Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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CIRCULAR CCR5 PEPTIDE CONJUGATES AND USES THEREOF
BACKGROUND OF THE INVENTION
Field of the Invention
[0001] The present invention is in the fields of medicine, public health,
immunology,
molecular biology and virology. The invention provides composition comprising
a virus-like
particle (VLP) linked to at least one antigen, wherein said antigen is CCR5
PNt domain
comprising one looped peptidic structure.
[0002] The invention also provides a process for producing the composition.
The
compositions of this invention are useful in the production of vaccines, in
particular, for the
prevention and treatment of HIV infection. Moreover, the compositions of the
invention
induce efficient immune responses, in particular antibody responses.
Related Art
[0003] HIV R5 strains use the cell surface molecules CD4 and CCR5 for
attachment
and entry into macrophages and CD4+ T cells. CCR5 is a 7-transmembrane
receptor with an
N-terminal sequence and three loops exposed to the extracellular space, which
are called
subsequently PNt, ECL-1, ECL-2, and ECL-3, respectively. The natural CCR5
ligands,
RANTES, MIP-la, MIP-1B and analogs thereof are able to block the virus-
coreceptor
interaction and further cause the internalization of CCR5 (Lederman et al.,
2004, Science 306,
p485). CCR5 specific auto-antibodies have been found in 12.5% women that were
repeatedly
exposed to HIV but remained uninfected (Lopalco et al., 2000, J. Immunology
164, 3426).
These antibodies were shown to bind the first extracellular loop (ECL-1) of
CCR5 and could
inhibit R5-tropic HIV infection of peripheral blood mononuclear cells (PBMC).
Alloimmunisation in women led to CCR5 specific antibodies that were capable of
inhibiting
R5-HIV infection in vitro (Wang et al., 2002, Clin. Exp. Immunol. 129, 493).
[0004] Monoclonal a-CCR5 antibodies are able to prevent HIV infection in vitro
(Olson et al., 1999, J. Virol. 73, 4145; Wu and LaRosa et al., 1997, J. Exp.
Med. 186, 1373).
Antibodies produced by immunizing monkeys with linear CCR5 peptides (from the
N-
terminal, the ECL-l, or the ECL-2 sequence) have viral inhibitory effect in
vitro (Lehner et
al., 2001, J. Immunology 166, 7446). The CCR5 PNt domain was displayed on
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papillomavirus like particles and immunized pig-tailed macaques. This vaccine
induced high-
avidity anti-CCR5 IgG autoantibody responses, and all five immunized macaques
generated
IgG that could block infection of CCR5-tropic simian/human immunodeficiency
virus SHIV
SF 162P3 in vitro (Chackerian et al., 2004, J. Virol. 78, 4037).
SUMMARY OF THE INVENTION
[0005] We have, now, surprisingly found that the inventive compositions and
vaccines, respectively, comprising at least one CCR5 PNt domain comprising one
looped
peptidic structure linked to a virus-like particle, are capable of inducing
immune responses, in
particular antibody responses, leading to high antibody titer against CCR5.
Moreover, we
have surprisingly found that inventive compositions and vaccines,
respectively, are capable of
inducing immune responses, in particular antibody responses, with protective
and/or
therapeutic effect against the HIV infection. This indicates that the immune
responses, in
particular the antibodies generated by the inventive compositions and
vaccines, respectively,
are, thus, capable of specifically recognizing HIV and/or HIV infected cells
in vivo, and
neutralizing and inhibiting the infection of the virus.
[0006] Thus, in the first aspect, the present invention provides a composition
comprising: (a) a virus-like particle with at least one first attachment site;
(b) at least one
antigenic peptide with at least one second attachment site, wherein said
antigenic peptide
comprises: (i) CCR5 PNt domain comprising an amino acid sequence KINVK (SEQ ID
NO:25); (ii) a first amino acid comprising a first reactive group and wherein
said first amino
acid is located at the N-terminal of KINVK; (iii) a second amino acid
comprising a second
reactive group and wherein said second amino acid is located at the C-terminal
of KINVK,
wherein said first and said second amino acid does not comprise said second
attachment site
linking said first attachment site; wherein said first reactive group binds to
said second
reactive group by at least one covalent bond so that the peptide starting from
said first amino
acid and ending with said second amino acid is looped; and wherein said virus-
like particle
and said at least one antigenic peptide are linked through said first and said
second attachment
site.
[0007] In one preferred embodiment of the invention, the virus-like particle
is a VLP
of an RNA-bacteriophage. In one preferred embodiment, the virus-like particle
suitable for
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use in the present invention comprises recombinant protein, preferably
recombinant coat
protein, mutants or fragments thereof, of a virus, preferably of an RNA
bacteriophage.
