lower respiratory tract infections in adults. 
5 The lipid A portion of the lipooligosaccharide 

surface antigen (LOS) of the bacterium is cleaved at 
the 3-deoxy-D-manno-octulosonic acid-glucosamine 
linkage. The cleavage product is treated with mild- 
alkali to remove ester-linked fatty acids while 
10 preserving amide-linked fatty acids to yield 



WO 99/40934 PCT/US99/03055 

-45- 

detoxified lipopolysaccharide (dLOS) from M. 
catarrhalis. The dLOS is not immunogenic until it is 
attached to a protein carrier ♦ Xin-Xing Gu, et al . 
Infection and Immunity 66(5) : 1891-1897 (1998) . 
5 Group B streptococci (GBS) is a cause of 

sepsis, meningitis, and related neurologic disorders 
in humans. The Capsular polysaccharide-specif ic 
antibodies are known to protect human infants from 
infection. Jennings et al . , U.S. 5,795,580. The 

10 repeating unit of the GBS capsular polysaccharide 

type II is: 4 ) -P-D-GlcpNAc- (1 3) - [p-D-Galp(l 6)]-{J- 
D-Galp(l 4) - P-D-Glcp- (1 3 ) - p-D-Glcp- (1 2)-[<x-D- 
NeupNAc(2 3) ]- p-D-Galp- (1 , where the bracketed 
portion is a branch connected to the immediately 

15 following unbracketed subunit. The repeating unit of 
GBS capsular polysaccharide type V is: 4)-[a-D- 
NeupNAc-(2 3 ) -p-D-Galp- (1 4) -p-D-GlcpNAc- (1 6) ] -a-D- 
Glcp-(1 4) - [p-D-Glcp- (1 3) ] -P-D-Galp- (1 4)-P-D-Glcp- 
(1 . 

20 European patent application No. EU-0 641 

568 -Al, Dr. Helmut Brade, discloses the method of 
obtaining ladder- like banding pattern antigen from 
Chlamydia trachomatis , pneumoniae and psittaci . 

25 D. Pathogen-Related Conjugate to the Modified HBc 
In one embodiment of the invention, the 
hapten that is conjugated to strategically modified 
HBc protein is a B cell determinant of a pathogen. B 
cell determinants of numerous pathogens are known in 

30 the art, and several were illustrated in the 



WO 99/40934 



PCT/US99/03055 



preceding discussions of polypeptide and carbohydrate 
haptens . 

In preferred embodiments, the hapten is a 
pathogen-related hapten. The use of a portion of a 
5 pathogen's protein sequence or carbohydrate sequence 
as a hapten has distinct advantages over the exposure 
to an actual pathogen, and even over a passivated or 
"killed" version of the pathogen. 

Exemplary pathogen- related haptens of 

10 particular importance are derived from bacteria such 
as B. pertussis, S. typosa, S. paratyphoid A and B, 
C. diptheriae, C. tetani, C. botulinum, C. 
perfringens, B. anthracis, P. pestis, P. multocida, 
V. cholerae, N. meningi tides , N. gonorrhea, H. 

15 influenzae, T. palladium, and the like. 

Other exemplary sources of pathogen-related 
haptens of particular importance are viruses such as 
poliovirus, adenovirus, parainfluenza virus, measles, 
mumps, respiratory syncytical virus, influenza virus, 

2 0 equine encephalomyelitis virus, hog cholera virus, 

Newcastle virus, fowl pox virus, rabies virus, feline 
and canine distemper viruses, foot and mouth disease 
virus (FMDV) , human and simian immunodeficiency 
viruses, and the like. Other important sources of 

25 pathogen-related haptens include rickettsiae, 
epidemic and endemic typhus, the spotted fever 
groups, and the like. 

Pathogen- related polypeptide haptens are 
well-known in art and are discussed in numerous 

30 disclosures such as U.S. Pat. Nos. 3,149,036, 

3,983,228, and 4 , 069 , 3 13 ; in Essential Immunology . 3 rd 



WO 99/40934 



PCTAJS99/03055 



Ed., by Roit, published by Blackwell Scientific 
Publications; in Fundamentals of Clinical Immunology , 
by Alexander and Good, published by W.B. Saunders; 
and in Immunology , by Bellanti, published by W.B. 
5 Saunders . 

Particularly preferred pathogen-related 
haptens are those described in U.S. Pat. Nos. 
4,625,015, 4,544,500, 4,545,931, 4,663,436, 
4,631,191, 4,62 9,783 and in Patent Cooperation Treaty 

10 International Publication No. WO87/02775 and No. 

WO87/02892, all of whose disclosures are incorporated 
herein by reference . 

Antibodies that immunoreact with the 
hepatitis B virus can be obtained by using modified 

15 HBc conjugated with a polypeptide hapten that 

corresponds to the sequence of a determinant portion 
of HBsAg; in particular, residues 110-137 of the n S H 
(surface) region disclosed in Gerin et al., Proc. 
Natl. Acad. Sci. USA, 80:2365 (1983). 

20 Another conjugate corresponds to amino 

acids 93-103 of capsid protein VPI of poliovirus type 
1 (PV1, Mahoney strain), analogous to the work by 
Delpeyroux et al . , Science, 233:472-475 (1986). Such 
a modified HBc conjugate provides antibodies that 

25 immunoreact with polio. Other potential haptens from 
poliovirus type 1, Mahoney and Sabin strains are 
described in European Patent No. 385610. 

In preferred embodiments, the hapten is a 
pathogen-related hapten that immunoreact s with; i.e., 

30 is immunologically bound by, antibodies induced by 

the pathogen. More preferably, the pathogen-related 



WO 99/40934 



PCT/US99/03055 



-48- 

hapten induces an antibody response that provides 
protection against infection by the pathogen. 

Methods for determining the presence of 
antibodies to an immunogen in a body sample from an 
5 immunized animal are well known in the art. Methods 
for determining the presence of both cross-reactive 
and protective antibodies are well known in the art. 

In another embodiment of the invention, the 
immune response to the B cell determinant is boosted 
10 by also providing a T cell determinant. 

For example, U.S. Patent No. 4,882,145 
describes T cell stimulating polypeptides derived 
from the HBV nucleocapsid protein. Other T cell 
determinants are known in the art and can be used as 
15 an operatively linked determinant in a contemplated 
modified HBc protein or particle. 

In a particularly preferred embodiment of 
the invention, such a T cell determinant is derived 
from the same pathogen as the B cell determinant that 
20 is conjugated to the modified HBc. The T cell 

determinants of various pathogens are reported in the 
art . 

Although it is preferred that the B and T 
cell determinants are derived from the same pathogen, 

25 it is not necessary that they be from the same 

protein of that pathogen. For example, the B cell 
determinant from the VP1 protein of the foot and 
mouth disease virus (FMDV) can be conjugated to a 
modified HBc particle, wherein the HBc protein is 

3 0 further modified by having a T cell determinant 
derived from the VP4 protein of FMDV. 



WO 99/40934 



PCT/US99/03055 



The additional T cell determinant can be 
introduced into a modified HBc protein on the genetic 
level using well-known methods within or at a 
terminus of the HBc protein sequence (amino or 
5 carboxy termini) , including as part of the DNA 

inserted to introduce the chemically-reactive amino 
acid residue, but preferably as a fusion protein at 
the carboxy terminus. Alternatively, the additional 
T cell determinant can be operatively linked to the 

10 conjugated B cell determinant or the strategically 
modified HBc protein. 

Exemplary disclosures that describe 
techniques for genetically engineering a DNA sequence 
that can be used to produce a fusion protein of the 

15 present invention can be found in: U.S. Pat. Nos. 

4,428,941 to Galibert et al . , 4,237,224 to Cohen et 
al.: 4,273,875 to Manis; 4,431,739 to Riggs; 
4,363,877 to Goodman et al . , and Rodriguez & Tait, 
Recombinant DNA Techniques: An Introduction, The 

20 Benjamin-Cummings Publishing Co., Inc. Menlo Park, 

Calif. (1983), whose disclosures are incorporated by 
reference. 

E. InoculU an3 V3cciry?g 

25 In yet another embodiment of this invention, 

a modified HBc protein or particle conjugated to a 
hapten (a HBc conjugate) is used as the immonogen of 
an inoculum that induces production of antibodies 
that immunoreact with the hapten or as a vaccine to 

3 0 provide protection against the pathogen from which 
the hapten is derived. 



WO 99/40934 



PCTAJS99/03055 



-50- 

A contemplated inoculum or vaccine 
comprises a HBcAg conjugate that is dissolved or 
dispersed in a pharmaceutical^ acceptable diluent 
composition that typically also contains water. When 
5 administered in an immunogenic effective amount to an 
animal such as a mammal (e.g., a mouse, dog, goat, 
sheep, horse, bovine, monkey, ape, or human) or bird 
(e.g., a chicken, turkey, duck or goose), an inoculum 
induces antibodies that immunoreact with the 

10 conjugated {pendent ly- linked) hapten. A vaccine is a 
type of inoculum in which the hapten is pathogen 
related and the induced antibodies not only 
immunoreact with the hapten, but also immunoreact 
with the pathogen or diseased cell, and neutralize 

15 the pathogen or diseased cell with which they 
immunoreact . 

The preparation of inocula and vaccines 
that contain proteinaceous materials as active 
ingredients is also well understood in the art. 

20 Typically, such inocula or vaccines are prepared as 
parenterals, either as liquid solutions or 
suspensions; solid forms suitable for solution in, or 
suspension in, liquid prior to injection can also be 
prepared. The preparation can also be emulsified. 

25 The immunogenic active ingredient is often mixed with 
excipients that are pharmaceutical ly acceptable and 
compatible with the active ingredient. Suitable 
excipients are, for example, water, saline, dextrose, 
glycerol, ethanol, or the like and combinations 

30 thereof. In addition, if desired, an inoculum or 
vaccine can contain minor amounts of auxiliary 



WO 99/40934 



PCTAJS99/03055 



-51- 

substances such as wetting or emulsifying agents , pH 
buffering agents or adjuvants which enhance the 
immunogenic effectiveness of the composition. 

Exemplary adjuvants include complete 
5 Freund's adjuvant (CFA) that is not used in humans, 
incomplete Freund's adjuvant (IFA) and alum, which 
are materials well known in the art, and are 
available commercially from several sources. The use 
of small molecule adjuvants is also contemplated 
10 herein. 

Exemplary of one group of small molecule 
adjuvants are the so-called muramyl dipeptide 
analogues described in U.S. Patent No. 4,767,842. 
Another type of small molecule adjuvant described in 

15 U.S. Patent No. 4,787,482 that is also useful herein 
is a 4 : 1 by volume mixture of squalene or squalane 
and Aracel™ A (mannide monooleate) . 

Yet another type of small molecule adjuvant 
useful herein is a 7-substituted-8-oxo or 8-sulfo- 

20 guanosine derivative described in U.S. Patents No. 
4,539,205, No. 4,643,992, No. 5,011,828 and No. 
5,093,318, whose disclosures are incorporated by 
reference. Of these materials, 7-allyl-8- 
oxoguanosine (loxoribine) is particularly preferred. 

2 5 That molecule has been shown to be particularly 

effective in inducing an antigen- (immunogen- ) specif ic 
response 

Inocula and vaccines are conventionally 
administered parenterally , by injection, for example, 

3 0 either subcutaneously or intramuscularly. Additional 

formulations that are suitable for other modes of 



WO 99/40934 



PCT/US99/03055 



-52- 

administration include suppositories and, in some 
cases, oral formulation or by nasal spray. For 
suppositories, traditional binders and carriers can 
include, for example, polyalkalene glycols or 
5 triglycerides; such suppositories may be formed from 
mixtures containing the active ingredient in the 
range of 0.5% to 10%, preferably 1-2%. Oral 
formulations include such normally employed 
excipients as, for example, pharmaceutical grades of 
10 mannitol, lactose, starch, magnesium stearate, sodium 
saccharine, cellulose, magnesium carbonate and the 
like. 

An inoculum or vaccine composition takes 
the form of a solution, suspension, tablet, pill, 

15 capsule, sustained release formulation or powder, and 
contains an immunogenic amount of strategically 
modified HBc protein conjugate or strategically 
modified HBc protein particle conjugate as active 
ingredient. In a typical composition, an immunogenic 

20 amount of strategically modified HBc protein 

conjugate or strategically modified HBc protein 
particle conjugate is about 50 jag to. about 2 mg of 
active ingredient per dose, and more preferably about 
100 ng to about 1 mg per dose. 

25 The particles and protein conjugates can be 

formulated into the vaccine as neutral or salt forms. 
Pharmaceutical ly acceptable salts, include the acid 
addition salts (formed with the free amino groups of 
the peptide) and which are formed with inorganic 

30 acids such as, for example, hydrochloric or 

phosphoric acids, or such organic acids as acetic, 



WO 99/40934 



PCT/US99/03055 



-53- 

oxalic, tartaric, mandelic, and the like. Salts 
formed with the free carboxyl groups may also be 
derived form inorganic bases such as, for example, 
sodium, potassium, ammonium, calcium, or ferric 
5 hydroxides, and such organic bases as isopropyl amine, 
trimethylamine, 2-ethylamino ethanol, histidine, 
procaine, and the like. 

The inocula or vaccines are administered in 
a manner compatible with the dosage formulation, and 

10 in such amount as are therapeutically effective and 

immunogenic. The quantity to be administered depends 
on the subject to be treated, capacity of the 
subject's immune system to synthesize antibodies, and 
degree of protection desired. .Precise amounts of 

15 active ingredient required to be administered depend 
on the judgment of the practitioner and are peculiar 
to each individual. However, suitable dosage ranges 
are of the order of several hundred micrograms active 
ingredient per individual . Suitable regimes for 

20 initial administration and booster shots are also 
variable, but are typified by an initial 
administration followed in intervals (weeks or 
months) by a subsequent injection or other 
administration. 

