U.S. patent application number 13/456996 was filed with the patent office on 2012-12-20 for hbv core antigen particles with multiple immunogenic components attached via peptide ligands.
This patent application is currently assigned to BIOGEN IDEC MA INC.. Invention is credited to Kenneth Murray.
Application Number | 20120321659 13/456996 |
Document ID | / |
Family ID | 22335607 |
Filed Date | 2012-12-20 |
United States Patent
Application |
20120321659 |
Kind Code |
A1 |
Murray; Kenneth |
December 20, 2012 |
HBV CORE ANTIGEN PARTICLES WITH MULTIPLE IMMUNOGENIC COMPONENTS
ATTACHED VIA PEPTIDE LIGANDS
Abstract
This invention relates to hepatitis B virus ("HBV") core antigen
particles that are characterized by multiple immunogen
specificities. More particularly, the invention relates to HBV core
antigen particles comprising immunogens, epitopes, or other related
structures, crosslinked thereto by ligands which are HBV
capsid-binding peptides that selectively bind to HBV core protein.
Such particles may be used as delivery systems for a diverse range
of immunogenic epitopes, including the HBV capsid-binding peptides,
which advantageously also inhibit and interfere with HBV viral
assembly by blocking the interaction between HBV core protein and
HBV surface proteins. Mixtures of different immunogens and/or
capsid-binding peptide ligands may be crosslinked to the same HBV
core particle. Such resulting multicomponent or multivalent HBV
core particles may be advantageously used in therapeutic and
prophylactic vaccines and compositions, as well as in diagnostic
compositions and methods using them.
Inventors: |
Murray; Kenneth; (Edinburgh,
GB) |
Assignee: |
BIOGEN IDEC MA INC.
Cambridge
MA
|
Family ID: |
22335607 |
Appl. No.: |
13/456996 |
Filed: |
April 26, 2012 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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11338397 |
Jan 24, 2006 |
8168190 |
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13456996 |
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10872550 |
Jun 21, 2004 |
7226603 |
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11338397 |
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10448546 |
May 29, 2003 |
6827937 |
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10872550 |
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09873459 |
Jun 4, 2001 |
6627202 |
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10448546 |
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PCT/US99/28755 |
Dec 3, 1999 |
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09873459 |
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60110911 |
Dec 4, 1998 |
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Current U.S.
Class: |
424/196.11 ;
436/501; 530/391.7; 530/395; 530/403; 530/404; 530/405 |
Current CPC
Class: |
A61P 31/04 20180101;
G01N 2469/20 20130101; C12N 2730/10134 20130101; C07K 2319/00
20130101; Y02A 50/30 20180101; A61P 31/00 20180101; A61P 33/00
20180101; C12N 7/00 20130101; G01N 2333/02 20130101; A61K 2039/6075
20130101; A61K 39/00 20130101; A61K 39/29 20130101; A61P 37/08
20180101; A61K 39/385 20130101; A61P 33/06 20180101; C07K 2319/70
20130101; A61K 39/292 20130101; G01N 33/56983 20130101; A61P 31/20
20180101; C12N 2730/10123 20130101; A61P 31/14 20180101; A61P 33/02
20180101; A61K 2039/64 20130101; A61P 31/12 20180101; C12N
2730/10122 20130101; A61K 2039/627 20130101; A61P 1/16 20180101;
Y10S 530/806 20130101; A61K 2039/5258 20130101; A61P 31/22
20180101; A61K 39/12 20130101; A61P 31/10 20180101; Y10S 530/81
20130101; C07K 14/005 20130101 |
Class at
Publication: |
424/196.11 ;
530/403; 530/404; 530/395; 530/391.7; 436/501; 530/405 |
International
Class: |
A61K 39/39 20060101
A61K039/39; C07K 17/10 20060101 C07K017/10; C07K 19/00 20060101
C07K019/00; G01N 33/53 20060101 G01N033/53; A61P 31/04 20060101
A61P031/04; A61P 33/02 20060101 A61P033/02; A61P 31/20 20060101
A61P031/20; A61P 33/06 20060101 A61P033/06; A61P 37/08 20060101
A61P037/08; A61P 31/22 20060101 A61P031/22; A61P 31/10 20060101
A61P031/10; A61P 33/00 20060101 A61P033/00; C07K 17/02 20060101
C07K017/02; A61P 31/14 20060101 A61P031/14 |
Claims
1. An HBV core antigen particle comprising at least one capsid
binding immunogen, said capsid binding immunogen comprising at
least one HBV capsid-binding peptide component and at least one
immunogenic component.
2. The HBV core antigen particle according to claim 1, wherein said
capsid binding immunogen is oriented on said particle such that it
permits said immunogenic component to elicit an immune response
when said particle is administered to an individual.
3. The HBV core antigen particle according to claim 1, wherein said
capsid binding immunogen is linked to said particle through any
amino acid residue of said HBV capsid-binding peptide
component.
4. The HBV core antigen particle according to claim 1, wherein said
capsid binding immunogen is linked to said particle through any
amino acid residue or other residue of said immunogenic
component.
5. The HBV core antigen particle according to claim 4, wherein said
other residue of said immunogenic component is a carbohydrate.
6. The HBV core antigen particle according to claim 1, wherein said
capsid binding immunogen is linked to said particle through the
amino terminus of said HBV capsid-binding peptide component.
7. The HBV core antigen particle according to claim 1, wherein said
capsid binding immunogen is linked to said particle through the
carboxy terminus of said HBV capsid-binding peptide component.
8. The HBV core antigen particle according claim 1, wherein said
capsid binding immunogen is crosslinked to said particle by a
crosslinker.
9. The HBV core antigen particle according to claim 1, wherein said
immunogenic component is linked to said HBV capsid-binding peptide
component directly or through a linker sequence.
10. The HBV core antigen particle according to claim 1, wherein
said immunogenic component is linked to the amino terminus of said
HBV capsid-binding peptide component directly or through a linker
sequence.
11. The HBV core antigen particle according to claim 1, wherein
said immunogenic component is linked to the carboxy terminus of
said HBV capsid-binding peptide component directly or through a
linker sequence.
12. The HBV core antigen particle according to any one of claims
9-11, wherein said immunogenic component is linked to said HBV
capsid-binding peptide component by a crosslinker.
13. The HBV core antigen particle according to claim 8, wherein
said crosslinker is a multifunctional crosslinker.
14. The HBV core antigen particle according to claim 12, wherein
said crosslinker is a multifunctional crosslinker.
15. The HBV core antigen particle according to claim 14, wherein
said multifunctional crosslinker is selected from the group
consisting of 1-ethyl-3-(3-dimethylaminopropyl) carbodimide
hydrochloride and N-hydroxy-sulphosuccinimide.
16. The HBV core antigen particle according to claim 1, wherein
said immunogenic component comprises one or more epitopes selected
from the group consisting of immunologic epitopes, immunogenic
epitopes and antigenic epitopes.
17. The HBV core antigen particle according to claim 16, wherein
said epitopes are selected from the group consisting of linear
epitopes, conformational epitopes, single epitopes and mixed
epitopes.
18. The HBV core antigen particle according to claim 1, wherein
said immunogenic component is selected from the group consisting of
antigens, allergens, antigenic determinants, proteins,
glycoproteins, antibodies, antibody fragments, peptides, peptide
mimotopes which mimic an antigen or antigenic determinant,
polypeptides, glycopeptides, carbohydrates, oligosaccharides,
polysaccharides, oligonucleotides and polynucleotides.
19. The HBV core antigen particle according to claim 1, wherein
said immunogenic component is targeted to or derived from a
pathogenic agent selected from the group consisting of viruses,
parasites, mycobacteria, bacteria, bacilli, fungi, protozoa,
plants, phage, animal cells and plant cells.
20. The HBV core antigen particle according to claim 19, wherein
said virus is selected from the group consisting of retroviruses,
herpesviruses, orthomyoxoviruses, paramyxoviruses, hepadnaviruses,
flaviviruses, picornaviruses, papoviruses, adenoviruses,
baculoviruses, hantaviruses, parvoviruses, enteroviruses,
rhinoviruses, tumor viruses, DNA viruses, RNA viruses, togaviruses,
rhabdoviruses and poxviruses.
21. The HBV core antigen particle according to claim 20, wherein
said virus is selected from the group consisting of human
immunodeficiency type 1 virus, human immunodeficiency type 2 virus,
T cell-leukemia virus, herpes simplex type 1 virus, herpes simplex
type 2 virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr
virus, influenza A virus, influenza B virus, influenza C virus,
respiratory syncytial virus, measles-like virus, mumps virus,
parainfluenza virus, hepatitis B virus, hepatitis C virus,
hepatitis A virus, hepatitis E virus, yellow fever virus, malaria,
dengue virus, tick-borne encephalitis virus, oncovirus,
poliomyelitis virus, papillomavirus, rubella virus, rabies virus
and vaccinia virus.
22. The HBV core antigen particle according to claim 19, wherein
said immunogenic component is targeted to or derived from bacillus,
enterobacteria, clostridium, listeria, mycobacterium, pseudomonas,
staphylococcus, eubacteria, mycoplasma, chlamydia, spirochetes,
neisseria or salmonella.
23. The HBV core antigen particle according to claim 19, wherein
said immunogenic component is targeted to diptheria, tetanus,
acellular pertussis, haemophilus influenza, polio, measles, mumps,
rubella, varicella, hepatitis B virus, hepatitis A virus,
pneumococcal pneumonia, yellow fever, malaria, hepatitis B virus,
hepatitis A virus, typhoid fever, meningococcal encephalitis or
cholera.
24. The HBV core antigen particle according to claim 18, wherein
said immunogenic component is selected from the group consisting of
animal allergens, insect allergens, plant allergens, atmospheric
allergens and inhalant allergens.
25. The HBV core antigen particle according to claim 1, wherein
said HBV core antigen is an HBV core antigen fusion protein.
26. The HBV core antigen particle according to claim 25, wherein
said HBV core antigen fusion protein comprises an immunologic
epitope, an immunogenic epitope or an antigenic epitope.
27. The HBV core antigen particle according to claim 26, wherein
said HBV core antigen fusion protein comprises an immunologic
epitope, an immunogenic epitope or an antigenic epitope fused to
HBV core antigen directly or through a linker sequence.
28. The HBV core antigen particle according to claim 26, wherein
said HBV core antigen fusion protein comprises an immunologic
epitope, an immunogenic epitope or an antigenic epitope fused to
the carboxy terminus of said HBV core antigen directly or through a
linker sequence.
29. The HBV core antigen particle according to claim 26, wherein
said HBV core antigen fusion protein comprises an immunologic
epitope, an immunogenic epitope or an antigenic epitope fused to
the amino terminus of said HBV core antigen directly or through a
linker sequence.
30. The HBV core antigen particle according to claim 25, wherein
said HBV core antigen fusion protein comprises truncated HBV core
antigen.
31. The HBV core antigen particle according to claim 25, wherein
said HBV core antigen fusion protein comprises HBV surface antigen
or portions thereof.