[0008] In another aspect, the present invention provides a method of
preventing and/or
treating HIV infection, wherein the method comprises administering the
inventive
composition or the inventive vaccine composition, respectively, to a human.
BRIEF DESCRIPTION OF THE FIGURES
[0009] FIG. 1 The antigenic peptides coupled to Q(3 shown by coomassie stained
SDS-PAGE. Lane 1: marker bands, Lane 2 and 4: Q(3-SMPH; lane 3: Q(3-P36; and
lane 5:
Q(3-P37.
[0010] FIG. 2 CCR5+ cells in a FACS staining assay. Individual sera from mice
immunized with Q(3-P36 (day 21) (FIG. 2A) or Q(3-P37 (day 65) (FIG. 2B) were
tested for
staining CCR5+ cells in a FACS staining assay. Analysed was the percentage of
live
(propidium negative) cells to which the sera bound. A serum was considered
positive, if more
than 10% cells were stained CCR5+ by the serum. Monoclonal antibodies 45531
and 2D7
were used as positive controls.
[0011] FIG. 3 HIV neutralisation assay. R5 tropic pseudotype HIV viruses were
used
to infect human CD8 depleted PBMC cells. The y-axis represents the
concentration of
antibodies needed for 50% (black bar), 70% (white bar) or 90% (stripe bar)
neutralization.
Mab PA14 was used as appositive control and Total IgG raised against Qb was
used as a
negative control.
[0012] FIG. 4 Sequence of P16 peptide indicating the reactive groups and the
looped
structures.
DETAILED DESCRIPTION OF THE INVENTION
[0013] Unless defined otherwise, all technical and scientific terms used
herein have
the same meanings as commonly understood by one of ordinary skill in the art
to which this
invention belongs.
[0014] Antigen: As used herein, the term "antigen" refers to a molecule
capable of being
bound by an antibody or a T cell receptor (TCR) if presented by MHC molecules.
The term
"antigen", as used herein, also encompasses T-cell epitopes. An antigen is
additionally
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capable of being recognized by the immune system and/or being capable of
inducing a
humoral immune response and/or cellular immune response leading to the
activation of B-
and/or T-lymphocytes. This may, however, require that, at least in certain
cases, the antigen
contains or is linked to a Th cell epitope and is given in adjuvant. An
antigen can have one or
more epitopes (B- and T- epitopes). The specific reaction referred to above is
meant to
indicate that the antigen will preferably react, typically in a highly
selective manner, with its
corresponding antibody or TCR and not with the multitude of other antibodies
or TCRs which
may be evoked by other antigens. Antigens as used herein may also be mixtures
of several
individual antigens.
[0015] Antigenic site: The term "antigenic site" and the term "antigenic
epitope",
which are used herein interchangeably, refer to continuous or discontinuous
portions of a
polypeptide, which can be bound immunospecifically by an antibody or by a T-
cell receptor
within the context of an MHC molecule. Immunospecific binding excludes non-
specific
binding but does not necessarily exclude cross-reactivity. Antigenic site
typically comprise 5-
amino acids in a spatial conformation which is unique to the antigenic site.
[0016] Associated: The term "associated" (or its noun association) as used
herein
refers to all possible ways, preferably chemical interactions, by which two
molecules are
joined together. Chemical interactions include covalent and non-covalent
interactions. Typical
examples for non-covalent interactions are ionic interactions, hydrophobic
interactions or
hydrogen bonds, whereas covalent interactions are based, by way of example, on
covalent
bonds such as ester, ether, phosphoester, amide, peptide, carbon-phosphorus
bonds, carbon-
sulfur bonds such as thioether, or imide bonds.
[0017] Attachment Site, First: As used herein, the phrase "first attachment
site" refers
to an element which is naturally occurring with the VLP or which is
artificially added to the
VLP, and to which the second attachment site may be linked. The first
attachment site may be
a protein, a polypeptide, an amino acid, a peptide, a sugar, a polynucleotide,
a natural or
synthetic polymer, a secondary metabolite or compound (biotin, fluorescein,
retinol,
digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a chemically
reactive group such
as an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, a
guanidinyl
group, histidinyl group, or a combination thereof. A preferred embodiment of a
chemically
reactive group being the first attachment site is the amino group of an amino
acid such as
lysine. The first attachment site is located, typically on the surface, and
preferably on the
outer surface of the VLP. Multiple first attachment sites are present on the
surface, preferably
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on the outer surface of virus-like particle, typically in a repetitive
configuration. In a preferred
embodiment the first attachment site is associated with the VLP, through at
least one covalent
bond, preferably through at least one peptide bond. In a further preferred
embodiment the first
attachment site is naturally occurring with the VLP. Alternatively, in another
preferred
embodiment the first attachment site is artificially added to the VLP.