25 Another embodiment of the invention is a 

process for inducing antibodies in an animal host 
comprising the steps of inoculating said animal host 
with an inoculum. The inoculum used in the process 
comprises an immunogenic amount of a strategically 

30 modified hepatitis B core protein conjugate dissolved 
or dispersed in a pharmaceutical ly acceptable 



WO 99/40934 



PCT/US99/03055 



-54- 

diluent. The strategically modified hepatitis B core 
protein conjugate used in the process comprises a 
hapten pendent ly linked to a strategically modified 
hepatitis B core protein. Preferably the 
5 strategically modified hepatitis B core protein is in 
particle form. The strategically modified hepatitis 
B core protein comprises an amino acid sequence 
corresponding to the hepatitis B core protein amino 
acid sequence of SEQ ID NO: 2 including the amino acid 

10 residues numbered about 10 to about 140 and 
additionally having an insert in the region 
corresponding to amino acid residues numbered about 
50 to about 100/ said insert (i) being 1 to about 40 
amino acid residues in length, and (ii) containing a 

15 chemically-reactive amino acid residue. The hapten 
is pendently linked to the strategically modified 
hepatitis B core protein through said chemically- 
reactive amino acid residue. Preferably, the hapten 
is pathogen-related. The animal is maintained for a 

20 time sufficient for antibodies to be induced. 

The invention is illustrated by the 
following non-limiting examples. 

25 Example 1: Construction of a Modified Hepatitis B 

core 'pyptQiyi Express A pn vector 

Using site-directed mutagenesis, a lysine 
codon (TTT) was introduced between amino acids D78 
and P79 of the HBc gene, along with EcoRI (GAATTC) 
30 and SacI (GAGCTC) restriction endonuclease sites to 

facilitate the genetic insertion of other condons for 



WO 99/40934 



PCTAJS99/03055 



-55- 

producing strategically modified hybrid HBc 
particles. The insert thus had an amino acid residue 
sequence GIQKEL, where the GIQ is an artifact of the 
EcoRI site and the EL is an artifact of the Sad 
5 site. The strategically modified hepatitis B core 
protein was therefore 155 amino acid residues long. 
The construction of the pKK322-HBcl55-K81 expression 
plasmid is described below. 

Oligonucleotide primers P1F (SEQ ID NO: 17) 

10 and P1R (SEQ ID NO: 18, on the complementary strand) 
were used to amplify the 5' end of the HBc gene 
(bases 1-234, amino acids 1-78), and simultaneously 
incorporate an Ncol restriction site (CCATGG) at the 
5' end, and an EcoRI restriction site (GAATTC) at the 

15 3' end of the amplified product. Oligonucleotide 

primers (SEQ ID NO: 19) P2F and P2R (SEQ ID NO:20, on 
the complementary strand) were used to amplify the 3' 
end of the HBc gene (bases 232-4 50, amino acids 79- 
149) , and simultaneously incorporate an EcoRI 

20 restriction site (GAATTC) at the 5' end, a SacI 

restriction site (GAGCTC) adjacent to it, an inserted 
lysine codon (AAA) between them, and a Hindi I I 
restriction site at (AAGCTT) the 3' end of the 
amplified product. 

25 The two PCR products (encoding amino acids 

1-78 and amino acids 7 9-14 9) were cleaved with EcoRI, 
ligated together at their common EcoRI overhangs, 
cleaved with Ncol and Hindi I I and cloned into the 
expression plasmid pKK332 (Pharmacia) , using standard 

30 techniques. The resulting plasmid was called pKK332- 
HBC-K81. This plasmid can be used for the expression 



WO 99/40934 



PCT/US99/03055 



-56- 

of a strategically modified HBc protein bearing a 
lysine in the immunodominant loop. The expressed 
strategically modified HBc protein spontaneously 
formed particles. The strategically modified HBc of 
5 this Example thus had an insert corresponding to 

position 78 of the HBc of SEQ ID NO:2, a chemically 
reactive lysine residue at a position corresponding 
to position 82 of the HBc of SEQ ID NO: 2, and was 
truncated at a position corresponding to position 149 
10 of the HBc of SEQ ID NO:2. 



WO 99/40934 PCT/US99/03055 

-57- 

Example 2: Modified Hepatitis B Core 

Particle Purification 

Strategically modified HBc particles of 
Example 1 were expressed in E. coli typically B, CPU 
5 BLR or BL21 from Novagen (Madison, Wisconsin) or JL. 
coli TB11 from Amersham (Arlington Heights, 
Illinois) . The transfected E. coli denoted HBcl55- 
K81, were expressed plasmid pKK332-HBcl55-K81 . The 
strategically modified HBc particles were purified 

10 via Sepharose CL-4B chromatography using established 
procedures. Because particles purify in a 
predictable manner, the monitoring of particle 
elution using simple spectroscopy (OD 280 ) , in concert 
with SDS-PAGE analysis to assess purity of individual 

15 fractions prior to pooling, was sufficient to enable 
the routine purification of electrophoretically pure 
particles in high yield (5-120 mg/L cell culture) . 
The spherical structure of the pure strategically 
modified hepatitis B core particles was clearly 

20 visible in an electron micrograph. 

Example 3: Chemical Coupling of Synthetic 

Peptides and Modified Hepatitis 

B Core Particles 

25 The strategically modified heptatitis B 

core particle product of the expression plasmid 
pKK332-HBcl55-K81 from Example 1 was assayed for its 
chemical reactivity compared with similarly expressed 
and purified "wild type" truncated hepatitis B core 
30 particle HBcl49, which is identical to HBcl55-K81 



WO 99/40934 



PCT/US99/03055 



-58- 

except that it lacks the introduced lysine residue 
and flanking five amino acids. 

Synthetic peptides (haptens) were 
chemically conjugated to HBc particles using 
5 succinimidyl 4- (N-maleimido-methyl) cyclohexane 
1-carboxylate (SMCC) , a water-soluble 
heterobifunctional cross-linking reagent. SMCC is 
reactive towards both sulfhydryl and primary amino 
groups, enabling the sequential conjugation of 

10 synthetic peptides to HBc particles whose primary 

amino groups have previously been modified with SMCC. 
Further, the 11.6 angstrom spacer arm afforded by 
SMCC helps to reduce steric hindrance between the 
hapten and the HBc carrier, thereby enabling higher 

15 coupling efficiencies. 

Briefly, HBcl55-K81 and HBcl49 particles 
were separately reacted with a 3 -fold excess of SMCC 
over total amino groups (native amino groups or 
native amino groups plus the one from the lysine 

20 residue of the insert) for 2 hours at room 

temperature in 50 mM sodium phosphate, pH 7.5, to 
form maleimide-activated HBc particles. Unreacted 
SMCC was removed by repeated dialysis against 50 mM 
sodium phosphate, pH 7.0. The SMCC derivitization of 

25 the HBc particles resulted in a minimal molecular 

weight increase which was not detectable by SDS-PAGE. 
However, the PAGE analysis did confirm the integrity 
of the HBc proteins prior to proceeding to the 
peptide conjugation step. 

30 Synthetic peptides to be coupled to the HBc 

particlrs were designed such that they had N- terminal 



WO 99/40934 



PCT/US99/03055 



-59- 

cysteine residues to enable directional conjugation 
of peptide haptens to the primary amine on the side 
chain of the introduced lysine residue via the 
cysteine sulfhydryl of the hapten. 

Table 2 shows the synthetic peptides 
derived from human cytochrome P450 enzymes that were 
chemically conjugated to HBc particles. The 
synthetic peptides were dissolved in 50 mM sodium 
phosphate, pH 7.0, to a concentration of 10 mg/ml. 
The synthetic peptides were then added, dropwise, to 
a 5- fold excess over total amino groups in maleimi de- 
activated strategically modified HBcl55-K81 
particles, and permitted to react at room temperature 
for 2 hours. Maleimide-activated HBcl49 particles 
were reacted with the two 2D6 peptides (206 and 206- 
C)as controls. 



WO 99/40934 



PCT/US99/03055 



-60- 

Table 5 
Cytochrome P-450 Haptens 





Peptide Name 


sequence bbu 


Tn ran 


5 


1A1 (289-302; 




Z 1 




1 A2 \Zy\- 6\)Z) 




ZZ 




2D6 (263-277) 


CLTEHRMTWDPAQPPRDLT 


23 




3A4 (253-273) 


CVKRMKESRLEDTQKHRVDFLQ 


24 




1A1-C 


CMQLRS 


106 


10 


1A2-C 


CRFSIN 


107 




2D6-C 


CAVPR 


108 




2E1-C 


CIPRS 


109 




2C-c 


CFIPV 


110 




3A3/4/7-C 


CTVSGA 


111 


15 


3A5-C 


CTLSGE 


112 



Example 4: Analysis of Modified Core 
Particles Conjugates 

20 Strategically modified HBc particles 

pendent ly linked to cytochrome P-450 determinant 
haptens of Example 3 were analyzed by SDS-PAGE and 
immunoblots to determine if synthetic peptides had 
been successfully conjugated to HBc. The denaturing 

25 conditions of the electrophoresis dissemble of 
particles into their constituent subunits, HBc 
monomers. Because HBc monomers have a molecular 
weight of approximately 16,000 Da / it was simple to 
resolve HBcl55-K81 particles chemically conjugated to 

30 either 1A1 (289-302), 1A2 (291-302), 2D6 (263-277) or 
3A4 (253-273) peptides, as those peptides have a 



WO 99/40934 



-61- 



PCT/US99/03055 



relative molecular mass of approximately 2,000 Da and 
therefore cause a visible increase in the molecular 
mass of the HBc protein monomers. From the relative 
intensities of the conjugated and non- conjugated 
5 bands on SDS-PAGE, it was revealed that approximately 
50 percent of the HBcl55-K81 monomers were 
operatively linked to hapten, whereas only about 5 
percent of the "wild type" HBcl4 9 particles were 
linked to hapten. The marked increase in the 

10 observed success in pendently linking hapten to the 
strategically modified hepatitis B core protein 
supports the conclusion that the observed linking 
occurs via the inserted lysine as opposed to a lysine 
residue that is also present in the "wild type". 

15 The shift in mobility of HBc particles 

conjugated to shorter C-terminal P450 derived 
peptides (5 and 6-mers) is not as pronounced in the 
SDS-PAGE as that of the longer inhibitory peptides, 
but shifts of approximately 700 Da were clearly 

20 evident in successfully coupled HBcl55-K81 monomers. 
The strategically modified HBc 155-K81 protein 
exhibited markedly enhanced ability to pendently link 
to a hapten over the "wild type" HBcl49 particles, 
which showed minimal conjugation. 

25 In the model of core particles propounded 

by Conway et al . of icosahedral particles of either 
180 or 240 associated core protein monomers [Mature, 
386:91-94 (1997)], dimers of the relatively exposed 
immunodominant loop regions of the core monomers 

30 extend out from the assembled core particle into 
solution like spikes on a mace. The "spikes" are 



WO 99/40934 PCT/US99/03055 

-62- 

closely arranged spatially on the HBc particles. The 
strategic location of the introduced lysine residue 
on the tip of the spike minimizes the propensity for 
steric constraints to reactions to link haptens to 
5 the assembled core particle. A maximum of 50 percent 
of the strategically modified HBc monomers were 
successfully conjugated to the synthetic peptides of 
Cyt P-450. That amount of pendent linkage 
corresponds to an average of one hapten attached per 

10 core particle spike. This proposed distribution of 
hapten linkage to the strategically modified HBc 
particle is supported by PAGE results under semi- 
denaturing conditions that dissemble the particle 
while maintaining the dimer association. 

15 HBC-2D6 particles, prepared by peptide 

coupling, were examined using immunoblots to confirm 
the presentation of the 2D6 epitope. When probed 
with anti-HBc antisera, the chemically coupled 
particle yielded two different monomers representing 

20 particles with and without the 2D6 peptide. Only the 
upper band of which blotted with anti-2D6 antisera, 
thereby confirming the correlation between mobility 
shift and attachment of the 2D6 peptide. 

25 Example 6: Strategic Lysine Insertions 

To construct HBc particles with inserted 
lysine residues at every position in the 
immunodominant, surface-exposed loop region (amino 
acids 75-85), PCR was used to amplify the 5* and 3' 

30 fragments of the HBc gene and a single lysine codon 
was introduced via the oligonucleotide primers. The 



WO 99/40934 



PCT/US99/03055 



-63- 

oligonucleotide primers and the resulting amino acid 
sequences are shown in SEQ ID NOs : 61-82. The "wild 
type" sequences are SEQ ID NOs : 59-60. 

In order to generate lysine inserts at 
5 positions 75 to 84 (HBC-K75 through HBC-K84) , the 
pairs of PCR fragments were digested with the 
restriction endonuclease Msel, which recognizes the 
sequence, AATT. The modified gene was restored by 
ligating the oligonucleotide primer (containing the 

10 lysine) at the convenient Msel restriction site 

located at nucleotides 221-224. For HBC-K85 (SEQ ID 
NOs : 85-86) it was necessary to generate two fragments 
that were ligated at a common Xhol restriction site 
(CTCGAG) that is not present in the wild type gene, 

15 but could be introduced at position 23 9-244 without 
altering any amino acids. 

Table 6 

Lysine insertion mutants of HBc 
20 in the immunodominant loop 



Name 


Sequence 


SEQ ID NO: 


Wild Type HBc 


TWVGVNLEDPASRDLWSYV 


60 


K75 


TWVGVJSNLEDPASRDLWSYV 


62 


K76 


TWVGVNKLEDPASRDLWSYV 


64 


K77 


TWVGVNLKEDPASRDLWSYV 


66 


K78 


TWVGVNLE&DPASRDLWSYV 


68 


K79 


TWVGVNLEDEPASRDLWSYV 


70 


K80 


TWVGVNLEDP&ASRDLWSYV 


72 


K81 


TWVGVNLEDPAKSRDLWSYV 


74 


K82 


TWVGVNLEDPAS&RDLWSYV 


76 



WO 99/40934 



PCT/US99/03055 



-64- 



K83 . 