32. The HBV core antigen particle according to claim 31, wherein
said HBV core antigen fusion protein comprises a sequence selected
from the group consisting of the pre-S1 region of HBV surface
antigen, the pre-S2 region of HBV surface antigen, the
immunodominant a region of HBV surface antigen and portions
thereof.
33. The HBV core antigen particle according to claim 1, wherein
said HBV core antigen is a full length HBV core antigen
polypeptide, or portions, truncates, mutations or derivatives
thereof which are capable of assembling in particulate form.
34. The HBV core antigen particle according to claim 1, wherein
said HBV capsid-binding peptide component is selected from the
group consisting of: SLLGRMKGA, GSLLGRMKGA, DGSLLGRMKGAA,
ADGSLLGRMKGAAG, SLLGRMKG(.beta.-A)C, RSLLGRMKGA, HRSLLGRMKGA,
ALLGRMKG, MHRSLLGRMKGA, RSLLGRMKGA(.beta.-A)C and
MHRSLLGRMKGAG(.beta.-A)GC.
35. A vaccine comprising a prophylactically effective amount of an
HBV core antigen particle according to claim 1.
36. A pharmaceutical composition comprising a therapeutically
effective amount of an HBV core antigen particle according to claim
1.
37. A method for producing an immune response in an individual
comprising the step of administering to said individual an HBV core
antigen particle according to claim 1 in an amount effective to
produce an immune response.
38. The method according to claim 37, wherein said HBV core antigen
particle is administered to said individual by parenteral
route.
39. A method for increasing the immunogencity of an immunogen by
linking said immunogen to an HBV core antigen particle through an
HBV capsid-binding peptide.
40. The HBV core antigen particle according to claim 1, wherein
said capsid binding immunogen comprises a diagnostic label or a
chemical marker.
41. A method for detecting the presence of antibodies to an
immunogen in a sample comprising the steps of: (a) contacting the
sample with an HBV core antigen particle according to claim 40, for
a time sufficient to permit any antibodies in said sample to form a
complex with said capsid binding immunogen and; (b) using detection
means to detect the complex formed between the capsid binding
immunogen and said antibodies in said sample.
42. An HBV capsid-binding peptide immunogen comprising at least one
capsid binding peptide component and at least one immunogenic
component.
43. The HBV capsid-binding peptide immunogen according to claim 42,
wherein said immunogenic component is linked to said HBV
capsid-binding peptide directly or through a linker sequence.
44. The HBV capsid-binding peptide immunogen according to claim 42,
wherein said immunogenic component is linked to the amino terminus
of said HBV capsid-binding peptide component directly or through a
linker sequence.
45. The HBV capsid-binding peptide immunogen according to claim 42,
wherein said immunogenic component is linked to the carboxy
terminus of said HBV capsid-binding peptide component directly or
through a linker sequence.
46. The HBV capsid-binding peptide immunogen according to any one
of claims 42-44, wherein said immunogenic component is crosslinked
to said HBV capsid-binding peptide component by a crosslinker.
47. The HBV capsid-binding peptide immunogen according to claim 46,
wherein said crosslinker is a multifunctional crosslinker.
48. The HBV capsid-binding peptide immunogen according to claim 47,
wherein said multifunctional crosslinker is selected from the group
consisting of 1-ethyl-3-(3-dimethylaminopropyl)carbodimide
hydrochloride and N-hydroxy-sulphosuccinimide.
49. The HBV capsid-binding peptide immunogen according to claim 42,
wherein said immunogenic component comprises one or more epitopes
selected from the group consisting of immunologic epitopes,
immunogenic epitopes and antigenic epitopes.
50. The HBV capsid-binding peptide immunogen according to claim 49,
wherein said epitopes are selected from the group consisting of
linear epitopes, conformational epitopes, single epitopes and mixed
epitopes.
51. The HBV capsid-binding peptide immunogen according to claim 42,
wherein said immunogenic component is selected from the group
consisting of antigens, allergens, antigenic determinants,
proteins, glycoproteins, antibodies, antibody fragments, peptides,
peptide mimotopes which mimic an antigen or antigenic determinant,
polypeptides, glycopeptides, carbohydrates, oligosaccharides,
polysaccharides, oligonucleotides and polynucleotides.
52. The HBV capsid-binding peptide immunogen according to claim 42,
wherein said immunogenic component is targeted to or derived from a
pathogenic agent selected from the group consisting of viruses,
parasites, mycobacteria, bacteria, bacilli, fungi, protozoa,
plants, phage, animal cells and plant cells.
53. The HBV capsid-binding peptide immunogen according to claim 52,
wherein said virus is selected from the group consisting of
retroviruses, herpesviruses, orthomyoxoviruses, paramyxoviruses,
hepadnaviruses, flaviviruses, picornaviruses, papoviruses,
adenoviruses, baculoviruses, hantaviruses, parvoviruses,
enteroviruses, rhinoviruses, tumor viruses, DNA viruses, RNA
viruses, togaviruses, rhabdoviruses and poxviruses.
54. The HBV capsid-binding peptide immunogen according to claim 53,
wherein said virus is selected from the group consisting of human
immunodeficiency type 1 virus, human immunodeficiency type 2 virus,
T cell-leukemia virus, herpes simplex type 1 virus, herpes simplex
type 2 virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr
virus, influenza A virus, influenza B virus and influenza C virus,
respiratory syncytial virus, measles-like virus, mumps virus,
parainfluenza virus, hepatitis B virus, hepatitis C virus,
hepatitis A virus, hepatitis E virus, yellow fever virus, dengue
virus, malaria, tick-borne encephalitis virus, poliomyelitis virus,
rubella virus, rabies virus and vaccinia virus.
55. The HBV capsid-binding peptide immunogen according to claim 42,
wherein said immunogenic component is targeted to or derived from
bacillus, enterobacteria, clostridium, listeria, mycobacterium,
pseudomonas, staphylococcus, eubacteria, mycoplasma, chlamydia,
spirochetes, neisseria or salmonella.
56. The HBV capsid-binding peptide immunogen according to claim 42,
wherein said immunogenic component is targeted to diptheria,
tetanus, acellular pertussis, haemophilus influenza, polio,
measles, mumps, rubella, varicella, hepatitis B virus, hepatitis A
virus, pneumococcal pneumonia, yellow fever, malaria, hepatitis B
virus, hepatitis A virus, typhoid fever, meningococcal encephalitis
or cholera.
57. The HBV capsid-binding peptide immunogen according to claim 42,
wherein said immunogenic component is selected from the group
consisting of animal allergens, insect allergens, plant allergens,
atmospheric allergens and inhalant allergens.
58. The HBV capsid-binding peptide immunogen according to claim 42,
wherein said HBV capsid-binding peptide component is selected from
the group consisting of: SLLGRMKGA, GSLLGRMKGA, DGSLLGRMKGAA,
ADGSLLGRMKGAAG, SLLGRMKG(.beta.-A)C, RSLLGRMKGA, HRSLLGRMKGA,
ALLGRMKG, MHRSLLGRMKGA, RSLLGRMKGA(.beta.-A)C and
MHRSLLGRMKGAG(.beta.-A)GC.
Description
[0001] This application is a continuation of co-pending application
Ser. No. 10/872,550, filed Jun. 21, 2004, which is a divisional of
application Ser. No. 10/448,546, filed May 29, 2003, now U.S. Pat.
No. 6,827,937, which is a divisional of application Ser. No.
09/873,459, filed Jun. 4, 2001, now U.S. Pat. No. 6,627,202, which
is a continuation of PCT application number PCT/US99/28755, filed
Dec. 3, 1999, which in turn claims the benefit of U.S. provisional
application No. 60/110,911, filed Dec. 4, 1998.
TECHNICAL FIELD OF THE INVENTION
[0002] This invention relates to hepatitis B virus ("HBV") core
antigen particles that are characterized by multiple immunogen
specificities. More particularly, the invention relates to HBV core
antigen particles comprising immunogens, epitopes, or other related
structures, crosslinked thereto by ligands which are HBV
capsid-binding peptides that selectively bind to HBV core protein.
Such particles may be used as delivery systems for a diverse range
of immunogenic epitopes, including the HBV capsid-binding peptides,
which advantageously also inhibit and interfere with HBV viral
assembly by blocking the interaction between HBV core protein and
HBV surface proteins. Mixtures of different immunogens, HBV
capsid-binding peptide ligands, or both, may be crosslinked to the
same HBV core particle. Such resulting multicomponent or
multivalent HBV core particles may be advantageously used in
therapeutic and prophylactic vaccines and compositions, as well as
in diagnostic compositions and methods using them.
BACKGROUND OF THE INVENTION
[0003] The front-line of clinical immunotherapeutic regimens
includes patient immunizations against infectious pathogens and
other health-threatening agents. Despite the plethora of
immunization agents, inoculations may afford, at best, partial
immunity, requiring frequent re-immunizations. Such is the case for
various conventional monovalent or polyvalent vaccines. And even
among such vaccines, the number of single agent inoculants capable
of eliciting immunity against a variety of immunogens is limited.
Furthermore, antigenic variation among pathogens may limit the
efficacy of conventional vaccines.
[0004] Due to such obstacles, efforts have focused on methodologies
for enhancing the immune system response to given immunogens. To
that end, immunogenic conjugates have been produced by linking
immunogens to hepatitis B virus ("HBV") core particles (also
referred to as nucleocapsids or nucleocapsid shells), in efforts to
enhance the immunogenicity of the linked immunogen, through the
operation of T cell dependent and T cell independent determinants
of HBV core antigen. See, for example, U.S. Pat. No. 4,818,527 and
R. Ulrich et al., "Core Particles of Hepatitis B Virus as Carrier
for Foreign Epitopes", Adv. Virus. Res., 50, pp. 141-82 (1998).
Enhanced immunogenicity of epitopes of interest has also been
approached via hybrid viral particle-forming proteins, comprising
at least a portion of a naturally occurring viral particle forming
protein, for example HBV surface antigen, and one or more epitopic
sites of interest. See U.S. Pat. No. 5,965,140. As evident from
such efforts, proteins of HBV have been used as platforms for
presenting immunogens of interest to the immune system.
[0005] Hepatitis B virus is a blood-borne virus, comprising a
small, partially double-stranded DNA genome, carrying four
extensively overlapping open reading frames, consisting of an inner
nucleocapsid, comprising the HBV core protein ("HBcAg"), viral
polymerase and viral DNA, surrounded by a membranous envelope
containing HBV surface antigens ("HBsAg"). The viral envelope
contains three different, but related surface antigen proteins,
long (L), medium (M) and short (S), which share a common carboxy
terminal region but have different amino termini, arising from
variable use of initiation triplets at different points within a
continuous open reading frame.
[0006] The long polypeptide (L polypeptide) consists of pre-S1,
pre-S2 and S regions. It is the product of the entire reading frame
and comprises the pre-S1 domain of 108 amino acids (or 199,
depending on the virus subtype) at its amino terminus, followed by
the pre-S2 domain of 55 amino acids, and the short polypeptide (S
polypeptide) region of 226 amino acids. The medium-length
polypeptide (M polypeptide) has the pre-S2 domain at its amino
terminus followed by the S region, whereas the S polypeptide, which
is the most abundant form, consists of only the S region. The pre-S
regions are believed to play an important role in both viral
assembly and attachment to the host cell. The S form is more
abundant than the M and L forms of HBsAg in the virus, and occurs
in both glycosylated and nonglycosylated forms [V. Bruss and D.