[0018] Attachment Site, Second: As used herein, the phrase "second attachment
site"
refers to an element which is naturally occurring with or which is
artificially added to the
antigenic peptide of the invention and to which the first attachment site may
be linked. The
second attachment site may be a protein, a polypeptide, a peptide, an amino
acid, a sugar, a
polynucleotide, a natural or synthetic polymer, a secondary metabolite or
compound (biotin,
fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonylfluoride),
or a chemically
reactive group such as an amino group, a carboxyl group, a sulfhydryl group, a
hydroxyl
group, a guanidinyl group, histidinyl group, or a combination thereof. A
preferred
embodiment of a chemically reactive group being the second attachment site is
a sulfhydryl
group, preferably a sulfhydryl group of cysteine. The term "antigenic peptide
with at least one
second attachment site", as used herein, refers, to a construct comprising the
antigenic peptide
and at least one second attachment site. In one preferred embodiment, the
second attachment
site is naturally occurring within the antigenic peptide. In another preferred
embodiment, the
second attachment site is artificially added to the antigenic peptide. In one
preferred
embodiment the second attachment site is associated with the antigenic peptide
through at
least one covalent bond, preferably through at least one peptide bond. In one
preferred
embodiment, the antigenic peptide with at least one second attachment site
further comprises
a linker, preferably said linker comprises at least one second attachment
site, preferably said
linker is fused to the antigenic peptide by a peptide bond.
[0019] Bound: As used herein, the term "bound" refers to binding or attachment
that
may be covalent, e.g., by chemically coupling, or non-covalent, e.g., ionic
interactions,
hydrophobic interactions, hydrogen bonds, etc. Preferably the term "bound"
refers to binding
or attachment that is covalent. Covalent bonds can be, for example, ester,
ether, phosphoester,
amide, peptide, imide, carbon-sulfur bonds, carbon-phosphorus bonds, and the
like.
[0020] CCR5 PNt domain: The term "CCR5 PNt domain", as used herein, should
encompass any polypeptide comprising, consisting essentially of, or
alternatively or
preferably consisting of human CCR5 PNt domain of SEQ ID NO:22. Moreover, the
term
"CCR5 PNt domain", as used herein, should also encompass any polypeptide
comprising,
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consisting essentially of, or alternatively or preferably consisting of, any
natural or genetically
engineered variant having more than 70%, preferably more than 80%, preferably
more than
85%, even more preferably more than 90%, even more preferably more than 93%,
again more
preferably more than 95%, and most preferably more than 97% amino acid
sequence identity
with the CCR5 PNt domain as defined above with the proviso that said
polypeptide comprises
the sequence KINVK (SEQ ID NO:25). The term "CCR5 PNt domain" as used herein
should
furthermore encompass post-translational modifications including but not
limited to
glycosylations, acetylations, phosphorylations of the CCR5 PNt domain as
defined above.
Preferably the CCR5 CCR5 PNt domain, as defined herein, consists of at most
50, even more
preferably at most 40 amino acids in length. Typically and preferably, CCR5
PNt domain,
preferably when linked to the virus-like particle of the invention, is capable
of inducing in
vivo the production of antibody specifically binding to CCR5.
[0021] Coat protein: The term "coat protein" and the interchangeably used term
"capsid protein" within this application, refers to a viral protein,
preferably a subunit of a
natural capsid of a virus, preferably of an RNA-bacteriophage, which is
capable of being
incorporated into a virus capsid or a VLP.
[0022] Linked: The term "linked" (or its noun: linkage) as used herein, refers
to all
possible ways, preferably chemical interactions, by which the at least one
first attachment site
and the at least one second attachment site are joined together. Chemical
interactions include
covalent and non-covalent interactions. Typical examples for non-covalent
interactions are
ionic interactions, hydrophobic interactions or hydrogen bonds, whereas
covalent interactions
are based, by way of example, on covalent bonds such as ester, ether,
phosphoester, amide,
peptide, carbon-phosphorus bonds, carbon-sulfur bonds such as thioether, or
imide bonds. In
certain preferred embodiments the first attachment site and the second
attachment site are
linked through at least one covalent bond, preferably through at least one non-
peptide bond,
and even more preferably through exclusively non-peptide bond(s). The term
"linked" as used
herein, however, shall not only encompass a direct linkage of the at least one
first attachment
site and the at least one second attachment site but also, alternatively and
preferably, an
indirect linkage of the at least one first attachment site and the at least
one second attachment
site through intermediate molecule(s), and hereby typically and preferably by
using at least
one, preferably one, heterobifunctional cross-linker.