TWVGVNLEDPASRKDLWSYV 


78 


K84 


TWVGVNLEDPASRDKLWSYV 


80 


K85 


TWVGVNLEDPASRDLKWSYV 


82 . 



To purify the strategically modified HBc 
proteins, cleared cell lysates from a 1L fermentation 
were precipitated with 45% ammonium sulfate and the 
5 resultant pellet subjected to gel filtration using 
Sepharose CL-4B chromatography (2.5cm x 100cm). 
Particulate HBc has a characteristic elution position 
when analyzed using this type of column, which was 
independent of the amino acid insertions made to the 
10 particle. The eleven strategically modified HBc 
particles generated for this study were analyzed 
using this procedure, and the elution profiles were 
measured spectrophotometrically at an absorbance of 
280 nm. 

15 Three of the constructs (HBc-K75, HBC-K77, 

and HBC-K79) were produced at levels of between 50 
and 100 mg/L, which is comparable with typical yields 
for wild- type, unmodified HBc particles, e.g. HBcl49 
particles. Four of the constructs (HBc-K76, HBc-K78, 

20 HBc-KSl, and HBC-K82) were produced at relatively low 
levels of between 1 and 2 0 mg/L. Finally, four of 
the particles (HBc-K80, HBC-K83, HBC-K84 , and HBc- 
K85) were produced at levels deemed to be barely 
detectable (< 1 mg/L) . 



WO 99/40934 



PCT/US99/03055 



-65- 
Table 7 

Yields of purified lysine-containing 
HBc particles from a 1L fermentation 



Particle 


Yield (mg/L) 


HBc-150 (K75) 


77 


HBc-150 (K76) 


5 


HBc-150 (K77) 


74 


HBc-150 (K78) 


10 


HBc-150 (K79) 


94 


HBc-150 (K80) 


0 


HBc-150 (K81) 


17 


HBc-150 (K82) 


1 


HBc-150 (K83) 


0 


HBc-150 (K84) 


0 


HBc-150 (K85) 


0 



The foregoing description of the invention, 
including the specific embodiments and examples, is 
intended to be illustrative of the present invention 
10 and is not to be taken as limiting. Numerous other 
variations and modifications can be effected without 
departing from the true spirit and scope of the 
present invention. 



WO 99/40934 



PCT/US99/03055 



-66- 

What is claimed: 

1. A strategically modified hepatitis B 
core protein conjugate comprising a strategically 
modified hepadnavirus nucleocapsid protein pendently 
linked to a hapten, wherein the strategically 
modified hepatitis B core protein comprises an amino 
acid sequence corresponding to the hepatitis B core 
protein amino acid sequence of SEQ ID NO: 2 including 
the amino acid residues numbered about 10 to about 
140 and additionally having an insert in the region 
corresponding to amino acid residues numbered about 
50 to about 100, said insert (i) being 1 to about 40 
amino acid residues in length, and (ii) containing a 
chemically-reactive amino acid residue, said hapten 
being pendently linked to said chemically-reactive 
amino acid residue. 

2. The stragetically modified hepatitis B 
20 core protein conjugate according to claim 1 wherein 

the insert is in the region corresponding to amino 
acid residues numbered about 70 to about 90. 

3. The stragetically modified hepatitis B 
25 core protein conjugate according to claim 1 wherein 

the insert is in the region corresponding to amino 
acid residues numbered 78 to 82 . 



10 



30 



4. The strategically modified hepatitis B 
core protein conjugate according to claim 1 wherein 



WO 99/40934 



PCT/US99/03055 



-67- 

the hapten is a polypeptide hapten, chemical hapten 
or carbohydrate hapten. 

5. The strategically modified hepatitis B 
5 core protein conjugate according to claim 1 wherein 

the hapten is a pathogen- related hapten. 

6. The strategically modified hepatitis B 
core protein conjugate according to claim 5 wherein 

10 the strategically modified hepatitis B core protein 
further comprises a T cell stimulating amino acid 
residue sequence operatively linked to the carboxy 
terminus of said hepatitis B amino acid sequence. 

15 7. The strategically modified hepatitis B 

core protein conjugate according to claim 6 wherein 
said T cell stimulating amino acid residue sequence 
is related to the same pathogen as the pendently 
linked pathogen -related hapten . 

20 

8. A strategically modified hepatitis B 
core protein particle comprising a strategically 
modified hepatitis B core protein subunit having an" 
insert containing a chemically-reactive amino acid 

25 side chain, said insert not itself an antigenic 
determinant . 

9. A strategically modified hepatitis B 
core protein particle conjugate comprising a 

30 plurality of strategically modified hepatitis B core 
protein subunits pendently linked to a hapten. 



WO 99/40934 



PCT/US99/03055 



-68- 

10. The strategically modified hepatitis B 
core protein particle conjugate according to claim 9 
wherein about 0.1 to about 0 . 5 of the strategically 

5 modified hepatitis B core protein subunits are 
pendent ly linked to a hapten. 

11. The strategically modified hepatitis B 
core protein particle conjugate according to claim 9 

10 having a plurality of strategically modified 

hepatitis B core protein subunits that are pendent ly 
linked to different haptens. 

12. The strategically modified hepatitis B 
15 core protein particle conjugate according to claim 9 

wherein the strategically modified hepatitis B core 
protein subunit further comprises a T cell 
stimulating amino acid residue sequence peptide- 
bonded to the carboxy terminal residue of the amino 
20 acid sequence corresponding to said hepatitis B amino 
acid sequence. 

13. The strategically modified hepatitis B 
core protein particle conjugate according to claim 12 

25 wherein the T cell stimulating amino acid residue is 
from the same pathogen as the pendent ly linked 
hapten. 



30 



14 . An immunogenic fusion protein 
conjugate comprising a polypeptide hapten pendently 
linked to a strategically modified hepatitis B core 



WO 99/40934 



PCT/US99/03055 



protein that comprises three peptide -linked domains, 
I, II and III from the N- terminus, wherein 

(a) Domain I corresponds to residues about 
10 through 50 of the amino acid sequence of hepatitis 

5 B core protein of SEQ ID NO: 2; 

(b) Domain II is bonded to the carboxy 
terminal residue of Domain I and corresponds to 
residues 50 to 100 of said amino acid sequence of 
hepatitis B core protein that further contains a 

10 heterologous insert 1 to about 40 amino acid residues 
in length that includes a chemically-reactive amino 
acid residue; and 

(c) Domain III is bonded to the carboxy 
terminal residue of Domain II and corresponds to 

15 residues 100 to about 140 of the amino acid sequence 
of hepatitis B core protein. 

15 . An immunogenic fusion protein 
conjugate according to claim 14 wherein the 

20 strategically modified hepatitis B core protein 
further comprises a fourth peptide- linked domain, 
Domain IV, wherein 

(d) Domain IV is bonded to the carboxy 
terminal residue of Domain III and comprises a T cell 

25 epitope. 

16. A strategically-modified hepatitis B 
core protein comprising an amino acid sequence 
corresponding to about amino acid residue 10 to about 

30 amino acid residue 140 of the hepatitis B amino acid 
sequence of SEQ ID NO: 2 and having an amino acid 



WO 99/40934 PCIYUS99/03055 

-70- 

sequence insert in the region corresponding to about 
residue 50 to about residue 100 of the hepatitis B, 
wherein the insert includes a chemically-reactive 
amino acid residue and the insert itself is not an 
5 antigenic determinant. 

17. An inoculum comprising an immunogenic 
amount of a strategically modified hepatitis B core 
protein conjugate dissolved or dispersed in a 

10 pharmaceutically acceptable diluent, said 

strategically modified hepatitis B core protein 
conjugate comprising a hapten pendently linked to a 
strategically modified hepatitis B core protein, 
wherein the strategically modified hepatitis B core 

15 protein comprises an amino acid sequence 

corresponding to the hepatitis B core protein amino 
acid sequence of SEQ ID NO: 2 including the amino acid 
residues numbered about 10 to about 14 0 and 
additionally having an insert in the region 

20 corresponding to amino acid residues numbered about 
50 to about 100, said insert (i) being 1 to about 40 
amino acid residues in length, and (ii) containing a 
chemically- reactive amino acid residue, said hapten 
being pendently linked to said chemically- reactive 

25 amino acid residue. 



18. The inoculum according to claim 17 
wherein said strategically modified hepatitis B core 
protein conjugate is present in the form of 
30 particles. 



WO 99/40934 PCT/US99/03055 

-71- 

19. The inoculum according to claim 18 
wherein said hapten is from a pathogen and wherein 
use of said inoculum in a mammalian host protects 
said mammalian host from said pathogen. 

5 

20. A process for inducing antibodies in 
an animal host comprising the steps of inoculating 
said animal host with an inoculum that comprises an 
immunogenic amount of a strategically modified 

10 hepatitis B core protein conjugate dissolved or 

dispersed in a pharmaceutically acceptable diluent, 
said strategically modified hepatitis B core protein 
conjugate comprising a hapten pendent ly linked to a 
strategically modified hepatitis B core protein, 

15 wherein the strategically modified hepatitis B core 
protein comprises an amino acid sequence 
corresponding to the hepatitis B core protein amino 
acid sequence of SEQ ID NO: 2 including the amino acid 
residues numbered about 10 to about 140 and 

20 additionally having an insert in the region 

corresponding to amino acid residues numbered about 
50 to about 100, said insert (i) being 1 to about 40 
amino acid residues in length, and (ii) containing a 
chemically- reactive amino acid residue, said hapten 

25 being pendently linked to said strategically modified 
hepatitis B core protein through said chemically- 
reactive amino acid residue, and maintaining said 
animal for a time sufficient for antibodies to be 
induced . 



30 



WO 99/40934 



PCT/US99/03055 



-72- 

21. The process according to claim 20 
wherein said strategically modified hepatitis B core 
protein conjugate is present in the form of 
particles - 

5 

22. process according to claim 21 wherein 
said hapten is from a pathogen and wherein the 
antibodies induced in said mammalian host protect 
said mammalian host from said pathogen. 



10 



WO 99/40934 



PCTAJS99/03055 




WO 99/40934 



PCT7US99/03055 



SEQUENCE LISTING 

<110> Birkett, Ashley J. 

Immune Complex Inc. 

<120> Strategically Modified Hepatitis B Core Proteins and 
their Derivatives 

<130> SYN-101 4564/69529 

<140> PCT/US99/ 
<141> 1999-02-11 

<150> 60/074537 
<151> 1998-02-12 

<160> 113 

<170> Pa tent In Ver. 2.0 

<210> 1 
<211> 553 
<212> DNA 

<213> Hepatitis B virus 

<220> 

<221> CDS 

<222> (1) . . (549) 

<300> 

<303> Nature 
<304> 281 
<306> 646- 
<307> 1979 

<400> 1 

atg gac ate gac cct tat aaa gaa ttt gga get act gtg gag tta etc 48 
Met Asp lie Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 
15 10 15 

teg ttt ttg cct tct gac ttc ttt cct tea gta cga gat ctt eta gat 96 
Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 
20 25 30 

acc gee tea get ctg tat egg gaa gee tta gag tct cct gag cat tgt 144 
Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 
35 40 45 



1 



WO 99/40934 



PCT/US99/03055 



tea cct cac cat act gca etc agg caa gca att ctt tgc tgg ggg gaa 192 
Ser Pro His His Thr Ala Leu Arg Gin Ala lie Leu Cys Trp Gly Glu 
50 55 60 

eta atg act eta get ace tgg gtg ggt gtt aat ttg gaa gat cca gcg 240 
Leu Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala 
65 70 75 80 

tct aga gac eta gta gtc agt tat gtc aac act aat atg ggc eta aag 288 
Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys 
85 90 95 

ttc agg caa etc ttg tgg ttt cac att tct tgt etc act ttt gga aga 336 
Phe Arg Gin Leu Leu Trp Phe His lie Ser Cys Leu Thr Phe Gly Arg 
100 105 110 

gaa aca gtt ata gag tat ttg gtg tct ttc gga gtg tgg att cgc act 384 
Glu Thr Val lie Glu Tyr Leu Val Ser Phe Gly Val Trp lie Arg Thr 
1X5 120 125 

cct cca get tat aga cca cca aat gec cct ate eta tea aca ctt ccg 432 
Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro lie Leu Ser Thr Leu Pro 
130 135 140 

gag act act gtt gtt aga cga cga ggc agg tec cct aga aga aga act 480 
Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr 
145 150 155 160 

ccc teg cct cgc aga cga agg tct caa teg ccg cgt cgc aga aga tct 528 
Pro Ser Pro Arg Arg Arg Arg Ser Gin Ser Pro Arg Arg Arg Arg Ser 
165 170 175 

caa tct egg gaa tct caa tgt tagt 553 
Gin Ser Arg Glu Ser Gin Cys 
180 



<210> 2 
<211> 183 
<212> PRT 

<213> Hepatitis B virus 
<400> 2 

Met Asp lie Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 
1 5 10 15 

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 
20 25 30 



2 



WO 99/40934 



PCTAJS99/03055 



Thr Ala Ser Ala Leu Tyr Arg Olu Ala Leu Olu Ser Pro Olu His Cys 
35 40 45 

Ser Pro His His Thr Ala Leu Arg Gin Ala lie Leu Cys Trp Gly Glu 
50 55 60 

Leu Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala 
65 70 75 80 

Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys 
85 90 95 

Phe Arg Gin Leu Leu Trp Phe His lie Ser Cys Leu Thr Phe Gly Arg 
100 105 110 

Glu Thr Val lie Glu Tyr Leu Val Ser Phe Gly Val Trp lie Arg Thr 
115 120 125 

Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro He Leu Ser Thr Leu Pro 
130 135 140 

Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr 
145 150 155 160 

Pro Ser Pro Arg Arg Arg Arg Ser Gin Ser Pro Arg Arg Arg Arg Ser 
165 170 175 

Gin Ser Arg Glu Ser Gin Cys 
180 



<210> 3 
<211> 552 
<212> DHA 

<213> Hepatitis B virus 

<220> 

<221> CDS 

<222> (1)..(549) 

<300> 

<303> Nucleic Acids Res* 
<304> 11 
<306> 1747- 
<307> 1983 

<400> 3 

atg gac att gac ccg tat aaa gaa 



ttt gga gca tct gtg gag tta etc 48 



3 



WO 99/40934 PCT/US99/03055 



Met Asp lie Asp Pro Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu 
15 10 15 

tct ttt ttg cct tct gac ttc ttt ccg tct att cga gat etc ctt gac 96 
Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser lie Arg Asp Leu Leu Asp 
20 25 30 

acc gec tct get ctg tat egg gag gee tta gag tct ccg gaa cat tgt 144 
Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 
35 40 45 

tea cct cac cat aca gca etc agg caa get att ctg tgt tgg ggt gag 192 
Ser Pro His His Thr Ala Leu Arg Gin Ala lie Leu Cys Trp Gly Glu 
50 55 60 

tta atg aat ctg gec acc tgg gtg gga agt aat ttg gaa gac cca gca 240 
Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala 
65 70 75 80 

tec agg gaa tta gta gtc age tat gtc aat gtt aat atg ggc eta aaa 288 
Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys 
85 90 95 

ate aga caa eta ttg tgg ttt cac att tec tgc ctt act ttt gga aga 336 
He Arg Gin Leu Leu Trp Phe His He Ser Cys Leu Thr Phe Gly Arg 
100 105 110 

gaa act gtt ttg gag tat ttg gta tct ttt gga gtg tgg att cgc act 384 
Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp He Arg Thr 
115 120 125 

cct ccc get tac aga cca cca aat gec cct ate tta tea aca ctt ccg 432 
Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro He Leu Ser Thr Leu Pro 
130 135 140 

gaa act act gtt gtt aga cga cga ggc agg tec cct aga aga aga act 480 
Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr 
145 150 155 160 

ccc teg cct cgc aga cga agg tct caa teg ccg cgt cgc aga aga tct 528 
Pro Ser Pro Arg Arg Arg Arg Ser Gin Ser Pro Arg Arg Arg Arg Ser 
165 170 175 

caa tct egg gaa tct caa tgt tag 552 
Gin Ser Arg Glu Ser Gin Cys 
180 



4 



WO 99/40934 



PCI7US99/03055 



<210> 4 
<211> 183 
<212>. PRT 

<213> Hepatitis B virus 
<400> 4 

Met Asp lie Asp Pro Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu 
15 10 15 

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser lie Arg Asp Leu Leu Asp 
20 25 30 

Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 
35 40 45 

Ser Pro His His Thr Ala Leu Arg Gin Ala lie Leu Cys Trp Gly Glu 
50 55 60 

Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala 
65 70 75 80 

Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys 
85 90 95 

lie Arg Gin Leu Leu Trp Phe His lie Ser Cys Leu Thr Phe Gly Arg 
100 105 110 

Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp He Arg Thr 
115 120 125 

Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro He Leu Ser Thr Leu Pro 
130 135 140 

Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr 
145 150 155 160 

Pro Ser Pro Arg Arg Arg Arg Ser Gin Ser Pro Arg Arg Arg Arg Ser 
165 170 175 

Gin Ser Arg Glu Ser Gin Cys 
180 



<210> 5 
<211> 558 
<212> DNA 

<213> Hepatitis B virus 



5 



WO 99/40934 



PCTAJS99/03055 



<220> 

<221> CDS 

<222> (1) . . (555) 

<300> 

<303> Nucleic Acids Res. 
<304> 11 
<306> 1747- 
<307> 1983 



<400> 5 

atg gac att gac cct tat aaa gaa 
Met Asp lie Asp Pro Tyr Lys Glu 
1 5 

teg ttt ttg cct tct gac ttc ttt 
Ser Phe Leu Pro Ser Asp Phe Phe 
20 



ttt gga get act gtg gag tta etc 48 
Phe Gly Ala Thr Val Glu Leu Leu 
10 15 

cct tec gta cga gat etc eta gac 96 
Pro Ser Val Arg Asp Leu Leu Asp 
25 30 



acc gee tea get ctg tat cga gaa gee tta gag tct cct gag cat tgc 144 
Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 
35 40 45 

tea cct cac cat act gca etc agg caa gee att etc tgc tgg ggg gaa 192 
Ser Pro His His Thr Ala Leu Arg Gin Ala lie Leu Cys Trp Gly Glu 
50 55 60 

ttg atg act eta get acc tgg gtg ggt aat aat ttg caa gat cca gca 240 
Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Gin Asp Pro Ala 
65 70 75 80 

tec aga gat eta gta gtc aat tat gtt aat act aac atg ggt tta aag 288 
Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys 
85 90 95 

ate agg caa eta ttg tgg ttt cat ata tct tgc ctt act ttt gga aga 336 
lie Arg Gin Leu Leu Trp Phe His lie Ser Cys I*eu Thr Phe Gly Arg 
100 105 HO 



gag act gta ctt gaa tat ttg gtc tct ttc gga gtg tgg att cgc act 
Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp He Arg Thr 
115 120 125 

cct cca gec tat aga cca cca aat gec cct ate tta tea aca ctt ccg 
Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro He Leu Ser Thr Leu Pro 
130 135 140 

gaa act act gtt gtt aga cga egg gac cga ggc agg tec cct aga aga 



384 



432 



480 



6 



WO 99/40934 



PCT/US99/03055 



Glu Thr Thr Val Val Arg Arg Arg Asp Arg Gly Arg Ser Pro Arg Arg 
145 150 155 160 

aga act ccc teg cct cgc aga cgc aga tct caa teg ccg cgt cgc aga 528 
Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gin Ser Pro Arg Arg Arg 
165 170 175 

aga tct caa tct egg gaa tct caa tgt tag 558 
Arg Ser Gin Ser Arg Glu Ser Gin Cys 
180 185 



<210> 6 
<211> 185 
<212> PRT 

<213> Hepatitis B virus 
<400> 6 

Met Asp He Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 
15 10 15 

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 
20 25 30 

Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 
35 40 45 

Ser Pro His His Thr Ala Leu Arg Gin Ala He Leu Cys Trp Gly Glu 
50 55 60 

Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Gin Asp Pro Ala 
65 70 75 80 

Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys 
85 90 95 

He Arg Gin Leu Leu Trp Phe His He Ser Cys Leu Thr Phe Gly Arg 
100 105 110 

Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp He Arg Thr 
115 120 125 

Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro He Leu Ser Thr Leu Pro 
130 135 140 

Glu Thr Thr Val Val Arg Arg Arg Asp Arg Gly Arg Ser Pro Arg Arg 
145 150 155 160 



7 



WO 99/40934 



PCT/US99/03055 



Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gin Ser Pro Arg Arg Arg 
165 170 175 

Arg Ser Gin Ser Arg Gin Ser Gin Cys 
180 185 



<210> 7 
<211> 188 
<212> PRT 

<213> Hepatitis B virus 
<300> 

<301> Schodel, Florian 
et al., 

<302> Animal Hepatitis B Viruses 
<303> Adv. Viral Oncol* 
<304> 8 
<306> 73-102 
<307> 1989 

<400> 7 

Met Asp lie Asp Pro Tyr Lys Glu Phe Gly Ser Ser Tyr Gin Leu Leu 
15 10 15 

Asn Phe Leu Pro Leu Asp Phe Phe Pro Asp Leu Asn Ala Leu Val Asp 
20 25 30 

Thr Ala Ala Ala Leu Tyr Glu Glu Glu Leu Thr Gly Arg Glu His Cys 
35 40 45 

Ser Pro His His Thr Ala lie Arg Gin Ala Leu Val Cys Trp Asp Glu 
50 55 60 

Leu Thr Lys Leu He Ala Trp Met Ser Ser Asn He Thr Ser Glu Gin 
65 70 75 80 

Val Arg Thr He He Val Asn His Val Asn Asp Thr Trp Gly Leu Lys 
85 90 95 

Val Arg Gin Ser Leu Trp Phe His Leu Ser Cys Leu Thr Phe Gly Gin 
100 105 110 

His Thr Val Gin Glu Phe Leu Val Ser Phe Gly Val Trp He Arg Thr 
115 120 125 

Pro Ala Pro Tyr Arg Pro Pro Asn Ala Pro He Leu Ser Thr Leu Pro 
130 135 140 



8 



WO 99/40934 



PCT/US99/03055 



Glu His Thr Val lie Arg Arg Arg Oly Gly Ala Arg Ala Ser Arg Ser 
145 150 155 160 

Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gin Ser Pro 
165 170 175 

Arg Arg Arg Arg Ser Gin Ser Pro Ser Ala Asn .Cys 
180 185 



<210> 8 
<211> 654 
<212> DNA 

<213> Hepatitis B virus 

<220> 

<221> CDS 

<222> (1) . . (651) 

<300> 

<302> Animal Hepatitis B Viruses 
<303> Adv. Viral Oncol. 
<304> 8 
<306> 73-102 
<307> 1989 

<400> 8 

atg tat ctt ttt cac ctg tgc ctt 
Met Tyr Leu Phe His Leu Cys Leu 
1 5 



gtt ttt gcc tgt gtt cca tgt cct 48 
Val Phe Ala Cys Val Pro Cys Pro 
10 15 



act gtt caa gcc tec aag ctg tgc ctt gga tgg ctt tgg gac atg gac 96 
Thr Val Gin Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Asp Met Asp 
20 25 30 

ata gat ccc tat aaa gaa ttt ggt tct tct tat cag ttg ttg aat ttt 144 
lie Asp Pro Tyr Lys Glu Phe Gly Ser Ser Tyr Gin Leu Leu Asn Phe 
35 40 45 

ctt cct ttg gac ttt ttt cct gat etc aat gca ttg gtg gac act get 192 
Leu Pro Leu Asp Phe Phe Pro Asp Leu Asn Ala Leu Val Asp Thr Ala 
50 55 60 



get get ctt tat gaa gaa gaa tta aca ggt agg gag cat tgt tct cct 240 
Ala Ala Leu Tyr Glu Glu Glu Leu Thr Gly Arg Glu His Cys Ser Pro 
65 70 75 80 



9 



WO 99/40934 PCTAJS99/03055 



cat cat act get att aga cag gec tta gtg tgt tgg gaa gaa tta act 288 
His His Thr Ala lie Arg Gin Ala Leu Val Cys Trp Glu Glu Leu Thr 
85 90 95 

aga tta att aca tgg atg agt gaa aat aca aca gaa gaa gtt aga aga 336 
Arg Leu He Thr Trp Met Ser Glu Asxi Thr Thr Glu Glu Val Arg Arg 
100 105 110 

att att gtt gat cat gtc aat aat act tgg gga ctt aaa gta aga cag 384 
lie lie Val Asp His Val Asn Asxi Thr Trp Gly Leu Lys Val Arg Gin 
115 120 125 

act tta tgg ttt cat tta tea tgt ctt act ttt gga caa cac aca gtt 432 
Thr Leu Trp Phe His Leu Ser Cys Leu Thr Phe Gly Gin His Thr Val 
130 135 140 

caa gaa ttt ttg gtt agt ttt gga gta tgg att aga act cca get cct 480 
Gin Glu Phe Leu Val Ser Phe Gly Val Trp He Arg Thr Pro Ala Pro 
145 150 155 160 

tat aga cca cct aat gca ccc att tta tea act ctt ccg gaa cat aca 528 
Tyr Arg Pro Pro Asn Ala Pro lie Leu Ser Thr Leu Pro Glu His Thr 
165 170 175 

gtc att agg aga aga gga ggt tea aga get get agg tec ccc cga aga 576 
Val He Arg Arg Arg Gly Gly Ser Arg Ala Ala Arg Ser Pro Arg Arg 
180 185 190 

cgc act ccc tct cct cgc agg aga agg tct caa tea ccg cgt cgc aga 624 
Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gin Ser Pro Arg Arg Arg 
195 200 205 

cgc tct caa tct cca get tec aac tgc tga 654 
Arg Ser Gin Ser Pro Ala Ser Asn Cys 
210 215 



<210> 9 
<211> 217 
<212> PRT 

<213> Hepatitis B virus 
<400> 9 

Met Tyr Leu Phe His Leu Cys Leu Val Phe Ala Cys Val Pro Cys Pro 
15 10 15 

Thr Val Gin Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Asp Met Asp 
20 25 30 



10 



WO 99/40934 PCT/US99/03055 



He Asp Pro Tyr Lys Glu Pbe Gly Ser Ser Tyr Gin Leu Leu Asn Phe 
35 40 45 

Leu Pro Leu Asp Phe Phe Pro Asp Leu Asn Ala Leu Val Asp Thr Ala 
50 55 60 - 

Ala Ala Leu Tyr Glu Glu Glu Leu Thr Gly Arg Glu His Cys Ser Pro 
65 70 75 80 

His His Thr Ala He Arg Gin Ala Leu Val Cys Trp Glu Glu Leu Thr 
65 90 95 

Arg Leu He Thr Trp Met Ser Glu Asn Thr Thr Glu Glu Val Arg Arg 
100 105 110 

He He Val Asp His Val Asn Asn Thr Trp Gly Leu Lys Val Arg Gin 
115 120 125 

Thr Leu Trp Phe His Leu Ser Cys Leu Thr Phe Gly Gin His Thr Val 
130 135 140 

Gin Glu Phe Leu Val Ser Phe Gly Val Trp He Arg Thr Pro Ala Pro 
145 150 155 160 

Tyr Arg Pro Pro Asn Ala Pro He Leu Ser Thr Leu Pro Glu His Thr 
165 170 175 

Val He Arg Arg Arg Gly Gly Ser Arg Ala Ala Arg Ser Pro Arg Arg 
180 185 190 

Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gin Ser Pro Arg Arg Arg 
195 200 205 