Ganem, "The Role of Envelope Protein in Hepatitis B Virus
Assembly", Proc. Natl. Acad. Sci. USA, 88, pp. 1059-63 (1991); V.
Bruss et al., "Post-translational Alteration in Transmembrane
Topology of Hepatitis B Virus Large Envelope Protein", EMBO J., 13,
pp. 2273-79 (1994); A. R. Neurath et al., "Identification and
Chemical Synthesis of a Host Cell Receptor Binding Site on
Hepatitis B Virus", Cell, 46, pp. 429-36 (1986); K. Ueda et al.,
"Three Envelope Proteins of Hepatitis B Virus: Large S, Middle S
and Major S Proteins Needed for the Formation of Dane Particles",
J. Viral., 65, pp. 3521-29 (1991)]. Specific interactions between
the outer surface of the core and the inner surface of the envelope
are likely to guide correct assembly of the virus and stabilize the
resulting particle
[0007] HBV core protein can be expressed efficiently in E. coli.
[M. Pasek et al., "Hepatitis B Virus Genes and Their Expression in
E. coli", Nature, 282, pp. 575-79 (1979)], where it assembles into
icosahedral shells of two sizes containing either 180 (T=3) or 240
(T=4) subunits [R. A. Crowther et al., "Three-Dimensional Structure
of Hepatitis B Virus Core Particles Determined by Electron
Microscopy", Cell, 77, pp. 943-50 (1994)]. The subunits are
clustered as dimers and each dimer forms a spike which protrudes on
the surface of the shell. Using electron cryomicroscopy and image
processing, a map of the T=4 shell was recently made at 7.4 .ANG.
resolution from images of more than 6000 individual particles [B.
Bottcher et al., "Determination of the Fold of the Core Protein of
Hepatitis B Virus by Electron Cryomicroscopy", Nature, 386, pp.
88-91 (1997)]. This revealed the fold of the polypeptide chain,
which was largely .alpha.-helical and quite unlike previously
solved viral capsids. Each dimer spike was formed by a pair of long
.alpha.-helical hairpins, one from each monomer in the dimer
[Bottcher et al. (1997); J. F. Conway et al., "Visualization of a
4-Helix Bundle in the Hepatitis B Virus Capsid by Cryo-electron
Microscopy", Nature, 386, pp. 91-94 (1997)]. A numbering scheme
which superimposed the amino acid sequence on the fold [Bottcher et
al. (1997)] placed the major immunodominant region of the HBV core
protein around amino acids 78-82 [J. Salfeld et al., "Antigenic
Determinants and Functional Domains in Core Antigen and E Antigen
from Hepatitis B Virus", J. Virol, 63, pp. 798-808 (1989); M.
Sallberg et al., "Characterisation of a Linear Binding Site for a
Monoclonal Antibody to Hepatitis B Core Antigen", J. Med. Virol.,
33, pp. 248-52 (1991)], at the tip of the spike.
[0008] Agents which inhibit HBV viral assembly include those that
bind to the core antigen of HBV, thereby blocking the interaction
between HBV core proteins and HBV surface proteins. Some such HBV
capsid-binding peptides are described in PCT patent application
WO98/18818 and in M. R. Dyson and K. Murray, "Selection of Peptide
Inhibitors of Interactions Involved in Complex Protein Assemblies:
Association of the Core and Surface Antigens of Hepatitis B Virus",
Proc. Natl. Acad. Sci. USA, 92, pp. 2194-98 (1995).
[0009] As will be apparent from the disclosure to follow, HBV
capsid-binding peptides may be advantageously used as ligands for
constructing HBV core antigen particles characterized by the
ability to elicit enhanced immune responses to single or multiple
immunogens.
DISCLOSURE OF THE INVENTION
[0010] The present invention addresses the problems referred to
above by providing HBV core antigen particles which elicit enhanced
immunogenicity to one or more component immunogens. Such
multicomponent or multivalent HBV core antigen particles comprise
immunogens, epitopes, or other related structures, crosslinked
thereto through ligands which are peptides that selectively bind to
HBV core antigen particles, in addition to immunogenic domains or
epitopes attached to or inserted into the HBV core antigen
polypeptide via genetic manipulation of the coding sequence or by
polypeptide synthesis. Such particles may be used as delivery
systems for a diverse range of immunogenic epitopes, including the
HBV capsid-binding peptides, which themselves, inhibit and
interfere with HBV viral assembly by blocking the interaction
between HBV core protein and HBV surface proteins. The resulting
multicomponent or multivalent HBV core particles may be
advantageously used in therapeutic and prophylactic vaccines and
compositions, as well as diagnostic compositions and methods using
them.
[0011] The present invention advantageously permits mixtures of
different immunogens, HBV capsid-binding peptide ligands, or both,
to be crosslinked to the same HBV core particle. The result is
single particles that are efficient stimulants of T cells and which
are immunologically multivalent. Thus, a single antigen-presenting
cell can stimulate the proliferation of multiple B cell clones of
differing specificity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 depicts the structure of an HBV core antigen particle
comprising various capsid binding immunogens. A "capsid binding
immunogen" comprises at least one HBV capsid-binding peptide
component and at least one immunogenic component. Each capsid
binding immunogen is linked to the HBV core antigen particle
through an HBV capsid-binding peptide.
[0013] FIG. 2 is a table summarizing various HBV core antigen
fusion proteins which may also serve as the HBV core antigen
particle to which various immunogens may be linked through HBV
capsid-binding peptides.
DETAILED DESCRIPTION OF THE INVENTION
[0014] In order that the invention herein described may be more
fully understood, the following detailed description is set
forth.
[0015] According to one embodiment of this invention, mixtures of
more than one type of immunogen, and one or more types of HBV
capsid-binding peptide ligands may be crosslinked to the same HBV
core particle. Alternatively, multiple copies of the same immunogen
may be linked to one type of HBV capsid-binding peptide and
crosslinked to various positions on the HBV core particle.
Multicomponent or multivalent HBV core antigen particles according
to this invention are particularly useful for inducing antibodies
to all component immunogens.
[0016] The use of HBV capsid-binding peptides to link immunogens to
the HBV core antigen particle permits enhanced immunogen
presentation, without destroying immunogenicity or stability of the
immunogen by denaturation, conformational disruption or other
destabilizing influences. For example, the HBV capsid-binding
peptide linkers reduce the risk that component immunogens will
interfere with each other to cause loss of functional material. As
a result, the HBV core antigen particle elicits an enhanced immune
response to its component immunogens. Therefore, it is possible to
achieve desired therapeutic or prophylactic effects with fewer
inoculations and/or less inoculant than that necessary, were each
immunogen administered as a single-agent.
[0017] Linkage of immunogens to the HBV core antigen particle
through HBV capsid-binding peptides also permits presentation of
immunogens which vary in size, conformation and nature. As a
result, the present invention allows inclusion in one vaccine or
composition, combinations of immunogens useful to elicit a broad
spectrum of immunity or treatment in a given individual.
Immunogens
[0018] Immunogens which may be linked to HBV capsid-binding
peptides and thus, incorporated into HBV core antigen particles,
include any molecule containing one or more immunologic,
immunogenic or antigenic epitopes. Such epitopes may be linear,
conformational, single, or mixed in nature.
[0019] More particularly, immunogens may be selected from any agent
capable of eliciting an immune response. Such agents include, but
are not limited to, antigens, antigenic determinants, proteins,
glycoproteins, antibodies, antibody fragments, peptides, peptide
mimotopes which mimic an antigen or antigenic determinant,
polypeptides, glycopeptides, carbohydrates, oligosaccharides,
polysaccharides, oligonucleotides and polynucleotides. Immunogens
may also be allergens, toxins or endotoxins.
[0020] Such agents also include those targeted to or derived from
various pathogenic agents, such as viruses, parasites, bacteria,
fungi, phages, protozoa and plants. Such viruses include
retroviruses, including human immunodeficiency type 1 and type 2
viruses and T cell leukemia virus; herpesviruses, such as herpes
simplex type 1 and type 2 viruses, varicella-zoster viruses,
cytomegaloviruses and Epstein-Barr virus, orthomyxoviruses, such as
influenza A, influenza B and influenza C viruses; paramyxoviruses,
such as respiratory syncytial virus, measles-like viruses, mumps
virus and parainfluenza viruses; hepadnaviruses, such as hepatitis
B viruses; flaviviruses, such as hepatitis C virus, hepatitis A
virus, hepatitis E virus, yellow fever virus, dengue virus and
tick-borne encephalitis viruses; picornaviruses, such as
enteroviruses, rhinoviruses, foot and mouth disease viruses and
poliomyelitis virus; togaviruses, such as rubella virus;
rhabdovirus, such as rabies virus; adenoviruses, ebolaviruses;
baculoviruses; hantaviruses; papovaviruses, such as
papillomaviruses; parvoviruses; DNA viruses; RNA viruses; RNA tumor
viruses, such as oncoviruses; and poxviruses, such as vaccinia
virus. In addition, immunogens may be those which are targeted to
or derived from bacillus, enterobacteria, clostridium, listeria,
mycobacterium, pseudomonas, staphylococcus, eubacteria, mycoplasma,
chlamydia, spirochetes, neisseria or salmonella. Immunogens may
also be selected from the following epitopes of human
immunodeficiency virus: GELDRWEKI (gag) (SEQ ID NO: 1); ELDKWAS (gp
40) (SEQ ID NO: 2); IGPGRAFYTTKN (V3 loop) (SEQ ID NO: 3); ELDKWA
(gp 41) (SEQ ID NO: 4) and DRFYKTLRA (gp 41) (SEQ ID NO: 5).
[0021] Glycoproteins which may be linked to HBV capsid-binding
peptides and thus, incorporated into HBV core antigen particles
include, for example, antibodies, glycopeptides from or resembling
surface components of animal cells or viruses or bacteria, such as
those causing meningitis, or fragments of such moieties.
[0022] As will be appreciated by those of skill in the art, the
size of the immunogen should not be large enough to allow a
functional group thereof to interfere with the HBV capsid-binding
peptide linker.
HBV Core Antigen Protein
[0023] Due to its particulate nature, HBV core antigen protein
constitutes an advantageous platform for the presentation of
multiple immunogens, of similar or dissimilar type, to the immune
system. According to the present invention, this advantage is
further enhanced by the use of HBV capsid-binding peptides as
ligands to attach desired immunogens to the HBV core particle. Such
particles contain either 90 or 120 ligand-binding sites--the capsid
spikes, each composed of an HBV core antigen dimer (see FIG. 1).
Thus, multiple immunogens may be physically linked to the HBV core
antigen particle by the HBV capsid-binding peptides as ligands. The
resulting particle is capable of inducing an immune response to all
of its component immunogens.
[0024] HBV core antigen particles may be formed upon expression of
recombinant coding sequences for HBV core antigen polypeptide in an
appropriate microbial, animal or plant system. See, for example,
Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd
Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989).