Arg Ser Gin Ser Pro Ala Ser Asn Cys 
210 215 



<210> 10 
<211> 918 
<212> DNA 

<213> Hepatitis B virus 

<220> 

<221> CDS 

<222> <1) . . (915) 

<400> 10 



11 



WO 99/40934 PCT/US99/03055 



atg tgg tct eta aga tta cac ccc tct cca ttc gga get gcg tgc caa 48 
Met Trp Ser Leu Arg Leu His Pro Ser Pro Phe Gly Ala Ala Cys Gin 
15 10 15 

ggt ate ttt acg teg ace teg ctg ttg ttc ctt gtg act gta cct ttg 96 
Gly lie Phe Thr Ser Thr Ser Leu Leu Phe Leu Val Thr Val Pro Leu 
20 25 30 

gta tgt acc att gtt tat gat tct tgc tta tat atg gat gtc aat get 144 
Val Cys Thr He Val Tyr Asp Ser Cys Leu Tyr Met Asp Val Asn Ala 
35 40 45 

tea aga get tta gca aat gta tat gat ctg cca gat gat ttc ttt cct 192 
Ser Arg Ala Leu Ala Asn Val Tyr Asp Leu Pro Asp Asp Phe Phe Pro 
50 55 60 

cag att gat gat ctt gtt aga gat get aag gat get tta gaa cct tat 240 
Gin He Asp Asp Leu Val Arg Asp Ala Lys Asp Ala Leu Glu Pro Tyr 
65 70 75 80 

tgg aaa gec gaa aca ata aag aaa cat gtt tta att get act cac ttt 288 
Trp Lys Ala Glu Thr lie Lys Lys His Val Leu He Ala Thr His Phe 
85 90 95 

gtg gat ttg att gag gac ttc tgg cag acc act cag ggt atg age caa 336 
Val Asp Leu He Glu Asp Phe Trp Gin Thr Thr Gin Gly Met Ser Gin 
100 105 110 

att gca gac gee etc cga gca gta att cca cct act acc gta cca gta 384 
He Ala Asp Ala Leu Arg Ala Val He Pro Pro Thr Thr Val Pro Val 
115 120 125 

ccg gag ggt ttt etc att act cat agt gag gca gaa gag ate ccc ttg 432 
Pro Glu Gly Phe Leu He Thr His Ser Glu Ala Glu Glu He Pro Leu 
130 135 140 

aac gat etc ttt tea aat caa gag gag agg ata gtc aat ttc caa cct 480 
Asn Asp Leu Phe Ser Asn Gin Glu Glu Arg He Val Asn Phe Gin Pro 
145 150 155 160 

gac tat ccc att aca get aga att cat acc cac tta cgt gtt tat act 528 
Asp Tyr Pro He Thr Ala Arg He His Thr His Leu Arg Val Tyr Thr 
165 170 175 

aaa ttg aat gaa caa get ttg gac aaa get cgc aga ttg ctt tgg tgg 576 
Lys Leu Asn Glu Gin Ala Leu Asp Lys Ala Arg Arg Leu Leu Trp Trp 
180 185 190 



12 



WO 99/40934 



PCT/US99/03055 



cat tac aat tgc etc etc tgg gga gaa gee act gtt aca aat tat att 624 
His Tyr Asn Cys Leu Leu Trp Gly Glu Ala Thr Val Thr Asn Tyr lie 
195 200 205 

tct cgc etc cgt act tgg ctt tct act ccc gaa aaa tat cga ggc aag 672 
Ser Arg Leu Arg Thr Trp Leu Ser Thr Pro Glu Lys Tyr Arg Gly Lys 
210 215 220 

gat gec cca acc att gaa gca ate act aga cca ate cag gtg get caa 720 
Asp Ala Pro Thr lie Glu Ala He Thr Arg Pro He Gin Val Ala Gin 
225 230 235 240 

gga ggc aga aat caa act aag gga act aga aaa cct cgt gga etc gaa 768 
Gly Gly Arg Asn Gin Thr Lys Gly Thr Arg Lys Pro Arg Gly Leu Glu 
245 , 250 255 

cct aga aga cga aag gtt aaa aec aca gtt gtc tat ggg aga aga cgt 816 
Pro Arg Arg Arg Lys Val Lys Thr Thr Val Val Tyr Gly Arg Arg Arg 
260 265 270 

tct aag tec cga ggc agg aga tec tct cca tec caa cgt gcg ggc tec 864 
Ser Lys Ser Arg Gly Arg Arg Ser Ser Pro Ser Gin Arg Ala Gly Ser 
275 280 285 

cct etc cca cgt aat egg gga aac cag aca cga tec ccc tea cct agg 912 
Pro Leu Pro Arg Asn Arg Gly Asn Gin Thr Arg Ser Pro Ser Pro Arg 
290 295 300 

gaa tag 918 

Glu 

305 



<210> 11 
<211> 305 
<212> PRT 

<213> Hepatitis B virus 
<400> 11 

Met Trp Ser Leu Arg Leu His Pro Ser Pro Phe Gly Ala Ala Cys Gin 
1 5 10 15 

Gly He Phe Thr Ser Thr Ser Leu Leu Phe Leu Val Thr Val Pro Leu 
20 25 30 

Val Cys Thr He Val Tyr Asp Ser Cys Leu Tyr Met Asp Val Asn Ala 
35 40 45 



13 



WO 99/40934 PCT/US99/03055 



Ser Arg Ala Leu Ala Asn Val Tyr Asp Leu Pro Asp Asp Phe Phe Pro 
50 55 60 

Gin lie Asp Asp Leu Val Arg Asp Ala Lys Asp Ala Leu Glu Pro Tyr 
65 70 75 80 

Trp Lys Ala Glu Thr lie Lys Lys His Val Leu lie Ala Thr His Phe 
85 90 95 

Val Asp Leu lie Glu Asp Phe Trp Gin Thr Thr Gin Gly Met Ser Gin 
100 105 110 

lie Ala Asp Ala Leu Arg Ala Val lie Pro Pro Thr Thr Val Pro Val 
115 120 125 

Pro Glu Gly Phe Leu lie Thr His Ser Glu Ala Glu Glu lie Pro Leu 
130 135 140 

Asn Asp Leu Phe Ser Asn Gin Glu Glu Arg lie Val Asn Phe Gin Pro 
145 150 155 160 

Asp Tyr Pro lie Thr Ala Arg lie His Thr His Leu Arg Val Tyr Thr 
165 170 175 

Lys Leu Asn Glu Gin Ala Leu Asp Lys Ala Arg Arg Leu Leu Trp Trp 
180 185 190 

His Tyr Asn Cys Leu Leu Trp Gly Glu Ala Thr Val Thr Asn Tyr lie 
195 200 205 

Ser Arg Leu Arg Thr Trp Leu Ser Thr Pro Glu Lys Tyr Arg Gly Lys 
210 215 220 

Asp Ala Pro Thr He Glu Ala lie Thr Arg Pro lie Gin Val Ala Gin 
225 230 235 240 

Gly Gly Arg Asn Gin Thr Lys Gly Thr Arg Lys Pro Arg Gly Leu Glu 
245 250 255 

Pro Arg Arg Arg Lys Val Lys Thr Thr Val Val Tyr Gly Arg Arg Arg 
260 265 270 

Ser Lys Ser Arg Gly Arg Arg Ser Ser Pro Ser Gin Arg Ala Gly Ser 
275 280 285 

Pro Leu Pro Arg Asn Arg Gly Asn Gin Thr Arg Ser Pro Ser Pro Arg 
290 295 300 



14 



WO 99/40934 



PCT/US99/03055 



Glu 
305 



<210> 12 
<211> 918 
<212> DNA 

<213> Hepatitis B virus 

<220> 

<221> CDS 

<222> <1)..<915) 

<400> 12 

atg tgg aac tta aga att aca ccc etc tec ttc gga get get tgc caa 48 

Met Trp Asn Leu Arg lie Thr Pro Leu Ser Phe Gly Ala Ala Cys Gin 

1 5 10 15 

ggt ate ttt acg tct aca ttg ctg ttg teg tgt gtg act gta cct ttg 96 
Gly He Phe Thr Ser Thr Leu Leu Leu Ser Cys Val Thr Val Pro Leu 
20 25 30 

gta tgt acc att gtt tat gat tct tgc tta tat atg gat ate aat get 144 
Val Cys Thr He Val Tyr Asp Ser Cys Leu Tyr Met Asp He Asn Ala 
35 40 45 

tct aga gec tta gee aat gtg tat gat eta cca gat gat ttc ttt cca 192 
Ser Arg Ala Leu Ala Asn Val Tyr Asp Leu Pro Asp Asp Phe Phe Pro 
50 55 60 

aaa ata gat gat ctt gtt aga gat get aaa gac get tta gag cct tat 240 
Lys lie Asp Asp Leu Val Arg Asp Ala Lys Asp Ala Leu Glu Pro Tyr 
65 70 75 80 

tgg aaa tea gat tea ata aag aaa cat gtt ttg att gca act cac ttt 288 
Trp Lys Ser Asp Ser lie Lys Lys His Val Leu He Ala Thr His Phe 
85 90 95 

gtg gat etc att gaa gac ttc tgg cag act aca cag ggc atg cat gaa 336 
Val Asp Leu He Glu Asp Phe Trp Gin Thr Thr Gin Gly Met His Glu 
100 105 110 

ata gee gaa tea tta aga get gtt ata cct ccc act act act cct gtt 384 
He Ala Glu Ser Leu Arg Ala Val He Pro Pro Thr Thr Thr Pro Val 
115 120 125 

cca ccg ggt tat ctt att cag cac gag gaa get gaa gag ata cct ttg 432 
Pro Pro Gly Tyr Leu lie Gin His Glu Glu Ala Glu Glu He Pro Leu 



15 



WO 99/40934 



PCMJS99/03055 



130 135 140 

gga gat tta ttt aaa cac caa gaa gaa agg ata gta agt ttc caa ccc 480 
Gly Asp Leu Phe Lys His Gin Glu Glu Arg lie Val Ser Phe Gin Pro 
145 150 155 160 

gac tat ccg att acg get aga att cat get cat ttg aaa get tat gca 528 
Asp Tyr Pro lie Thr Ala Arg He His Ala His Leu Lys Ala Tyr Ala 
165 170 175 

aaa att aac gag gaa tea ctg gat agg get agg aga ttg ctt tgg tgg 576 
Lys He Asn Glu Glu Ser Leu Asp Arg Ala Arg Arg Leu Leu Trp Trp 
180 185 190 

cat tac aac tgt tta ctg tgg gga gaa get caa gtt act aac tat att 624 
His Tyr Asn Cys Leu Leu Trp Gly Glu Ala Gin Val Thr Asn Tyr He 
195 200 205 

tct cgt ttg cgt act tgg ttg tea act cct gag aaa tat aga ggt aga ^672^ 
Ser Arg Leu Arg Thr Trp Leu Ser Thr Pro Glu Lys Tyr Arg Gly Arg 
210 215 220 

gat gec ccg acc att gaa gca ate act aga cca ate cag gtg get cag 720 
Asp Ala Pro Thr He Glu Ala He Thr Arg Pro He Gin Val Ala Gin 
225 230 235 240 

gga ggc aga aaa aca act acg ggt act aga aaa cct cgt gga etc gaa 7 68 
Gly Gly Arg Lys Thr Thr Thr Gly Thr Arg Lys Pro Arg Gly Leu Glu 
245 250 255 

cct aga aga aga aaa gtt aaa acc aca gtt gtc tat ggg aga aga cgt 816 
Pro Arg Arg Arg Lys Val Lys Thr Thr Val Val Tyr Gly Arg Arg Arg 
260 265 270 

tea aag tec egg gaa agg aga gee cct aca ccc caa cgt gcg ggc tec 864 
Ser Lys Ser Arg Glu Arg Arg Ala Pro Thr Pro Gin Arg Ala Gly Ser 
275 280 285 

cct etc cca cgt agt teg age age cac cat aga tct ccc teg cct agg 912 
Pro Leu Pro Arg Ser Ser Ser Ser His His Arg Ser Pro Ser Pro Arg 
290 295 300 

aaa taa 918 

Lys 

305 



<210> 13 



16 



WO 99/40934 PCT/US99/03055 



<211> 305 
<212> PRT 

<213> Hepatitis B virus 
<400> 13 

Met Trp Asn Leu Arg He Thr Pro Leu Ser Phe Gly Ala Ala Cys Qln 
15 10 15 

Gly He Phe Thr Ser Thr Leu Leu Leu Ser Cys Val Thr Val Pro Leu 
20 25 30 

Val Cys Thr He Val Tyr Asp Ser Cys Leu Tyr Met Asp He Asn Ala 
35 40 45 

Ser Arg Ala Leu Ala Asn Val Tyr Asp Leu Pro Asp Asp Phe Phe Pro 
50 55 60 

Lys He Asp Asp Leu Val Arg Asp Ala Lys Asp Ala Leu Glu Pro Tyr 
65 70 75 80 

Trp Lys Ser Asp Ser He Lys Lys His Val Leu He Ala Thr His Phe 
85 90 95 

Val Asp Leu He Glu Asp Phe Trp Gin Thr Thr Gin Gly Met His Glu 
100 105 110 

He Ala Glu Ser Leu Arg Ala Val He Pro Pro Thr Thr Thr Pro Val 
115 120 125 

Pro Pro Gly Tyr Leu He Gin His Glu Glu Ala Glu Glu He Pro Leu 
130 135 140 

Gly Asp Leu Phe Lys His Gin Glu Glu Arg He Val Ser Phe Gin Pro 
145 150 155 160 

t 

Asp Tyr Pro He Thr Ala Arg He His Ala His Leu Lys Ala Tyr Ala 
165 170 175 

Lys He Asn Glu Glu Ser Leu Asp Arg Ala Arg Arg Leu Leu Trp Trp 
180 185 190 

His Tyr Asn Cys Leu Leu Trp Gly Glu Ala Gin Val Thr Asn Tyr He 
195 200 205 

Ser Arg Leu Arg Thr Trp Leu Ser Thr Pro Glu Lys Tyr Arg Gly Arg 
210 215 220 

Asp Ala Pro Thr He Glu Ala He Thr Arg Pro He Gin Val Ala Gin 



17 



WO 99/40934 



PCT/US99/03055 



225 230 235 240 

Gly Gly Arg Lys Thr Thr Thr Gly Thr Arg Lys Pro Arg Gly Leu Glu 
245 250 255 

Pro Arg Arg Arg Lys Val Lys Thr Thr Val Val Tyr Gly Arg Arg Arg 
260 265 270 

Ser Lys Ser Arg Glu Arg Arg Ala Pro Thr Pro Gin Arg Ala Gly Ser 
275 280 285 

Pro Leu Pro Arg Ser Ser Ser Ser His His Arg Ser Pro Ser Pro Arg 
290 295 300 

Lys 
305 



<210> 14 
<211> 37 , 
<212> PRT 

<213> Influenza virus 
<400> 14 

Ser lie Met Arg Ser Asp Ala Pro He Gly Thr Cys Ser Ser Glu Cys 
15 10 15 

He Thr Pro Asn Gly Ser He Pro Asn Asp Lys Pro Phe Gin Asn Val 
20 25 30 

Asn Lys He Thr Tyr 
35 



<210> 15 
<211> 29 
<212> PRT 

<213> Influenza virus 
<400> 15 

Arg Gly He Phe Gly Ala He Ala Gly Phe He Glu Asn Gly Trp Glu 
15 10 15 

Gly Met He Asp Gly Trp Tyr Gly Phe Arg His Gin Asn 
20 25 



<210> 16 



18 



WO 99/40934 



<211> 10 
<212> PRT 

<213> Influenza virus 
<400> 16 

Glu Lys Gin Thr Arg Gly He Phe Gly Ala 
15 10 



<210> 17 
<211> 23 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Hepatitis B 
virus PCR primer with an Ncol restriction site 

<400> 17 

ttgggccatg gacatcgacc tta 

<210> 18 
<211> 31 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Hepatitis B 
virus PCR primer with an EcoRI restriction site 

<400> 18 

gcggaattcc atcttccaaa ttaacaccca c 

<210> 19 
<211> 41 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Hepatitis B 
virus PCR primer with EcoRI and Sad restriction 
sites and an inserted lysine codon 

<400> 19 

cgcgaattca aaaagagctc ccagcgtcta gagagaccta g 



19 



WO 99/40934 



PCT/US99/03055 



<210> 20 
<211> 31 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Hepatitis B 
virus PCR primer with HindlXX restriction site 

<400> 20 

cgcaagctta aacaacagta gtctccggaa g 31 



<210> 21 
<211> 14 
<212> PRT 

<213> Hepatitis B virus 
<400> 21 

Cys Gin Olu Lys Gin Leu Asp 61u Asn Ala Asn Val Gin Leu 
15 10 



<210> 22 
<211> 13 
<212> PRT 

<213> Hepatitis B virus 
<400> 22 

Cys Ser Lys Lys Gly Pro Arg Ala Ser Gly Asn Leu lie 
15 10 



<210> 23 
<211> 19 
<212> PRT 

<213> Hepatitis B virus 



<400> 23 

Cys Leu Thr Glu His Arg Met Thr Trp Asp Pro Ala Gin Pro Pro Arg 
15 10 15 

Asp Leu Thr 



<210> 24 



WO 99/40934 



PCT/US99/03055 



<211> 22 
<212> PRT 

<213> Hepatitis B virus 
<400> 24 

Cys Val Lys Arg Met Lys Glu Ser Arg Leu Glu Asp Thr Gin Lys His 
15 10 15 

Arg Val Asp Phe Leu Gin 
20 



<210> 25 
<211> 15 
<212> PRT 

<213> Streptococcus pneumoniae 
<300> 

<310> BP-0 786 521-A 
<400> 25 

Lys Leu Glu Glu Leu Ser Asp Lys He Asp Glu Leu Asp Ala Glu 
15 10 15 



<210> 26 
<211> 35 
<212> PRT 

<213> Streptococcus pneumoniae 
<300> 

<310> EP-0 786 521-A 
<400> 26 

Gin Lys Lys Tyr Asp Glu Asp Gin Lys Lys Thr Glu Glu Lys Ala Ala 
15 10 15 

Leu Glu Lys Ala Ala Ser Glu Glu Met Asp Lys Ala Val Ala Ala Val 
20 25 30 

Gin Gin Ala 
35 



<210> 27 
<211> 31 
<212> PRT 

<213> Cryptosporidium parvum 



21 



WO 99/40934 



PCT/US99/03055 



<300> 

<310> WO 98/07320 
<400> 27 

Gin Asp Lys Pro Ala Asp Ala Pro Ala Ala Glu Ala Pro Ala Ala Glu 
15 10 15 

Pro Ala Ala Glu Pro Ala Ala Gin Gin Asp Lys Pro Ala Asp Ala 
20 25 30 



<210> 28 
<211> 16 
<212> PRT 

<213> Human immunodeficiency virus 
<300> 

<310> US 5,639,854 
<400> 28 

Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Cys 
1 5 10 15 



<210> 29 
<211> 17 
<212> PRT 

<213> Human immunodeficiency virus 
<300> 

<310> WO 98/07320 ' 
<400> 29 

Arg Lys Arg He His He Gly Pro Gly Arg Ala Phe Tyr He Thr Lys 
15 10 15 

Asn 



<210> 30 
<211> 31 
<212> PRT 

<213> Foot-and-mouth disease virus 
<300> 

<310> US 4,544,500 



22 



WO 99/40934 



PCT/US99/03055 



<400> 30 

Tyr Asn Gly Glu Cys Arg Tyr Asn 
1 5 

Oly Asp Leu Gin Val Leu Ala Gin 
20 



Arg Asn Ala Val Pro Asn Leu Arg 
10 15 

Lys Val Ala Arg Thr Leu Pro 
25 30 



<210> 31 
<211> 17 
<212> PRT 

<213> Corynebacterium diphtheriae 
<300> 

<310> EP-0 399 011-B1 
<400> 31 

Phe Gin Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly 
1 5 10 15 

Cys 



<210> 32 
<211> 25 
<212> PRT 

<213> Borrelia burgdorferi 
<300> 

<301> Bockenstedt, L. K. 

et aL, 
<303> J. Immunol. 
<304> 157 
<305> 12 
<306> 5496- 
<307> (1966) 

<400> 32 

Val Glu lie Lys Glu Gly Thr Val 
1 5 

Asn Gly Lys Val Thr Val Ser Leu 
20 



Thr Leu Lys Arg Glu lie Asp Lys 
10 15 

Cys 
25 



<210> 33 



23 



WO 99/40934 



PCT/US99/03055 



<211> 19 
<212> PRT 

<213> Borrelia burgdorferi 
<300> 

<301> Zhong, W. 

et al . , 
<303> Eur. J. Immunol. 
<304> 26 
<305> 11 
<306> 2749- 
<307> 1996 

<400> 33 

Thr Leu Ser Lys Asn lie Ser Lys Ser Gly Glu Val Ser Val Glu Leu 
15 10 15 

Asn Asp Cys 



<210> 34 
<211> 11 
<212> PRT 

<213> Influenza A virus 
<300> 

<301> Brumeanu, T. D. 
<303> Immuno technology 
<304> 2 
<305> 2 
<306> 85- 
<307> (1996) 

<400> 34 

Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Cys 
15 10 



<210> 35 
<211> 10 
<212> PRT 

<213> Influenza A virus 
<300> 

<301> Brumeanu, T. D. 
<303> Immuno technology 
<304> 2 



24 



WO 99/40934 



PCT/US99/03055 



<305> 2 
<306> 85- 
<307> (1996) 

<400> 35 

Tyr Arg Asn Leu Lou Trp Leu Thr Glu Lys 
1 5 10 



<210> 36 
<211> 142 
<212> PRT 

<213> Yersinia pestis 
<300> 

<301> Hill, J. 

et al . , 
<303> Infect. Immun. 
<304> 65 
<305> 11 
<306> 4476- 
<307> (1997) 

<400> 36 

Asp lie Leu Lys Val He Val Asp Ser Met Asn His His Gly Asp Ala 
1 5 10 15 

Arg Ser Lys Leu Arg Glu Glu Leu Ala Glu Leu Thr Ala Glu Leu Lys 
20 25 30 

He Tyr Ser Val He Gin Ala Glu He Asn Lys His Leu Ser Ser Ser 
35 40 45 

Gly Thr He Asn He His Asp Lys Ser He Asn Leu Met Asp Lys Asn 
50 55 60 

Leu Tyr Gly Tyr Thr Asp Glu Glu He Phe Lys Ala Ser Ala Glu Tyr 
65 70 75 80 

Lys He Leu Glu Lys Met Pro Gin Thr Thr He Gin Val Asp Gly Ser 
85 90 95 

Glu Lys Lys He Val Ser He Lys Asp Phe Leu Gly Ser Glu Asn Lys 
100 105 110 

Arg Thr Gly Ala Leu Gly Asn Leu Lys Asn Ser Tyr Ser Tyr Asn Lys 
115 120 125 



25 



WO 99/40934 



PCT/US99/03055 



Asp Asn Asn Glu Leu Ser His Phe Ala Thr Thr Cys Ser Asp 
130 135 140 



<210> 37 
<211> 19 
<212> PRT 

<213> Haemophilus influenzae 
<300> 

<310> EP-0 432 220-B1 
<400> 37 

Cys Ser Ser Ser Asn Asn Asp Ala Ala Gly Asn Gly Ala Ala Gin Phe 
15 10 15 

Gly Gly Tyr 



<210> 38 
<211> 11 
<212> PRT 

<213> Haemophilus influenzae 
<300> 

<310> EP-0 432 220-B1 
<400> 38 

Asn Lys Leu Gly Thr Val Ser Tyr Gly Glu Glu 
15 10 



<210> 39 
<211> 16 
<212> PRT 

<213> Haemophilus influenzae 
<300> 

<310> EP-0 432 220-B1 
<400> 39 

Asn Asp Glu Ala Ala Tyr Ser Lys Asn Arg Arg Ala Val Leu Ala Tyr 
15 10 15 



<210> 40 
<211> 28 



26 



WO 99/40934 



PCTAJS99/03055 



<212> PRT 

<213> Moraxella catarrhalis 
<300> 

<310> WO 98/06851 
<400> 40 

Leu Asp lie Glu Lys Asp Lys Lys Lys Arg Thr Asp Glu Gin Leu Gin 
15 10 15 

Ala Glu Leu Asp Asp Lys Tyr Ala Gly Lys Gly Tyr 
20 25 



<210> 41 
<211> 28 
<212> PRT 

<213> Moraxella catarrhalis 
<300> 

<310> WO 98/06851 
<400> 41 

Leu Asp lie Glu Lys Asn Lys Lys 
1 5 

Ala Glu Leu Asp Asp Lys Tyr Ala 
20 



Lys Arg Thr Glu Ala Glu Leu Gin 
10 15 

Gly Lys Gly Tyr 
25 



<210> 42 
<211> 28 
<212> PRT 

<213> Moraxella catarrhalis 
<300> 

<310> WO 98/06851 
<400> 42 

lie Asp lie Glu Lys Lys Gly Lys lie Arg Thr Glu Ala Glu Leu Leu 
1 5 10 . 15 

Ala Glu Leu Asn Lys Asp Tyr Pro Gly Gin Gly Tyr 
20 25 



<210> 43 
<211> 25 



27 



WO 99/40934 



PCT/US99/03055 



<212> PRT 

<213> Porphyromonas gingival is 

<300> 
<304> 110 
<305> 2 
<306> 285- 
<307> 1997 

<400> 43 

Gly Val Ser Pro Lys Val Cys Lys 
1 5 

Glu Phe Ala Pro Val Gin Asn Leu 
20 



Asp Val Thr Val Glu Gly Ser Asn 
10 15 

Thr 
25 



<210> 44 

<211> 20 

<212> PRT 

<213> Porphyromonas gingivalis 

<300> 
<304> 110 
<305> 2 
<306> 285- 
<307> 1997 

<400> 44 

Arg lie Gin Ser Thr Trp Arg Gin Lys Thr Val Asp Leu Pro Ala Gly 
15 10 15 

Thr Lys Tyr Val 
20 



<210> 45 
<211> 20 
<212> PRT 

<213> Trypanosoma cruzi 

<300> 
<304> 159 
<305> 9 
<306> 4444- 
<307> (1997) 