The polypeptide to be expressed may comprise the full-length HBV
core antigen sequence, or mutations, derivatives, truncations, or
portions thereof, which retain the ability to assemble in
particulate form in the cells of the expression system. Recombinant
methods for producing such HBV core antigen particles are known in
the art. See, for example, U.S. Pat. No. 4,710,463.
[0025] Alternatively, chemical synthesis methods may be used to
produce HBV core antigen polypeptide. Based on the amino acid
sequence of the HBV core antigen polypeptides, chemical synthesis
may be carried out using solid phase synthesis [R. B. Merrifield,
Fed. Proced., 21, p. 412 (1964); R. B. Merrifield, Biochemistry, 3,
pp. 1385-90 (1964) and D. R. Milich et al., J. Immunol., 139, pp.
1223-31 (1987)].
[0026] Those skilled in the art will appreciate that since mutated
or variant HBV core antigen sequences may influence reactions with
binding or ligand peptides, the present invention applies equally
to natural variants or mutations introduced by manipulation of
coding sequences, or other procedures, in the HBV core antigen
subunits or the corresponding binding or ligand peptides.
Mutation of Core Protein Residues
Important for Peptide Binding
[0027] Methods used to determine the fold of the core protein have
been applied to locate by cryomicroscopy the binding sites on the
core protein of SLLGRMKGA (SEQ ID NO: 6), an HBV capsid-binding
peptide that inhibits binding to L-HBsAg. This approach has now
shown the peptide bound to the tips of the spikes, in both T=3 and
T=4 shells. Image analysis shows that the peptide binding sites lie
at the tip of the spikes which in the proposed numbering scheme for
the polypeptide fold corresponds to residues in the region of amino
acids 78-82. There are two acidic residues (glu77 and asp78) close
to the tip of the core protein and the selected binding peptide
contained two conserved basic residues. The importance of these
oppositely charged residues in the binding reaction was confirmed
when mutation of either of the acidic residues in the protein to
alanine was found to greatly reduce the affinity of the peptides
for the altered core shells. Changing aspartic acid 78 to alanine
reduced the affinity 160-fold and changing glutamic acid 77 to
alanine reduced the affinity 1000-fold. This suggests that either
or both acidic residues on the HBV core antigen protein may provide
at least part of the binding site for HBV capsid-binding
peptides.
[0028] These results also illustrate the importance of the amino
acid sequence of HBV core antigen in the region of the tip of the
spike for ligand binding. Those of skill in the art will appreciate
that HBV core antigen from some HBV strains may require mutation
for effective binding of a particular ligand-immunogen peptide, or
the selection and adaptation of variants of the ligand for
effective binding to that specific HBV core antigen variant.
HBV Core Antigen Fusion Proteins
[0029] According to one embodiment of this invention, the HBV core
antigen particle to which immunogens may be linked via HBV
capsid-binding peptides may be one already displaying one or more
immunogens, as a result of genetic fusion techniques. In one such
technique, relevant coding sequences are incorporated at
appropriate positions in plasmids or other vectors carrying that
for HBV core antigen polypeptide.
[0030] Fusions to the .beta.-galactosidase gene of E. coli used to
enhance expression levels of HBV core antigen polypeptide
demonstrated that replacement of the first two amino acids of the
antigen with a sequence of eleven amino acids (eight from the amino
terminus of .beta.-galactosidase and three further residues
resulting from translation of a linker sequence introduced in the
gene fusion) had no adverse impact upon the ease of recovery,
antigenicity, or morphology of the product [S. Stahl et al.,
"Hepatitis B Virus Core Antigen. Synthesis in Escherichia Coli and
Application in Diagnosis", Proc. Natl. Acad. Sci. USA, 79, pp.
1606-10 (1982); B. J. Cohen and J. E. Richmond, "Electron
Microscopy of Hepatitis B Core Antigen Synthesized in E. Coli",
Nature, 296, pp. 677-78 (1982)].
[0031] HBV core antigen fusion proteins useful in the present
invention may be produced as exemplified in S. J. Stahl and K.
Murray, "Immunogenicity of Peptide Fusions to Hepatitis B Virus
Core Antigen", Proc. Natl. Acad. Sci. USA, 86, pp. 6283-87 (1989).
Alternatively, fusions of polypeptide sequences to the major
segment of HBV core antigen to give highly immunogenic particles
are exemplified with a number of viral coding sequences, as
enumerated in FIG. 2. These include a particulate product with high
immunogenicity, produced by expression via a vaccinia virus vector
of the VP1 peptide (residues 142-160) fused through a heptapeptide
linker sequence to the six amino acids of the pre-core sequence
immediately preceding the amino terminus of HBV core antigen
polypeptide [B. E. Clarke et al., "Improved Immunogenicity of a
Peptide Epitope after Fusion to Hepatitis B Core Protein", Nature,
330, pp. 381-84 (1987)]. See also Ulrich et al. (1998) for other
useful HBV core antigen fusion proteins.
[0032] A series of other fusion proteins are characterized by
replacement of the arginine-rich region at the carboxy terminus of
HBV core antigen polypeptide by other alternate coding sequences.
Peptides that included the immunodominant a region of HBV surface
antigen (residues 111-165), the pre-S1 and pre-S2 epitopes, and
various segments of the envelope protein of human immunodeficiency
virus (HIV) were attached to residue 144 of HBV core antigen
polypeptide. All were expressed efficiently in E. coli to give
particulate products displaying essentially the same morphology as
that of HBV core antigen itself [Stahl and Murray, 1989]. The
products displayed the antigenic reactivity of HBV core antigen
and, like preparations of HBV core antigen polypeptide truncated at
residue 144, those tested also exhibited HBV e antigen reactivity,
whereas full-length HBV core antigen shows very little such
activity. Fusion proteins carrying residues 111-156 or 111-165 from
HBV surface antigen displayed no significant HBV surface antigen
reactivity, a result not inconsistent with the conformation
dependence of this major epitope, or the likelihood of the
sequences being buried within the particles. Immunogenic responses
to the fusion proteins, however, reflected their various component
epitopes.
[0033] Immune responses to HBV surface antigen are complex, for in
addition to epitopes residing in the pre-S1 and pre-S2 regions of
L-HBsAg and the major immunodominant a region, a number of variable
subtype determinants have been assigned to other regions of the
short, or S polypeptide of HBV surface antigen [G. L. Le Bouvier,
"The Heterogeneity of Australia Antigen", J. Infect. Dis., 123, pp.
671-75 (1971); W. H. Bancroft et al., "Detection of Additional
Antigenic Determinants of Hepatitis B Antigen", J. Immunol., 109,
pp. 842-48 (1972); A.-M. Courouce-Pauty P. V. and Holland, "Summary
of Workshop A2: HBsAg and its Subtypes", in Viral Hepatitis. G. N.
Vyas, S. N. Cohen and R. Schmid, eds. (Philadelphia, USA: Franklin
Institute Press), pp. 649-54 (1978)]. The HBV surface antigen
coding sequences determined on HBV DNA cloned from sera of
differing subtypes display differences in the corresponding protein
sequences. However, specific single mutations of apparently
critical residues did not effect a switch from one serological
subtype (y) to another (d), but additional single mutations induced
a gradual change with both y and d reactivities and
immunogenicities being displayed from the same molecule [P. G.
Ashton-Rickardt and K. Murray, "Mutations that Change the
Immunological Subtype of Hepatitis B Virus Surface Antigen and
Distinguish Between Antigenic and Immunogenic Determination", J.
Med. Virol., 29, pp. 204-14 (1989)]. The mutations involved were
made within or close to the conformation-sensitive immunodominant a
region, and were all within the segment of HBV core antigen used in
the fusions to HBV core antigen described above.
[0034] The impact of the mutations upon the subtype specificity of
the antibodies induced prompted the suggestion that fusion proteins
might also provide a means for changing the specificity of the
response to epitopes of interest, particularly if they are
dependent on conformation. Mutations of glycine.sub.145 to
arginine, to mimic the natural escape mutant, and to other
positively or negatively charged residues (lysine and glutamic
acid) were therefore made at this residue in HBcS.sub.111-156 for
comparative studies of humoral and cellular immune responses [A. L.
Shiau and K. Murray, "Mutated Epitopes of Hepatitis B Surface
Antigen Fused to the Core Antigen of the Virus Induce Antibodies
That React with the Nature Surface Antigen", J. Med. Virol., 51,
pp. 159-66 (1997)]. All were expressed efficiently in E. coli,
yielding the anticipated particulate products showing strong HBV
core antigenicity and all induced high titers of antibody to HBV
core antigen in rabbits.
[0035] Like their parent protein HBcS.sub.111-156, the three
residue 145 mutants showed minimal interaction with antibody to HBV
surface antigen in solid phase radio-immune assays (AUSRIA; Abbott
Laboratories) or antibody precipitation assays in solution.
However, they all showed strong reactions with a rabbit anti-HBV
surface serum in immunoblotting experiments after electrophoresis
in acylamide gels under denaturing conditions [A. L. Shiau,
"Immunological Aspects of Hepatitis B Virus Core Antigen and its
Derivatives", PhD Thesis, University of Edinburgh, UK. (1993)]. At
high concentrations, the parent and mutant proteins also gave
weakly positive reactions with antibody to HBV surface antigen when
captured on a solid phase coated with antibody to HBV core antigen,
possibly as a result of some disruption of the particles affording
access for anti-HBsAg molecules [Shiau and Murray, 1997].
[0036] Immunized rabbits were used to examine T-cell responses to
the fusion proteins, as well as antibody production. Peripheral
blood mononuclear cells (PBMC) taken at various times after
immunization were used for proliferation assays based upon
[.sup.3H]-thymidine incorporation in response to exposure to the
HBV core antigen, or fusion protein used for immunization. In all
cases, strong responses were found, with the fusion protein
exhibiting a higher stimulation index than HBV core antigen, and
HBV surface antigen being a poor stimulant, as expected. A double
antibody radio-immunoprecipitation assay [C. J. Burrell et al.,
"Rapid Detection of Hepatitis B Surface Antigen by Double Antibody
Radioimmunoassay", J. Med. Virol., 3, pp. 1926 (1978)] with
[.sup.125I]-HBV surface antigen was used to measure anti-HBs in the
serum samples and showed the anticipated positive response to
HBcS.sub.111-156. The arginine mutant also gave a positive response
in this assay, although somewhat less than that of its parent
molecule, and a weak response was obtained from the glutamic acid
mutant, but none from the lysine mutant. Thus, the results showed
that the fusion protein (designated HBcS.sub.145R) carrying the
arginine 145 mutant was a strong T-cell stimulant and induced
antibodies with a broader reaction specificity.
[0037] A further group of fusions of various portions of the HBV
surface antigen polypeptide, including residue 145 mutants, to HBV
core antigen polypeptide was made to explore the effect of the
overall size and the number and position of the various additional
components on the immunogenicity of the products [Shiau (1993)].
These constructs are included in FIG. 2 and, as with the other
fusions, all gave particulate products displaying the morphology of
HBV core antigen, although fusions with the HBs.sub.111-156
fragment at the amino terminus of HBV core antigen were less
satisfactory, giving products that formed insoluble aggregates.