<400> 45 



28 



WO 99/40934 



PCT/US99/03055 



Ser His Asn Phe Thr I*eu Val Ala Ser Val lie lie Glu Ala Pro Ser 
15 10 15 

Gly Asn Thr Cys 
20 



<210> 46 
<211> 21 
<212> PRT 

<213> Trypanosoma cruzi 
<300> 

<310> WO 97/18475 
<400> 46 

Lys Ala Ala lie Ala Pro Ala Lys Ala Ala Ala Ala Pro Ala Lys Ala 
1 5 10 15 

Ala Thr Ala Pro Ala 
20 



<210> 47 
<211> 16 
<212> PRT 

<213> Plasmodium falciparum 
<300> 

<303> Int. Arch. Allergy Appl. Immunol. 
<304> 114 
<305> 1 
<306> 15- 

<400> 47 

Ser Val Gin lie Pro Lys Val Pro Tyr Pro Asn Gly lie Val Tyr Cys 
15 10 15 



<210> 48 
<211> 16 
<212> PRT 

<213> Plasmodium falciparum 
<300> 

<303> Int. Arch. Allergy Appl . Immunol. 
<304> 114 
<305> 1 



29 



WO 99/40934 



PCT/US99/03055 



<306> 15 



<400> 48 

Asp Phe Asn His Tyr Tyr Thr Leu Lys Thr Gly Leu Glu Ala Asp Cys 
15 10 15 



<210> 49 
<211> 16 
<212> PRT 

<213> Streptococcus sobrinus 
<300> 

<303> Arch. Oral Biol. 
<304> 35 

<306> Suppl. 475- 
<307> (1990) 

<400> 49 

Lys Pro Arg Pro He Tyr Glu Ala 
1 5 



Lys Leu Ala Gin Asn Gin Lys Cys 
10 15 



<210> 50 
<211> 17 
<212> PRT 

<213> Streptococcus sobrinus 
<300> 

<303> Arch. Oral Biol. 
<304> 35 

<306> Suppl. 475- 
<307> (1990) 

<400> 50 

Ala Lys Ala Asp Tyr Glu Ala Lys Leu Ala Gin Tyr Glu Lys Asp Leu 
15 10 15 

Cys 



<210> 51 
<211> 16 
<212> PRT 

<213> Lymphocytic choriomeningitis virus 
<300> 



30 



WO 99/40934 



PCT/US99/03055 



<303> Proc. Natl. Acad. Sci. U.S.A. 
<304> 94 
<305> 7 
<306> 3314- 
<307> (1997) 

<400> 51 

Arg Pro Gin Ala Ser Qly Val Tyr Met Gly Asn Leu Thr Ala Gin Cys 
15 10 15 



<210> 52 
<211> 9 
<212> PRT 

<213> Shigella flexneri 
<300> 

<303> J. Biol. Chem. 
<304> 271 
<305> 52 
<306> 33670- 
<307> (1996) 

<400> 52 

Lys Asp Arg Thr Leu lie Glu Gin Lys 
1 5 



<210> 53 
<211> 15 
<212> PRT 

<213> respiratory syncytial virus 

<300> 
<304> 234 
<305> 1 
<306> 118- 
<307> 1997 

<400> 53 

Cys Ser lie Cys Ser Asn Asn Pro Thr Cys Trp Ala lie Cys Lys 
1 5 10 15 



<210> 54 
<211> 19 
<212> PRT 

<213> Plasmodium vivax 



31 



WO 99/40934 



PCT/US99/03055 



<300> 

<303> Vaccine 
<304> 15 
<305> 4 
<306> 377- 
<307> 1997 

<400> 54 

Gly Asp Arg Ala Asp Oly Gin Pro Ala Gly Asp Arg Ala Asp Gly Gin 
15 10 15 

Pro Ala Gly 



<210> 55 
<211> 16 
<212> PRT 

<213> Clostridium tetani 
<300> 

<303> Vaccine 
<304> 15 
<305> 4 
<306> 377- 
<307> 1997 

<400> 55 

Gin Tyr lie Lys Ala Asn Ser Lys 
1 5 



Phe lie Gly lie Thr Glu Leu Cys 
10 15 



<210> 56 
<211> 25 
<212> PRT 

<213> Entamoeba histolytica 
<300> 

<303> J. Exp. Med. 
<304> 185 
<305> 10 
<306> 1793- 
<307> 1997 

<400> 56 

Val Glu Cys Ala Ser Thr Val Cys Gin Asn Asp Asn Ser Cys Pro lie 
15 10 15 



32 



WO 99/40934 



PCT/US99/03055 



lie Ala Asp Val Glu Lys Cya Asn Oln 
20 25 



<210> 57 
<211> 34 
<212> PRT 

<213> Schistosoma japonicum 
<300> 

<303> Vaccine 
<304> 15 
<305> 1 
<306> 79- 
<307> 1997 

<400> 57 

Asp Leu Gin Ser Glu lie Ser Leu Ser Leu Glu Asn Gly Glu Leu lie 
15 10 15 

Arg Arg Ala Lys Ser Ala Glu Ser Leu Ala Ser Glu Leu Gin Arg Arg 
20 25 30 

Val Asp 



<210> 58 
<211> 34 
<212> PRT 

<213> Schistosoma mansoni 
<300> 

<303> Vaccine 
<304> 15 
<305> 1 
<306> 79- 
<307> 1997 

<400> 58 

Asp Leu Gin Ser Glu lie Ser Leu Ser Leu Glu Asn Ser Glu Leu lie 
15 10 15 

Arg Arg Ala Lys Ala Ala Glu Ser Leu Ala Ser Asp Leu Gin Arg Arg 
20 25 30 

Val Asp 



33 



WO 99/40934 



PCT/US99/03055 



<210> 59 
<211> 63 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Msel 

restriction endonuclease site inserted into wild 
type Hepatitis B sequence at position 75 

<220> 
<221> CDS 
<222> (1) . . (63) 

<400> 59 

get acc tgg gtg ggt gtt aat ttg gaa gat cca gcg tct aga gac eta 48 

Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp Leu 
15 10 15 

gta gtc agt tat gtc 63 
Val Val Ser Tyr Val 
20 



<210> 60 
<211> 21 
<212> PRT 

<213> Artificial Sequence 
<400> 60 

Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp Leu 
15 10 15 

Val Val Ser Tyr Val 
20 



<210> 61 
<211> 41 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: K inserted at 
amino acid position 75 of Hetatitis B core 



34 



WO 99/40934 PCT/US99/03055 



sequence 

<220> 
<221> CDS 
<222> <1)..(39) 

<400> 61 

get acc tgg gtg ggt gtt aaa aat ttg gaa gat cca gcg tc 41 
Ala Thr Trp Val Gly Val Lya Asn Leu Olu Asp Pro Ala 
15 10 



<210> 62 
<211> 13 
<212> PRT 

<213> Artificial Sequence 
<400> 62 

Ala Thr Trp Val Gly Val Lys Asn Leu Glu Asp Pro Ala 
15 10 



<210> 63 
<211> 27 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: K inserted at 
amino acid position 76 of hepatitis B core 

<220> 
<221> CDS 
<222> (3).. (26) 

<400> 63 

tt aat aaa ttg gaa gat cca gcg tct a 27 
Asn Lys Leu Glu Asp Pro Ala Ser 
1 5 



<210> 64 
<211> 8 
<212> PRT 

<213> Artificial Sequence 
<400> 64 

Asn Lys Leu Glu Asp Pro Ala Ser 



35 



WO 99/40934 



PCTAJS99/03055 



1 5 



<210> 65 
<211> 27 
<212> SNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: K inserted at 
position 77 of hepatitis B virus core 

<220> 
<221> CDS 
<222> (3).- (26) 

<400> 65 

tt aat ttg aaa gaa gat cca gcg tct a 

Asn Leu Lys Olu Asp Pro Ala Ser 

1 5 



<210> 66 
<211> 8 
<212> PRT 

<213> Artificial Sequence 
<400> 66 

Asn Leu Lys Glu Asp Pro Ala Ser 
1 5 



<210> 67 
<211> 32 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: X inserted at 
position 78 of hepatitis B core 

<220> 
<221> CDS 
<222> (3).. (32) 

<400> 67 

tt aat ttg gaa aaa gat cca gcg tct aga gac 
Asn Leu Glu Lys Asp Pro Ala Ser Arg Asp 

36 



WO 99/40934 



PCT/US99/03055 



15 10 



<210>, 68 
<211> 10 
<212> PRT 

<213> Artificial Sequence 
<400> 68 

Asn Leu Glu Lys Asp Pro Ala Ser Arg Asp 
15 10 



<210> 69 
<211> 36 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: K inserted at 
amino acid position 79 of hepatitis B core 

<220> 
<221> CDS 
<222> (3).. (35) 

<400> 69 

tt aat ttg gaa gat aaa cca gcg tct aga gac eta g 36 
Asn Leu Glu Asp Lys Pro Ala Ser Arg Asp Leu 
15 10 



<210> 70 
<211> 11 
<212> PRT 

<213> Artificial Sequence 
<400> 70 

Asn Leu Glu Asp Lys Pro Ala Ser Arg Asp Leu 
1 5 10 



<210> 71 
<211> 39 
<212> DNA 

<213> Artificial Sequence 
<220> 



37 



WO 99/40934 



PCT/US99/03055 



<223> Description of Artificial Sequence: K inserted at 
amino acid position 80 of hepatitis B core 

<220> 
<221> CDS 
<222> (3).. (38) 

<400> 71 

tt aat ttg gaa gat cca aaa gcg tct aga gac eta gta g 39 

Asn Leu Glu Asp Pro Lye Ala Ser Arg Asp Leu Val 
15 10 



<210> 72 
<211> 12 
<212> PRT 

<213> Artificial Sequence 
<400> 72 

Asn Leu Glu Asp Pro Lys Ala Ser Arg Asp Leu Val 
15 10 



<210> 73 
<211> 43 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: K inserted at 
amino acid position 81 of hepatitis B core 

<220> 
<221> CDS 
<222> (3).. (41) 

<400> 73 

tt aat ttg gaa gat cca gcg aaa tct aga gac eta gta gtc ag 43 
Asn Leu Glu Asp Pro Ala Lys Ser Arg Asp Leu Val Val 
15 10 



<210> 74 
<211> 13 
<212> PRT 

<213> Artificial Sequence 
<400> 74 



38 



WO 99/40934 



PCT/US99/03055 



Asn Leu Glu Asp Pro Ala Lys Ser Arg Asp Leu Val Val 
15 10 



<210> 75 
<211> 45 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: K inserted at 
amino acid position 82 of hepatitis B core 

<220> 
<221> CDS 
<222> (3) . . (44) 

<400> 75 

tt aat ttg gaa gat cca gcg tct aaa aga gac eta gta gtc agt t 
Asn Leu Glu Asp Pro Ala Ser Lys Arg Asp Leu Val Val Ser 
15 10 



<210> 76 
<211> 14 
<212> PRT 

<213> Artificial Sequence 
<400> 76 

Asn Leu Glu Asp Pro Ala Ser Lys Arg Asp Leu Val Val Ser 
15 10 



<210> 77 
<211> 50 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: K inserted at 
amino acid position 83 of hepatitis B core 

<220> 
<221> CDS 
<222> (3) . . (50) 

<400> 77 

tt aat ttg gaa gat cca gcg tct aga aaa gac eta gta gtc agt tat 



39 



WO 99/40934 



PCT/US99/03055 1 



Asn Leu Glu Asp Pro Ala Ser Arg Lys Asp Leu Val Val Ser Tyr 
1 5 10 15 



gtc 
val 



50 



<210> 78 
<211> 16 
<212> PRT 

<213> Artificial Sequence 
<400> 78 

Asn Leu Glu Asp Pro Ala Ser Arg Lys Asp Leu Val Val Ser Tyr Val 
15 10 15 



<210> 79 
<211> 50 
<212> DKA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: K inserted at 
amino acid position 84 of hepatitis B core 

<220> 
<221> CDS 
<222> (3) . . (50) 

<400> 79 

tt aat ttg gaa gat cca gcg tct aga gac aaa eta gta gtc agt tat 47 

Asn Leu Glu Asp Pro Ala Ser Arg Asp Lys Leu Val Val Ser Tyr 
15 10 15 

gtc 50 
Val 



<210> 80 
<211> 16 
<212> PRT 

<213> Artificial Sequence 
<400> 80 

Asn Leu Glu Asp Pro Ala Ser Arg Asp Lys Leu Val Val Ser Tyr Val 
15 10 15 



40 



WO 99/40934 



PCTAJS99/03055 



<210> 81 
<211> 31 
<212> DNA 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence t K inserted at 
amino acid position 85 of hepatitis B core 

<220> 
<221> CDS 
<222> (2).. (31) 

<400> 81 

c teg aga gac eta aaa gta gtc agt tat gtc 31 
Ser Arg Asp Leu Lys Val Val Ser Tyr Val 
1 5 .10 