[0038] This group of products, like the earlier ones with the
HBcS.sub.111-156 segments, showed little or no reaction with
antibody to HBV surface antigen on solid phase or in solution, but
when captured by antibody to HBV core antigen on solid phase, they
showed similar reactivity with antibody to HBV surface antigen and
this was somewhat higher (about two-fold), with fusions carrying
pre-S1 and pre-S2 segments in addition to HBcS.sub.111-156. The
stimulation indices for lymphocyte proliferation inhibition were
again strong for all the fusion proteins and those that included
pre-S segments as well as native or mutant HBcS.sub.111-156
sequences gave the stronger responses. Inclusion of the pre-S1 and
pre-S2 sequences between HBc.sub.144 and the HBcS.sub.111-156
sequences (either wild type or mutant) gave higher antibody levels
in the double antibody radio-immunoprecipitation assay than the
fusions lacking the pre-S segments, but the introduction of a
second HBcS.sub.111-156 sequence between HBc.sub.144 and the pre-S
sequences produced no further enhancement in any of the responses.
The longest of these sequences attached to HBV core antigen
polypeptide at proline.sub.144--165 amino acids--had no obviously
adverse impact on the yield or physical properties of the fusion
protein.
[0039] The core protein (HCc) of hepatitis C virus (HCV) has also
been fused, in part and in multiple full-length copies, to HBV core
antigen polypeptide truncated at valine 149 [A. Yoshikawa et al.,
"Chimeric Hepatitis B Virus Core Particles with Parts of Copies of
the Hepatitis C Virus Core Protein", J. Virol., 67, pp. 6064-70
(1993)]. Fusions carrying HCc residues 39-75 showed negligible HCc
antigenicity, but residues 1-91 or the full sequence of 180 amino
acids gave positive reactions and the antigenicity increased almost
arithmetically with the addition of further copies (up to four) of
the 1-180 sequence via short linkers. Electronmicroscopy showed
that the fusion carrying a single copy of HCc residues 1-91 formed
particles morphologically equivalent to HBV core antigen
polypeptide, but three full-length HCc copies greatly distorted
this structure and the product was very sensitive to proteolysis
giving, however, material that retained HBV core antigenicity.
While the largest fusion protein carried more than 720 additional
amino acids, the limit for a particle of the HBV core antigen type
appears to be appreciatively less.
[0040] PreS sequences have been used in other studies of the effect
of the position of fusion to HBcAg on immunogenicity. Borisova et
al. (1989) made fusions with segments of pre-S1 (residues 20-68,
20-69, or 69-106) or the whole of pre-S2 linked to HBV core antigen
truncated at proline 144 or inserted at this position within the
full length HBV core antigen sequence. In these and analogous
constructions with residues 56-103 of the envelope protein of
bovine leukaemic virus (BLV) or residues 78-129 of the HIV
transmembrane protein (gp41), the sequences fused to HBV core
antigen were believed to be exposed on the particle surfaces, for
all were reported to be both antigenic and immunogenic and the
C-terminal arginine-rich domain apparently had little adverse
effect.
[0041] F. Schodel et al., "The Position of Heterologous Epitopes
Inserted in Hepatitis B Virus Core Particles Determines Their
Immunogenicity", J. Virol., 6, pp. 106-14 (1992) explored the
impact of position of fusion on antigenicity and the immune
response in inbred mice, when pre-S1 or pre-S2 segments were
attached at the amino terminus of full-length HBV core antigen
(either directly or via part of the pre-core sequence) or the
carboxy terminus of truncated HBV core antigen; a further
construction carried a pre-S1 segment between HBV core antigen
residues 75 and 83 as well as the pre-S2 fragment at the truncated
carboxy terminus (proline 156).
[0042] The comprehensive analysis showed that the pre-S1 sequence
fused to the amino terminus of HBV core antigen via the short
pre-core sequence was antigenic, but that fused directly to the
amino terminus was not and, while both had the same HBV core
antigen immunogenicity, the fusion via the pre-core sequence
stimulated a much higher anti-pre-S1 response. The pre-S2 sequence
at the truncated HBV core antigen terminus was antigenic and
immunogenic to a similar degree in both contexts, but the pre-S1
sequence fused internally so as to replace residues 76-82 (which
include the major HBV core antigen epitope) was substantially more
antigenic and dramatically more immunogenic than in the N-terminal
fusions. As anticipated, HBV core antigenicity and immunogenicity
were greatly reduced in the internal fusion proteins. Replacement
of an internal sequence of HBV core antigen (residues 78-82) with a
fragment of HBV surface antigen containing the immunodominant a
epitope also gave a product exhibiting positive HBV core
antigenicity and immunogenicity [G. Borisova et al., "Hybrid
Hepatitis B Virus Nucleocapsid Bearing an Immunodominant Region
from Hepatitis B Surface Antigen", J. Virol., 67, pp. 3696-3701
(1993)].
[0043] As a further alternative, or as an addition to the fusion
proteins described above, immunogenic components may be attached to
HBV core antigen by chemical cross-linking procedures.
[0044] Superimposition of the amino acid sequence of HBV core
antigen on the physical structure suggested by Bottcher et al.
(1997) helps to explain the low antigenicity of sequences fused at
or near the carboxy terminus of HBV core antigen, since such
sequences are likely to be buried within the HBV core antigen
particles, while N-terminal fusions may benefit from flexible
linker sequences, to bring the immunogen further from the
relatively confined space at the foot of the spikes. Location of
the immunodominant HBV core antigen epitope [residues 78-82;
Salfeld et al. (1989)] at the tip of the spike shows the attraction
of this position for insertion or attachment of the HBV
capsid-binding peptide-immunogen. In principle, all these positions
may be used simultaneously to increase the number and/or diversity
of epitopes presented by a given HBV core antigen particle.
HBV Capsid-Binding Peptides Used to Ligate Immunogens to HBV Core
Antigen Particles
[0045] As described above, immunogens of interest may be linked to
a HBV core particle using a ligand which is an HBV capsid-binding
peptide. Such HBV capsid-binding peptides are isolated, purified
peptides. These HBV capsid-binding peptides advantageously inhibit
and interfere with HBV viral assembly by blocking the interaction
between HBV core protein and HBV surface proteins.
[0046] Preferably, HBV capsid-binding peptides include peptides,
fragments, analogs and homologs thereof, which are between about 2
and about 20 amino acids in length. More preferably, the peptides
are between about 3 to about 15 amino acids in length. Such
peptides include those listed in the tables below, as well as
fragments and analogs thereof.
[0047] As used herein, the term "fragment" refers to an amino acid
sequence which is shorter than the peptide from which it is
derived, but which retains biological activity substantially
similar to that of the original peptide. Such a fragment is at
least two amino acids in length.
[0048] As used herein, the term "analog" refers to variations in
the amino acid sequences of the peptides, which may typically
include analogs that differ only by one to about four amino acid
changes. Other examples of analogs include peptides with minor
amino acid variations from the peptides exemplified herein. In
particular, peptides containing conservative amino acid
replacements, i.e., those that take place within a family of amino
acids that are related in their side chains, constitute
analogs.
[0049] Genetically encoded amino acids are generally divided into
four families: (1) acidic: aspartate, glutamate; (2) basic: lysine,
arginine, histidine; (3) nonpolar: alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan; and (4)
uncharged polar: glycine, asparagine, glutamine, cysteine, serine,
threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are
sometimes classified jointly as aromatic amino acids. With respect
to HBV capsid-binding peptides, it may be beneficial to change one
or more amino acids. Those of skill in the art may readily evaluate
the impact of such a change.
[0050] The term "homolog" includes peptide fragments which share at
least 60 percent identity at the amino acid level, and preferably
75 percent identity, and substantially similar biological activity
to a reference peptide. These preferred percentages reflect the
small size of the peptides.
[0051] Useful HBV capsid-binding peptides include those based on
the peptides disclosed in Dyson and Murray (1995). Such peptides
were synthesized following random mutagenesis of residues flanking
the peptide LLGRMK (SEQ ID NO: 7) in the fusion phage B1 and
re-selection against HBV core antigen in a bio-panning reaction to
obtain derivatives that bind the antigen with improved affinity.
High resolution electron cryomicroscopy demonstrated that such HBV
capsid-binding peptides bind at the tips of the spikes of the HBV
core protein shell. The inhibitory effect of the peptides on the
interaction between HBV core antigen and HBV surface antigen
proteins in infected cells was examined through transfection of
permeabilized hepatoma Hep G2 cells with a replication-competent
plasmid carrying a head-to-tail dimer of the HBV genome in the
presence or absence of the peptide. See Bottcher et al. (1998).
[0052] HBV capsid-binding peptides carrying the LLGRMK (SEQ ID NO:
7) sequence reduced the yield of HBV in transfected hepatoma cell
cultures in a dose-dependent manner and with relative efficiencies
that reflect the IC.sub.50 values for the peptides in their
inhibition of reactions between HBV core antigen and L-HBV surface
antigen in solution.
[0053] HBV capsid-binding peptides preferably have a half maximal
concentration (IC.sub.50) less than about 10, preferably less than
5, more preferably less than about 2, and most preferably less than
about 0.5 .mu.M. Preferred peptides include, but are not limited
to: SLLGRMKG (.beta.-A)C (SEQ ID NO: 8), RSLLGRMKGA (SEQ ID NO: 9),
HRSLLGRMKGA (SEQ ID NO: 10), and RSLLGRMKGAO-A)C (SEQ ID NO: 11),
or peptides derived therefrom. Alternatively, such a peptide may be
peptide ALLGRMKG (SEQ ID NO: 12), which inhibits the interaction
between the long hepatitis B virus surface antigen (L HBsAg) and
HBcAg, with a half maximal concentration (IC.sub.50) of 10.0
.mu.M.
[0054] HBV capsid-binding peptides are exemplified by the
following, wherein K.sub.D.sup.Rel (nm) represents a relative
dissociation constant for reactions between HBV core antigen and fd
fusion phage carrying the peptide sequences in the amino terminal
region of the gpIII protein:
TABLE-US-00001 Sequence K.sub.D .sup.Rel (nm) ADGALLGRMKGA (SEQ ID
NO: 13) 152 .+-. 5 ADGALLGRMKPA (SEQ ID NO: 14) 767 .+-. 8
ADGSLLGRMKPA (SEQ ID NO: 15) 322 .+-. 50 ADGALLGRMKRA (SEQ ID NO:
16) 181 .+-. 12 ADGTLLGRMKLA (SEQ ID NO: 17) 20 .+-. 2 ADGSLLGRMKGA
(SEQ ID NO: 18) 1.7 .+-. 0.3 ADRSLLGRMKGA (SEQ ID NO: 19) 1.09 .+-.
0.02 ADGSRSSLLGRMKGA (SEQ ID NO: 20) 1.96 .+-. 0.32 ADGAHSSLLGRMKGA
(SEQ ID NO: 21) 1.72 .+-. 0.17 ADGHRSSLLGRMKGA (SEQ ID NO: 22) 1.40
.+-. 0.13 ADGPRSSLLGRMKGA (SEQ ID NO: 23) 0.84 .+-. 0.07
ADGAHRSLLGRMKGA (SEQ ID NO: 24) 0.94 .+-. 0.12 ADGYQRSLLGRMKGA (SEQ
ID NO: 25) 0.88 .+-. 0.08 ADGTQRSLLGRMKGA (SEQ ID NO: 26) 0.84 .+-.
0.06 ADGMHRSLLGRMKGA (SEQ ID NO: 27) 0.55 .+-. 0.03.
[0055] These peptides, which mimic cytoplasmic regions of L HBsAg,
were identified by selection from a random hexapeptide library
displayed on filamentous phage and their affinities for HBV core
antigen in solution determined in the phage associated form. The
following related peptides (listed below), are also examples of HBV
capsid-binding peptides and the IC.sub.50 .mu.M values represent
the concentration of peptide required to inhibit binding of L HBsAg
to HBV core antigen at a half maximal level, N/D represents no
observable inhibition and .beta.-A represents beta alanine:
TABLE-US-00002 Sequence IC.sub.50 .mu.M ALLGRMKG (SEQ ID NO: 12)
11.0 .+-. 0.8 LLGRMKG (SEQ ID NO: 28) 46.2 .+-. 7.4 LGRMKG (SEQ ID
NO: 29) 980 .+-. 157 GRMKG (SEQ ID NO: 30) N/D LLGRM (SEQ ID NO:
31) N/D CLLGRMKC (SEQ ID NO: 32) 652 .+-. 74 ALLPRMKG (SEQ ID NO:
33) N/D SLLGRMKG (SEQ ID NO: 34) 6.4 .+-. 0.7 SLLGRMK (SEQ ID NO:
35) 40.7 .+-. 4.8 SLLGRMKGA (SEQ ID NO: 6) 2.4 .+-. 0.2 GSLLGRMKGA
(SEQ ID NO: 36) 0.79 .+-. 0.23 DGSLLGRMKGAA (SEQ ID NO: 37) 3.0
.+-. 0.4 ADGSLLGRMKGAAG (SEQ ID NO: 38) 4.5 .+-. 0.8 ACSLLGRMKG
(SEQ ID NO: 39) 26.2 .+-. 5.0 SLLGRMKG(.beta.-A)C (SEQ ID NO: 8)
1.8 .+-. 0.4 SLLGRMKGA (SEQ ID NO: 9) 0.29 .+-. 0.02 HRSLLGRMKGA
(SEQ ID NO: 10) 0.50 .+-. 0.04 MHRSLLGRMKGA (SEQ ID NO: 40) 0.80
.+-. 0.10 RSLLGRMKGA(.beta.-A)C (SEQ ID NO: 11) 0.29 .+-. 0.03
MHRSLLGRMKGAG(.beta.-A)GC (SEQ ID NO: 41) 3.80 .+-. 0.69.
[0056] The HBV capsid-binding peptides, fragments, analogs and
homologs thereof, which may serve as ligands to bind immunogens to
HBV core antigen particles are preferably synthesized using
conventional synthesis techniques, e.g., by chemical synthesis
techniques. Alternatively, the skilled artisan may synthesize any
of the peptides by using an automated peptide synthesizer using
standard chemistry such as, for example, t-BOC chemistry. See, for
example, L. A. Carpino, J. Am. Chem. Soc., 79, pp. 4427 (1957). And
the peptides may be prepared by chemical cleavage of a protein or
other methods. The peptides are isolated such that they are
substantially free of chemical precursors or other chemicals when
synthesized chemically, or obtained by chemical cleavage of a
protein.
[0057] Alternatively, HBV capsid-binding peptides may be prepared
by conventional genetic engineering techniques, e.g., recombinant
DNA techniques in a host cell transformed with a nucleic acid
sequence coding for the peptide, by cloning and expressing within a
host microorganism or cell a DNA fragment carrying a coding
sequence for the selected peptide. When produced by recombinant
techniques, in appropriately transformed cells, the peptides may be
purified from the cell culture medium, host cells, or both, using
conventional methods. The recombinant peptides are isolated such
that the peptide is substantially free of cellular material or
culture medium when produced by recombinant DNA techniques. Coding
sequences for the peptides may be prepared synthetically, or
derived from viral RNA by known techniques, or from available
cDNA-containing plasmids.
[0058] For use in the methods of this invention, the
above-described peptides may be designed into conventionally known,
or alternative constructs, to enhance production of the peptide, or
its binding to HBV core antigen. For example, the peptides may be
optionally fused to a protein or peptide fusion partner. Thus, one
of skill in the art may design the peptide in association with a
selected fusion partner, such as another peptide, or other peptides
or proteins which impart desirable characteristics to it.
[0059] Systems for cloning and expressing HBV capsid-binding
peptides in various microorganisms and cells, including, for
example, E. coli, bacillus, streptomyces, saccharomyces, mammalian,
yeast, insect cells and plant cells, and suitable vectors therefor,
are known and available from private and public laboratories and
depositories and from commercial vendors.
[0060] Whether produced recombinantly or synthesized, the HBV
capsid-binding peptides may be purified using conventional
purification means. One of skill in the art can readily determine
the appropriate level of purity required for the desired
application for which the peptides are to be used.
[0061] It should be understood that the choice of HBV
capsid-binding peptide linker will depend, to some extent, on the
nature of the particular HBV core antigen polypeptide forming the
HBV core antigen particle. For example, HBV core antigen particles
of different original virus strains may require different HBV
capsid-binding peptide ligands, due to differing amino acid
sequences at or near the ligand binding sites of the given HBV core
antigen polypeptide.
Linkage of HBV Capsid-Binding Peptides to Immunogens
[0062] HBV capsid-binding peptides may be linked to immunogens of
interest to form a capsid-binding immunogen through a peptide bond.
Where the immunogen is itself a peptide, this will usually be
achieved conveniently by a single synthesis, or by expression of a
corresponding coding sequence, in transformed cells, of a peptide
comprising the HBV capsid-binding sequence linked to the immunogen
sequence, usually and preferably through two to five (and often
three) glycine residues, to impart a degree of flexibility between
the two components of this longer peptide. Alternatively, the
peptides may be crosslinked to the immunogens.
[0063] The orientation of the linkage between the binding component
of the peptide and immunogen may affect the efficiency of the
ultimate process for crosslinking the capsid-binding immunogen to
the HBV core antigen particle. Alternatively, among a number of
capsid-binding immunogens to be crosslinked to an HBV core antigen
particle, a given immunogen may be placed at the amino terminus of
the peptide to which it is linked, while another immunogen may be
placed at the carboxy terminus of the peptide to which it is
linked. In some instances, it may be advantageous to place the same
or different immunogens at each end of the HBV capsid-binding
peptide. Such variation in organization of the HBV capsid-binding
peptide-immunogen complexes to be crosslinked to a given HBV core
antigen particle advantageously provides highly multicomponent or
multivalent HBV core antigen particles. See FIG. 1.
[0064] Thus, orientation of the capsid-binding immunogen to be
crosslinked to the HBV core antigen particle is important to the
ultimate immunogenicity or multivalency of the resulting particle.
Higher immunogenicity or multivalency are expected when the
immunogen is oriented to the amino terminus of the HBV
capsid-binding peptide. Such orientation, which affords greater
flexibility, is also preferred for large size immunogens.
Linkage of Capsid-Binding Immunogens to the HBV Core Antigen
Particle
[0065] Capsid-binding immunogens may be crosslinked to an HBV core
antigen particle using any conventional crosslinking agent. Such
crosslinking agents include, for example, multifunctional
crosslinking agents, for example, glutaraldehyde, succinaldehyde,
octanedialdehyde and glyoxol. Additional crosslinking agents are
listed in the Pierce Catalog and Handbook, Pierce Chemical Company,
Rockford, Ill. (1997). Other crosslinking agents include those such
as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC) and N-hydroxysulphosuccinimide (sulpho-NHS), which link
adjacent primary amino and carboxyl groups to form an amide bond.
When added to a capsid-binding immunogen/HBV core antigen particle
mixture, such agents covalently crosslink an available lysine
component of the peptide to a neighboring aspartate or glutamate
from HBV core antigen.
[0066] It should also be understood that the proportion of
different immunogens attached to a given HBV core antigen particle
may, of course, be varied by the relative proportions of respective
immunogens in the mixture used for linking the capsid-binding
immunogen to the HBV core antigen particle.
Therapeutic Compositions According to this Invention
[0067] The present invention also provides compositions useful for
the therapeutic or prophylactic treatment of individuals with
multicomponent or multivalent HBV core antigen particles disclosed
herein. Any individual, including humans and other mammals, as well
as any animals, may be treated with the HBV core antigen particles
disclosed herein. Therapeutic compositions comprise a
pharmaceutically effective amount of the HBV core antigen
particles, i.e., an amount which is effective to immunize against
one or more infectious agents or to treat one or more conditions in
an individual to whom they are administered over some period of
time. Prophylactic compositions comprise a prophylactically
effective amount of the HBV core antigen particles, i.e., an amount
which is effective to prevent one or more conditions in an
individual to whom they are administered over some period of
time.
[0068] In cases in which the HBV core antigen particles contain
multiple immunogens of different types, compositions and vaccines
comprising them may be used to elicit an enhanced immune response
in an individual to each component immunogen. In cases in which the
HBV core antigen particles contain multiple immunogens of a common
type, compositions and vaccines comprising them may be used to
elicit an enhanced immune response in an individual to the common
immunogen. The latter compositions and vaccines are characterized
by enhanced monovalency and potency, as compared with conventional
monotherapies.
[0069] Compositions comprising multicomponent or multivalent HBV
core antigen particles of the invention may be administered alone,
or as part of a pharmaceutical or prophylactic preparation, with or
without adjuvant, including controlled release formulations. They
may additionally contain pharmaceutically acceptable carriers or
diluents suitable for administration for the treatment of such
infections. Suitable pharmaceutically acceptable carriers are
physiologically inert and/or non-toxic. Numerous carriers are known
in the art and may be chosen based upon the desired application.
Exemplary carriers include, but are not limited to, sterile saline,
lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, alum,
alumina, aluminum hydroxide, peptin, peanut oil, olive oil, sesame
oil and water. Additionally, the carrier or diluent may include a
time delay material, such as glycerol monosterate or glycerol
disterate, alone, or in combination with a wax. In addition,
conventional slow release polymer formulations including, for
example, soluble glasses, may be used.
[0070] Potentially, compositions comprising multicomponent or
multivalent HBV core antigen particles may contain other
therapeutic or prophylactic agents. For example, such compositions
may comprise a "cocktail" of multiple reagents useful in the
treatment, or prevention, of infection. One such cocktail may
include other reagents such as interferons, nucleoside analogs
and/or N-acetyl-cysteine.
[0071] Optionally, compositions comprising immunogenic HBV core
antigen particles may further contain immune system modifiers, such
as adjuvants or cytokines which are useful to further induce
antibody and T cell responses in the patient. Such modifiers
include conventional alum based adjuvants, or muramyl dipeptides,
preservatives, chemical stabilizers or other antigenic proteins.