<210> 82 
<2U> 10 
<212> PRT 

<213> Artificial Sequence 
<400> 82 

Ser Arg Asp Leu Lys Val Val Ser Tyr Val 
15 10 



<210> 83 
<211> 18 
<212> PRT 

<213> Plasmodium vivax 
<400> 83 

Asp Arg Ala Ala Gly Gin Pro Ala Gly Asp Arg Ala Asp Gly Gin Pro 
15 10 15 



Ala Gly 



<210> 84 
<211> 8 
<212> PRT 

<213> Influenza virus 



41 



WO 99/40934 



PCT/US99/03055 



<400> 84 

Cys Asn Asn Pro His Arg lie Leu 
1 5 



<210> 85 
<211> 25 
<212> PRT 

<213> Influenza virus 
<400> 85 

Cys Pro Lys Tyr Val Lys Gin Asn Thr Leu Lys Leu Ala Thr Gly Met 
1 5 10 15 

Arg Asn Val Pro Glu Lys Gin Thr Arg 
20 25 



<210> 86 
<211> 15 
<212> PRT 

<213> Mycobacterium tuberculosis 
<400> 86 

Ala Val Leu Glu Asp Pro Tyr lie Leu Leu Val Ser Ser Lys Val 
1 5 10 15 



<210> 87 
<211> 15 
<212> PRT 

<213> Mycobacterium tuberculosis 
<400> 87 

Leu Leu Val Ser Ser Lys Val Ser Thr Val Lys Asp Leu Leu Pro 
1.5 10 15 



<210> 88 
<211> 15 
<212> PRT 

<213> Mycobacterium tuberculosis 
<400> 88 

Leu Leu Pro Leu Leu Glu Lys Val lie Gly Ala Gly Lys Pro Leu 
15 10 15 



42 



WO 99/40934 



PCT/US99/03055 



<210> 89 
<211> 15 
<212> PRT 

<213> Mycobacterium tuberculosis 
<400> 89 

Ala lie Leu Thr Gly Gly Gin Val He Ser Glu Glu Val Gly Leu 
1 5 10 15 



<210> 90 
<211> 15 
<212> PRT 

<213> Mycobacterium tuberculosis 
<400> 90 

He Ala Phe Asn Ser Gly Leu Glu Pro Gly Val Val Ala Glu Lys 
15 10 15 



<210> 91 
<211> 18 
<212> PRT 

<213> Mycobacterium tuberculosis 
<400> 91 

Ala Arg Arg Gly Leu Glu Arg Gly Leu Asn Ala Leu Ala Asp Ala Val 
1 5 10 15 

Lys Val 



<210> 92 
<211> 14 
<212> PRT 

<213> Mycobacterium tuberculosis 
<400> 92 

Glu Lys He Gly Ala Glu Leu Val Lys Glu Val Ala Lys Lys 
15 10 



<210> 93 
<211> 17 
<212> PRT 

<213> Mycobacterium tuberculosis 



43 



WO 99/40934 



PCT/US99/03055 



<400> 93 

Gly Leu Lys Arg Gly He Glu Lys Ala Val Glu Lys Val Thr Glu Thr 
15 10 15 

Leu 



<210> 94 
<211> 15 
<212> PRT 

<213> Mycobacterium tuberculosis 
<400> 94 

He Glu Asp Ala Val Arg Asn Ala Lys Ala Ala Val Glu Glu Gly 
15 10 15 



<210> 95 
<211> 20 
<212> PRT 

<213> Feline leukemia virus 
<400> 95 

Cys Asp He He Gly Asn Thr Trp Asn Pro Ser Asp Gin Glu Pro Phe 
15 10 15 

Pro Gly Tyr Gly 
20 



<210> 96 
<211> 21 
<212> PRT 

<213> Feline leukemia virus 

t 

<400> 96 

Cys He Gly Thr Val Pro Lys Thr His Gin Ala Leu Cys Asn Glu Thr 
15 10 15 

Gin Gin Gly His Thr 
20 



<210> 97 
<211> 13 
<212> PRT 

<213> Feline leukemia virus 



44 



WO 99/40934 



PCIYUS99/03055 



<400> 97 

Gly Asn Tyr Ser Asn Gin Thr Asn Pro Pro Pro Ser Cya 
15 10 



<210> 98 
<211> 22 
<212> PRT 

<213> Feline leukemia virus 
<400> 98 

Thr Asp He Gin Ala Leu Glu Glu Ser lie Ser Ala Leu Glu Lys Ser 
15 10 15 

Leu Thr Ser Leu Ser Glu 
20 



<210> 99 
<211> 14 
<212> PRT 

<213> Feline leukemia virus 
<400> 99 

Ala Lys Leu Arg Glu Arg Leu Lys Gin Arg Gin Gin Leu Phe 
1 5 10 



<210> 100 
<211> 23 
<212> PRT 

<213> Feline leukemia virus 
<400> 100 

Asp Ser Gin Gin Gly Trp Phe Glu Gly Trp Phe Asn Lys Ser Pro Trp 
15 10 15 

Phe Thr Thr Leu He Ser Ser 
20 



<210> 101 
<211> 18 
<212> PRT 

<213> Feline leukemia virus 
<400> 101 



45 



WO 99/40934 



PCT/US99/03055 



Gin Val Met Thr lie Thr Pro Pro Gin Ala Met Gly Pro Asn Leu Val 
15 10 15 



Leu Pro 



<210> 102 
<211> 17 
<212> PRT 

<213> Feline leukemia virus 
<400> 102 

Asp Gin Lys Pro Pro Ser Arg Gin Ser Gin lie Glu Ser Arg Val Thr 
15 10 15 

Pro 



<210> 103 
<211> 15 
<212> PRT 

<213> Feline leukemia virus 
<400> 103 

Arg Arg Gly Leu Asp lie Leu Phe Leu Gin Glu Gly Gly Leu Cys 
15 10 15 



<210> 104 
<211> 25 
<212> PRT 

<213> Feline leukemia virus 
<400> 104 

Gin Glu Gly Gly Leu Cys Ala Ala 
1 5 

Leu Cys Ala Ala Leu Lys Glu Glu 
20 



Leu Glu Glu Cys Gin He Gly Gly 
10 15 

Cys 
25 



<210> 105 
<211> 12 
<212> PRT 

<213> Artificial Sequence 



46 



WO 99/40934 



PCT/US99/03055 



<220> 

<223> Description of Artificial Sequences Cytochrome 
P-450 fragment 

<300> 

<303> Science 
<304> 228 
<306> 1436-1440 
<307> 1985 

<400> 105 

Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 
15 10 



<210> 106 
<211> 6 
<212> PRT 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Cytochrome 
P-450 fragment 

<400> 106 

Cys Met Gin Leu Arg Ser 
1 5 



<210> 107 
<211> 6 
<212> PRT 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Cytochrome 
P-450 fragment 

<400> 107 

Cys Arg Phe Ser lie Asn 
1 5 



<210> 108 

<211> 5 

<212> PRT 

<213> Artificial Sequence 



47 



WO 99/40934 



PCT/US99/03055 



<220> 

<223> Description of Artificial Sequences Cytochrome 
P-450 fragment 

<400> 108 

Cys Ala Val Pro Arg 
1 5 



<210> 109 
<211> 5 
<212> PRT 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Cytochrome 
P-450 fragment 

<400> 109 

Cys He Pro Arg Ser 
1 5 



<210> 110 
<211> 5 
<212> PRT 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Cytochrome 
P-450 fragment 

<400> 110 

Cys Phe He Pro Val 
1 5 



<210> 111 
<211> 6 
<212> PRT 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Cytochrome 
P-450 fragment 

<400> 111 

Cys Thr Val Ser Oly Ala 



48 



WO 99/40934 



1 5 



<210> 112 
<211> 6 
<212> PRT 

<213> Artificial Sequence 
<220> 

<223> Description of Artificial Sequence: Cytochrome 
P-450 fragment 

<400> 112 

Cys Thr Leu Ser Gly Glu 
1 5 



<210> 113 
<211> 12 
<212> PRT 

<213> Plasmodium knowlesi 
<400> 113 

Gin Ala Gin Gly Asp Gly Ala Asn Ala Gly Gin Pro 
15 10 



49 



INTERNATIONAL SEARCH REPORT 



International application No. 
PCT/US99/03055 



CLASSIFICATION OP SUBJECT MATTER 
IPC(6) :A61K39/00 f 39/38. 39/29; C07K 7/00, 15/00 
US CL : 424/184.1, 189.1; 530/324, 325, 326; 536723.72 
According to International Patent Clarification (IPC) or to both national classification and IPC 



FIELDS SEARCHED 



Minimum documentation searched (classification system followed by classification symbols) 
U.S. : 424/184.1, 189.1; 530/324, 325, 326; 536723.72 



Documentation searched other than minimum documentation to the extent that such documents arc included in the fields searched 



Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) 
MEDLINE, APS, CAS ONLINE 

search terms: hepatitis B, core protein, hapten, fusk>7, pendent?, antigenic, Ab, immunoglo?, antibodies. 



C DOCUMENTS CONSIDERED TO BE RELEVANT 



Category* 



Citation of document, with indication, where appropriate, of the relevant passages 



KOLETZKI et al. Mosaic hepatitis B virus core particles allow 
insertion of extended foreign protein segments. Journal of General 
Virology. 1997, Vol. 78, pages 2049-2053, see the entire document. 

SHIAU et al. Mutated Epitopes of Hepatitis B Surface Antigen 
Fused to the Core Antigen of the Virus Induce Antibodies That React 
With the Native Surface Antigen. Journal of Medical Virology. 
1997, Vol. 51, pages 159-166, see the entire document 

US 5,143,726 A (THORNTON et al) 01 September 1992, see the 
claims. 

US 4,818,527 A ( THORNTON et al) 04 April 1989, see the claims 



Relevant to claim No. 



1-22 



1-22 



1-22 



1-22 



fx] Further documents are listed in the continuation of Box C. Q See patent family annex. 



Special oatagoriae of cited documents: "T* 

document «ur-img the general state of tha art which b not considered 
to be of ptftioultf relevance 

earlier document published on or after lb* blftroabonal filing data 

document wbioh may throw doubti on priority claim (•) or which ia 
oitad to Ttfrfrfoh tha publioatioo data of another ohation or other 
special feeaoo (ta specified) 

dooumeot referring to as oral disclosure, tiae, exhibkioo or other 
means 

ifrM n"» -tn< publ»n+d prior to international filing data but later than 
tha priority data claim ad 



later document published after the international filing data or priority 
data and not in conflict with tha application but cited to understand 
the principle or theory underlying the invention 

document of particular relevance; tha claim ad tor cation cannot be 
considered novel or cannot be considered to involve an inventive itep 
when the document is taken alone 

document of particular relevance; the churned inventioo cannot be 
considered to invobe an inventive atop when the document is 
combined with one or mote other such documents, such combination 
being obvious to a peraon skilled in the art 

document member of the same patent funDy 



Date of the actual completion of the international search 



Name and mailing address of the ISA/US 
Commissioner of Talents and Trademark* 

Box PCT 

Washington, D.C 20231 
Facsimile No. 



Date of mailing of the international search report 

.M. fltf* IS99 



Authorized officer 



Telephone N 




Form PCT/ISA/210 (second sheet)(July 1992)* 



INTERNATIONAL SEARCH REPORT 



International application No. 
PCT/US99/03055 



C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT 



Category* 



A.E 



US 4,882,145 A (THORNTON et al ) 21 November 1989, see 
the claims. 

US 5,840,303 A (CHISARI et al) 24 November 1998, see the 
abstract, and the claims. 



Citation of document, with indication, where appropriate, of the relevant passages 



Relevant to claim No. 



1-22 



1-22 



ponn PCT/ISA/210 (continuation of teoond iheet)(July 1992)* 



INTERNATIONAL SEARCH REPORT 



International application No. 
PCT/US99/03055 



Box I Observations where certain claims were found unsearchable (Continuation of Item 1 of first sheet) 



This international report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons: 

□ Claims Nos.: . 
because they relate to subject matter not required to bo tearched by this Authority, namely: 



□ Claims Nos.: . , _ , t . . 

because they relate to parts of the mternational application that do not comply with the prescribed requirements to such 
an extent that no meaningful international search can be carried out, specifically: 



3 ' ^ Sc^^y arc Dependent claims and are a* drafted in accordarK* wim the second and third sentences of Rule 6.4<a). 



Box II Observations where unity of invention is lacking (Continuation of item 1 of first sheet) 



This International Searching Authority found multiple inventions in this international application, as follows: 
Please See Extra Sheet. 



1. [x[ As all required additional search fees were timely paid by the applicant, this international search report covers all searchable 
claims. 



2. □ As all searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment 

of any additional fee. 

3. PI As only some of the required additional search foe. were timely paid by the applicant, this mtcrnarional search report covers 
1 — 1 only those claims for which fees were paid, specifically claims Nos.: 



4. |—| No required additional search fees were timely paid by the applicant. Consequently, this international search report is 
' — ■ restricted to the invention first mentioned in the claims; it is covered by claims Nos.: 



Remark on Protest Q The additional search fees were accompanied by the applicant's protest. 

j j No protest accompanied the payment of additional search fees. 



Form PCT/ISA/210 (continuation of first shcet(l))(July 1992)* 



INTERNATIONAL SEARCH REPORT 



Internationa] application No. 
PCT/US99/03055 



BOX II. OBSERVATIONS WHERE UNITY OF INVENTION WAS LACKING 
This ISA found multiple inventions as follows: 

Group 1, claim(s)l-7, drawn to hepatitis B core protein conjugated to a hapten wherein hapten is pendenUy linkc 
wherein hepatitis protein is identified with a specific sequence. 

Group II, claim(s) 8, drawn to hepatitis B core protein particles comprising an insert that is antigenic. 
Group HI, claim(s) 9-13, drawn to hepatitis B core protein pendenUy linked to a hapten . 
Group IV, claim(s)14, 15, drawn to an immunogenk fusion protein conjugated to hapten pendenUy Imked to thi 
peptide domains of hepatitis B core protein. 

Group V, claim(s) 16, drawn to hepatitis B core protein particles comprising an insert that is non-antigemc. 

Group VI, claim(s)17-19, drawn to a vaccine composition of hepatitis B core protein and a hapten. 

Group VII, claim(s) 20-22, drawn to process of inducing antibodies against hepatitis B core protein and hapten. 



The inventions listed as Groups I-VII do not relate to a single inventive concept under PCT Rule 13. 1 because, un< 
PCT Rule 13 2, they lack the same or corresponding special technical features for the following reasons: Groups 1 
II-VII are directed to different seta of hepatitis B core proteins with different hapten(s) and multitude of different 
characteristics, different sets of nucleotide and domains, that arc not so linked by a special technical feature within 
meaning of PCT Rule 13.2 so as to form a single inventive concept, accordingly, the unity of invention is lacking 
among the groups. 



Form PCT/ISA/210 (extra sheet)(July 1992)*