Typically, stabilizers, adjuvants and preservatives, etc., are
optimized to determine the best formulation for efficacy in the
desired application. Suitable preservatives may include
chlorylbutynol, potassium sorbate, sorbic acid, sulfur dioxide,
propyl gallade, parabens, glycerine and phenol.
[0072] Suitable amounts of these compositions may be determined
based upon the level of response desired. In general, compositions
comprising immunogenic HBV core antigen particles may contain
between about 5 .mu.g and about 200 .mu.g of the particles. Such
compositions may be administered as one or a series of
inoculations, for example, three inoculations at intervals of two
to six months. Suitable dosages may also be determined by judgment
of the treating physician, taking into account factors, such as the
patient's health status, weight or age, as well as the conventional
dosage of a component immunogen, when administered as a
monotherapy. Upon improvement of a patient's condition or
likelihood of increase exposure to a given pathogen, a maintenance
dose of a composition comprising immunogenic HBV core antigen
particles may be administered, if necessary. Subsequently, the
dosage or frequency of administration, or both, may be reduced to a
level at which the desired effect is retained. At that point,
treatment should cease. Individuals may, however, require
intermittent treatment on a long-term basis upon recurrence of a
given unwanted condition.
[0073] Compositions comprising multicomponent or multivalent HBV
core antigen particles may be administered by any suitable route,
such as, for example, parenteral administration, particularly
intramuscular or subcutaneous, as well as oral administration.
Other routes, may be used, such as pulmonary, nasal, aural, anal,
dermal, ocular, intravenous, intraarterial, intraperitoneal,
mucosal, sublingual, subcutaneous and intracranial.
[0074] Immunogenic HBV core antigen particles according to this
invention may be used in the active therapy of HBV infected
individuals to inhibit, decrease, or slow the proliferation of the
virus within the body. Therapeutic compositions comprise the
immunogenic HBV core antigen particles capable of disabling,
inhibiting, or preventing the assembly mechanism of the virus. Such
therapeutic compositions may be formulated to contain carriers or
diluents, and one or more of the immunogenic HBV core antigen
particles of the invention. Such carriers and diluents are
discussed above in connection with certain other compositions, and
are identifiable by those of skill in the art.
[0075] Preparation of compositions or vaccines which contain
immunogenic HBV core antigen particles as active ingredients may be
carried out to formulate injectable compositions or vaccines,
either as liquid solutions or suspensions. Solid forms suitable for
solution or suspension in liquid prior to injection may also be
prepared. Preparations also may, in certain embodiments, be
emulsified or encapsulated in liposomes, or in soluble glasses, for
gradual release and/or prolonged delivery. Alternatively,
preparations may be in aerosol or spray form. They may also be
included in transdermal patches. The active ingredient may be mixed
with any number of excipients which are pharmaceutically acceptable
and compatible with the active ingredient or ingredients. Such
excipients include, for example, Freund's incomplete, bacterial
lipopolysaccharides, ion exchangers, alumina, aluminum stearate,
muramyl dipeptide, lecithin, buffer substances, cellulose-based
substances and polyethylene glycol.
[0076] Advantageously, vaccines comprising HBV core antigen
particles according to this invention may be combination vaccines,
comprising a number of different immunogens. Such vaccines,
include, for example, combination vaccines comprising immunogens
against two or more of: diphtheria, tetanus, acellular pertussis,
Haemophilus influenza, polio, measles, mumps, rubella, varicella,
hepatitis B virus, hepatitis A virus or pneumococcal pneumonia.
Other vaccines include those for inoculation of individuals prior
to international travel. Such vaccines include, for example,
vaccines comprising immunogens against two or more of: yellow
fever, hepatitis B virus, hepatitis A virus, typhoid fever,
meningococcal encephalitis or cholera.
[0077] Compositions comprising HBV core antigen particles according
to this invention may also be used in immunotherapeutic regiments
for desensitizing individuals to one or more allergens, such as
animal allergens, insect allergens, plant allergens, atmospheric
allergens and inhalant allergens.
[0078] According to an alternate embodiment of the present
invention, HBV core antigen particles may be used to elicit
antibodies against immunogens of interest, for use in immunotherapy
or diagnostics. For example, antibodies raised in individuals
inoculated with HBV core antigen particles may be isolated and used
in purified form. Alternatively, such antibodies or B cells from
the individual may be employed to produce monoclonal antibodies,
using conventional techniques.
Detection Methods According to this Invention
[0079] The HBV core antigen particles of the present invention may
also be used in number of conventional assay formats, particularly
immunoassay formats for diagnosis of infection or exposure to
infectious agents. Such utility is realized when the HBV
capsid-binding peptide components of the constructs of the present
invention are associated with a diagnostic label, a chemical
marker, a toxin or another protein or peptide. For example, the HBV
capsid-binding peptides may be associated with conventional labels
which are capable, alone or in combination, with other compositions
or compounds, of providing a detectable signal which would indicate
the presence of a target analyte in a sample, upon exposure to the
immunogen attached to a given HBV core antigen-binding peptide.
Such detectable labels may be selected from among numerous
compositions known and readily available to those skilled in the
art of diagnostic assays.
[0080] The invention, therefore, is not limited by the selection of
the particular assay format, and is believed to encompass assay
formats that are known to those of skill in the art. For
convenience, reagents for assays may be provided in the form of
kits. These kits can include microtiter plates to which the HBV
core antigen particles of this invention have been preadsorbed,
various diluents and buffers, labeled conjugates for the detection
of specifically bound capsid binding peptide immunogens and other
signal generating reagents, such as enzyme substrates, cofactors
and chromagens. Other components may be easily determined by those
of skill in the art.
[0081] Alternatively, HBV core antigen particles according to this
invention may be used in the immunological diagnostic tests
currently available for pathogen detection, that is
radioimmunoassay or ELISA (enzyme linked immunosorbent assay).
[0082] In one embodiment of the present invention, a sample to be
tested for the presence of antibodies to various immunogens may be
contacted with an HBV core antigen particle comprising detectably
labelled HBV capsid binding immunogens having different immunogenic
components, for a time sufficient to permit any antibodies in said
sample to form a complex with one or more of the HBV capsid binding
immunogens. Detection means may then be used the complex formed
between the capsid binding immunogen(s) and said antibodies in said
sample. A second screen may then be carried out on the sample based
on each component immunogen, to identify the specificity of the
antibodies in the sample.
[0083] In an alternate embodiment of this invention, a sample to be
tested for the presence of antibodies to a specific immunogen may
be contacted with an HBV core antigen particle comprising
detectably labelled HBV capsid binding immunogens having that
specific immunogen as their immunogenic component, for a time
sufficient to permit any antibodies in said sample to form a
complex with one or more of the HBV capsid binding immunogens. Due
to the high valency of the specific immunogen demonstrated by the
HBV core antigen particle, such a diagnostic assay is characterized
by higher sensitivity than conventional assays.
EXAMPLES
[0084] In order that the invention described herein be more fully
understood, the following examples are set forth. It should be
understood that these examples are for illustrative purposes only
and are not to be construed as limiting this invention in any
manner.
Example 1
HBV Core Antigen Preparations
[0085] Expression of either HBV core antigen (aa3-183) or
C-terminally truncated HBV core antigen aa3-148) in E. coli and
purification were performed as described in Dyson and Murray
(1995). Protein preparations were stored at 4.degree. C. as sucrose
gradient fractions in a buffer containing TBS, sucrose (20%) and
NaN.sub.3 (0.02%). Preparations were stable in this form for at
least six months.
Chemical Cross Linking of HBV Capsid-Binding Peptides to HBV Core
Antigen
[0086] The HBV capsid-binding peptide MHRSLLGRMKGA (SEQ ID NO: 40)
(Albachem, University of Edinburgh) was crosslinked to HBV core
antigen particles using 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDC) and N-hydroxysulphosuccinimide
(sulpho-NHS) (both from Pierce Europe B.V.) These reagents link
adjacent primary amino and carboxyl groups to form an amide bond.
When added to an HBV capsid-binding peptide/HBV core antigen
mixture, they should covalently crosslink the lysine from the
peptide to a neighboring aspartate or glutamate from HBV core
antigen, causing its molecular weight to increase.
[0087] More specifically, truncated HBV core antigen (15 .mu.g) was
incubated at room temperature in a buffer (30 .mu.l) containing
potassium phosphate (25 mM, pH7), NaCl (150 mM), (EDC 1.8 mM) and
sulpho-NHS (1.8 mM) in the presence or absence of the peptide
MHRSLLGRMKGA (SEQ ID NO: 40) (1 mM). After 18 h, the reaction was
analyzed by SDS/PAGE (15% w/v) as described [Sambrook et al.
(1989)]. Addition of EDC and sulpho-NHS to the peptide-HBV core
antigen particle complex resulted in a band shift corresponding to
about 1 kd, occurring on SDS-PAGE for a fraction of the HBV core
antigen. Despite runs of the reaction under various conditions, no
yield of more than 50% of the shifted protein band was obtained.
This is consistent with one peptide binding to a dimer of HBV core
antigen close to the local 2-fold axis and thus sterically blocking
binding of another peptide to the 2-fold related site.
Example 2
HBV Core Antigen Preparations
[0088] In addition to the two HBV core antigen samples prepared in
Example 1, samples of HBV core antigens with the HBV pre-S1
sequence 1-36 or the HBV surface antigen sequence 111-156 or
111-165 attached to the truncated HBV core antigen polypeptide
(truncated at residue 144) via a short linker peptide sequence were
also prepared as described by Stahl and Murray (1989).
Chemical Cross Linking of HBV Capsid-Binding Peptide to HBV Core
Antigen
[0089] The following capsid-binding immunogens, made by solid phase
synthesis, were obtained from Albachem, University of
Edinburgh:
TABLE-US-00003 AS-151: (SEQ ID NO: 42) GSLLGRMKGA GGG LDPAFRG
AS-152: (SEQ ID NO: 43) GSLLGRMKGA GGG EQKLISEEDL AS-163: (SEQ ID
NO: 44) LDPAFR GG GSLLGRMKGA AS-164: (SEQ ID NO: 45) EQKLISEEDL GG
GSLLGRMKGA,
in which the sequence GSLLGRMKGA (SEQ ID NO: 36) is the HBV
capsid-binding peptide, the sequence LDPAFR (SEQ ID NO: 46) is the
HBV pre-S1 epitope or immunogen, and the sequence EQKLISEEDL (SEQ
ID NO: 47) is the myc oncogene epitope or immunogen. These
peptides, and the basic HBV capsid-binding peptide GSLLGRMKGA (SEQ
ID NO: 36), were bound to an HBV core antigen particle separately,
or in combination, at differing concentrations, and crosslinked
with EDC or sulpho-NHS, as described in Example 1.
Properties of the Resulting HBV Core Antigen Particles
[0090] The products were analyzed by electrophoresis in acrylamide
gels, in the presence of SDS (SDS-PAGE), followed by staining by
Coomassie blue and Western blot analysis with monoclonal antibodies
and polyclonal rabbit sera raised against HBV core antigen
particles or denatured HBV surface antigen particles. Monoclonal
antibodies to each of the HBV pre-S1 epitope and the myc oncogene
epitope are available. These are, respectively, monoclonal antibody
18/7 [K. H. Heermann et al., J. Virol., 52, pp. 396-402 (1984)] and
monoclonal antibody 9E10 [Invitrogen, Catalog #R950--25].
[0091] These experiments demonstrated that products from all the
crosslinking reactions exhibited positive reactions with antibodies
against each of the constituent epitopes in the ligation reaction
components. Positive reactions were obtained with the immunogen
linked through the amino or the carboxy terminus of the ligand
peptide.
[0092] As detailed below, preparations of purified HBV core antigen
particles from reactions involving crosslinking with two or more
different immunogens, using a common HBV capsid-binding peptide
ligand, react with antibodies to all the component immunogens.
Furthermore, HBV core antigen particles precipitated with antibody
specific for one of the immunogens exhibit cross-reactivity with
antibodies to the other peptides(s) included in the ligand
crosslinking procedure.
[0093] The products of the ligation were subjected to
ultracentrifugation through sucrose gradients. They were
precipitated with one of the antibodies, the anti-myc antibody,
then analyzed by SDS-PAGE and Western blotting.
[0094] Material precipitated with one of the antibodies, for
example, anti-myc antibody, showed strong cross-reactivity with
both anti-myc and anti-pre-S1 antibody, in the Western blot.
Products precipitated with the other antibody, the anti-pre-S1
antibody, also showed the same.
[0095] In reactions in which two HBV capsid-binding peptides,
carrying different immunogens, were mixed in different proportions
for binding and crosslinked to the core particles, analysis by
SDS-PAGE and Western blotting showed that the relative intensities
of staining with the two monoclonal antibodies reflected the
proportion of the two immunogens in the mixture used for
crosslinking.
[0096] These experiments showed that at least some of the HBV core
particles resulting from the reactions had both immunogens
covalently attached to them. Since the ligand peptide binds to the
tips of the HBV core antigen particles (nucleocapsids), such
preparations will display high immunogenic potency for both
components and would be expected to elict high antibody titers in
individuals to whom they are administered.
[0097] While we have hereinbefore presented a number of embodiments
of this invention, it is apparent that our basic construction can
be altered to provide other embodiments which utilize the process
of this invention. Therefore, it will be appreciated that the scope
of this invention is to be defined by the claims appended hereto
rather than the specific embodiments which have been presented
hereinbefore by way of example.
Sequence CWU 1
1
4719PRTHuman immunodeficiency virus 1Gly Glu Leu Asp Arg Trp Glu
Lys Ile1 527PRTHuman immunodeficiency virus 2Glu Leu Asp Lys Trp
Ala Ser1 5312PRTHuman immunodeficiency virus 3Ile Gly Pro Gly Arg
Ala Phe Tyr Thr Thr Lys Asn1 5 1046PRTHuman immunodeficiency virus
4Glu Leu Asp Lys Trp Ala1 559PRTHuman immunodeficiency virus 5Asp
Arg Phe Tyr Lys Thr Leu Arg Ala1 569PRTArtificial
SequenceDescription of Artificial SequenceHBV capsid binding
peptide 6Ser Leu Leu Gly Arg Met Lys Gly Ala1 576PRTArtificial
SequenceDescription of Artificial SequenceHBV capsid binding
peptide 7Leu Leu Gly Arg Met Lys1 5810PRTArtificial
SequenceMOD_RES(9)bAla 8Ser Leu Leu Gly Arg Met Lys Gly Xaa Cys1 5
10910PRTArtificial SequenceDescription of Artificial SequenceHBV
capsid-binding peptide 9Arg Ser Leu Leu Gly Arg Met Lys Gly Ala1 5
101011PRTArtificial SequenceDescription of Artificial SequenceHBV
capsid-binding peptide 10His Arg Ser Leu Leu Gly Arg Met Lys Gly
Ala1 5 101112PRTArtificial SequenceDescription of Artificial
SequenceHBV capsid-binding peptide 11Arg Ser Leu Leu Gly Arg Met
Lys Gly Ala Xaa Cys1 5 10128PRTArtificial SequenceDescription of
Artificial SequenceHBV capsid-binding peptide 12Ala Leu Leu Gly Arg
Met Lys Gly1 51312PRTArtificial SequenceDescription of Artificial
SequenceHBV capsid-binding peptide 13Ala Asp Gly Ala Leu Leu Gly
Arg Met Lys Gly Ala1 5 101412PRTArtificial SequenceDescription of
Artificial SequenceHBV capsid-binding peptide 14Ala Asp Gly Ala Leu
Leu Gly Arg Met Lys Pro Ala1 5 101512PRTArtificial
SequenceDescription of Artificial SequenceHBV capsid-binding
peptide 15Ala Asp Gly Ser Leu Leu Gly Arg Met Lys Pro Ala1 5
101612PRTArtificial SequenceDescription of Artificial SequenceHBV
capsid-binding peptide 16Ala Asp Gly Ala Leu Leu Gly Arg Met Lys
Arg Ala1 5 101712PRTArtificial SequenceDescription of Artificial
SequenceHBV capsid-binding peptide 17Ala Asp Gly Thr Leu Leu Gly
Arg Met Lys Leu Ala1 5 101812PRTArtificial SequenceDescription of
Artificial SequenceHBV capsid-binding peptide 18Ala Asp Gly Ser Leu
Leu Gly Arg Met Lys Gly Ala1 5 101912PRTArtificial
SequenceDescription of Artificial SequenceHBV capsid-binding
peptide 19Ala Asp Arg Ser Leu Leu Gly Arg Met Lys Gly Ala1 5
102015PRTArtificial SequenceDescription of Artificial SequenceHBV
capsid-binding peptide 20Ala Asp Gly Ser Arg Ser Ser Leu Leu Gly
Arg Met Lys Gly Ala1 5 10 152115PRTArtificial SequenceDescription
of Artificial SequenceHBV capsid-binding peptide 21Ala Asp Gly Ala
His Ser Ser Leu Leu Gly Arg Met Lys Gly Ala1 5 10
152215PRTArtificial SequenceDescription of Artificial SequenceHBV
capsid-binding peptide 22Ala Asp Gly His Arg Ser Ser Leu Leu Gly
Arg Met Lys Gly Ala1 5 10 152315PRTArtificial SequenceDescription
of Artificial SequenceHBV capsid-binding peptide 23Ala Asp Gly Pro
Arg Ser Ser Leu Leu Gly Arg Met Lys Gly Ala1 5 10
152415PRTArtificial SequenceDescription of Artificial SequenceHBV
capsid-binding peptide 24Ala Asp Gly Ala His Arg Ser Leu Leu Gly
Arg Met Lys Gly Ala1 5 10 152515PRTArtificial SequenceDescription
of Artificial SequenceHBV capsid-binding peptide 25Ala Asp Gly Tyr
Gln Arg Ser Leu Leu Gly Arg Met Lys Gly Ala1 5 10
152615PRTArtificial SequenceDescription of Artificial SequenceHBV
capsid-binding peptide 26Ala Asp Gly Thr Gln Arg Ser Leu Leu Gly
Arg Met Lys Gly Ala1 5 10 152715PRTArtificial SequenceDescription
of Artificial SequenceHBV capsid-binding peptide 27Ala Asp Gly Met
His Arg Ser Leu Leu Gly Arg Met Lys Gly Ala1 5 10
15287PRTArtificial SequenceDescription of Artificial SequenceHBV
capsid-binding peptide 28Leu Leu Gly Arg Met Lys Gly1
5296PRTArtificial SequenceDescription of Artificial SequenceHBV
capsid-binding peptide 29Leu Gly Arg Met Lys Gly1 5305PRTArtificial
SequenceDescription of Artificial SequenceHBV capsid-binding
peptide 30Gly Arg Met Lys Gly1 5315PRTArtificial
SequenceDescription of Artificial SequenceHBV capsid-binding
peptide 31Leu Leu Gly Arg Met1 5328PRTArtificial
SequenceDescription of Artificial SequenceHBV capsid-binding
peptide 32Cys Leu Leu Gly Arg Met Lys Cys1 5338PRTArtificial
SequenceDescription of Artificial SequenceHBV capsid-binding
peptide 33Ala Leu Leu Pro Arg Met Lys Gly1 5348PRTArtificial
SequenceDescription of Artificial SequenceHBV capsid-binding
peptide 34Ser Leu Leu Gly Arg Met Lys Gly1 5357PRTArtificial
SequenceDescription of Artificial SequenceHBV capsid-binding
peptide 35Ser Leu Leu Gly Arg Met Lys1 53610PRTArtificial
SequenceDescription of Artificial SequenceHBV capsid-binding
peptide 36Gly Ser Leu Leu Gly Arg Met Lys Gly Ala1 5
103712PRTArtificial SequenceDescription of Artificial SequenceHBV
capsid-binding peptide 37Asp Gly Ser Leu Leu Gly Arg Met Lys Gly
Ala Ala1 5 103814PRTArtificial SequenceDescription of Artificial
SequenceHBV capsid-binding peptide 38Ala Asp Gly Ser Leu Leu Gly
Arg Met Lys Gly Ala Ala Gly1 5 103910PRTArtificial
SequenceDescription of Artificial SequenceHBV capsid-binding
peptide 39Ala Cys Ser Leu Leu Gly Arg Met Lys Gly1 5
104012PRTArtificial SequenceDescription of Artificial SequenceHBV
capsid-binding peptide 40Met His Arg Ser Leu Leu Gly Arg Met Lys
Gly Ala1 5 104116PRTArtificial SequenceMOD_RES(14)bAla 41Met His
Arg Ser Leu Leu Gly Arg Met Lys Gly Ala Gly Xaa Gly Cys1 5 10
154220PRTArtificial SequenceDescription of Artificial SequenceHBV
capsid-binding peptide 42Gly Ser Leu Leu Gly Arg Met Lys Gly Ala
Gly Gly Gly Leu Asp Pro1 5 10 15Ala Phe Arg Gly 204323PRTArtificial
SequenceDescription of Artificial SequenceHBV capsid-binding
peptide 43Gly Ser Leu Leu Gly Arg Met Lys Gly Ala Gly Gly Gly Glu
Gln Lys1 5 10 15Leu Ile Ser Glu Glu Asp Leu 204418PRTArtificial
SequenceDescription of Artificial SequenceHBV capsid-binding
peptide 44Leu Asp Pro Ala Phe Arg Gly Gly Gly Ser Leu Leu Gly Arg
Met Lys1 5 10 15Gly Ala4522PRTArtificial SequenceDescription of
Artificial Sequencemyc oncogene epitope or immunogen 45Glu Gln Lys
Leu Ile Ser Glu Glu Asp Leu Gly Gly Gly Ser Leu Leu1 5 10 15Gly Arg
Met Lys Gly Ala 20466PRTArtificial SequenceDescription of
Artificial SequenceHBV pre-S1 epitope or immunogen 46Leu Asp Pro
Ala Phe Arg1 54710PRTArtificial SequenceDescription of Artificial
Sequencemyc oncogene epitope or immunogen 47Glu Gln Lys Leu Ile Ser
Glu Glu Asp Leu1 5 10
* * * * *