[0023] Linker: A "linker", as used herein, either associates the second
attachment site
with the antigenic peptide or already comprises, essentially consists of, or
consists of the
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second attachment site. Preferably, a "linker", as used herein, already
comprises the second
attachment site, typically and preferably - but not necessarily - as one amino
acid residue,
preferably as a cysteine residue. A "linker" as used herein is also termed
"amino acid linker",
in particular when a linker according to the invention contains at least one
amino acid residue.
Thus, the terms "linker" and "amino acid linker" are interchangeably used
herein. However,
this does not imply that such a linker consists exclusively of amino acid
residues, even if a
linker consisting of amino acid residues is a preferred embodiment of the
present invention.
The amino acid residues of the linker are, preferably, composed of naturally
occurring amino
acids or unnatural amino acids known in the art, all-L or all-D or mixtures
thereof. Further
preferred embodiments of a linker in accordance with this invention are
molecules comprising
a sulfhydryl group or a cysteine residue and such molecules are, therefore,
also encompassed
within this invention. Further linkers useful for the present invention are
molecules
comprising a Cl-C6 alkyl-, a cycloalkyl such as a cyclopentyl or cyclohexyl, a
cycloalkenyl,
aryl or heteroaryl moiety. Moreover, linkers comprising preferably a Cl-C6
alkyl-,
cycloalkyl- (C5, C6), aryl- or heteroaryl- moiety and additional amino acid(s)
can also be
used as linkers for the present invention and shall be encompassed within the
scope of the
invention. Association of the linker with the antigenic peptide is preferably
by way of at least
one covalent bond, more preferably by way of at least one peptide bond. In
case of a second
attachment site not naturally occurring with the antigenic peptide, the linker
is associated to
the at least one second attachment site, for example, a cysteine, preferably,
by way of at least
one covalent bond, more preferably by way of at least one peptide bond.
[0024] Ordered and repetitive antigen array: As used herein, the term "ordered
and
repetitive antigen array" generally refers to a repeating pattern of antigen
or, characterized by
a typically and preferably high order of uniformity in spacial arrangement of
the antigens with
respect to virus-like particle, respectively. In one embodiment of the
invention, the repeating
pattern may be a geometric pattern. Certain embodiments of the invention, such
as VLP of
RNA-bacteriophage s, are typical and preferred examples of suitable ordered
and repetitive
antigen arrays which, moreoever, possess strictly repetitive paracrystalline
orders of antigens,
preferably with spacings of 1 to 30 nanometers, preferably 2 to 15 nanometers,
even more
preferably 2 to 10 nanometers, even again more preferably 2 to 8 nanometers,
and further
more preferably 1.6 to 7 nanometers.
[0025] Polypeptide: The term "polypeptide" as used herein refers to a molecule
composed of monomers (amino acids) linearly linked by amide bonds (also known
as peptide
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bonds). It indicates a molecular chain of amino acids and does not refer to a
specific length of
the product. Thus, peptides, dipeptides, tripeptides, oligopeptides and
proteins are included
within the definition of polypeptide. Post-translational modifications of the
polypeptide, for
example, glycosylations, acetylations, phosphorylations, and the like are also
encompassed.
[0026] Recombinant VLP: The term "recombinant VLP", as used herein, refers to
a VLP
that is obtained by a process which comprises at least one step of recombinant
DNA
technology. The term "VLP recombinantly produced", as used herein, refers to a
VLP that is
obtained by a process which comprises at least one step of recombinant DNA
technology.
Thus, the terms "recombinant VLP" and "VLP recombinantly produced" are
interchangeably
used herein and should have the identical meaning.
[0027] Reactive group first/second: the term "the first/second reactive group"
refers to
part of the side chain of the first/second amino acid of the antigenic peptide
of the invention,
which associates to "the second/first reactive group" of the second/first
amino acid of the
antigenic peptide of the invention, either directly or preferably through at
least one, preferably
only one intermediate molecule. Part of the side chain that may serve as the
first or second
reactive group include thiol (C), amine (K), amido (QQ, arginine (R),
carboxylic acid (DE),
alcohol (ST), thioether (M), imidazol (K), phenyl (F), phenol (Y), indole (W),
and aliphatic
(AVILP).
[0028] Virus particle: The term "virus particle" as used herein refers to the
morphological form of a virus. In some virus types it comprises a genome
surrounded by a
protein capsid; others have additional structures (e.g., envelopes, tails,
etc.).
[0029] Virus-like particle (VLP), as used herein, refers to a non-replicative
or non-
infectious, preferably a non-replicative and non-infectious virus particle, or
refers to a non-
replicative or non-infectious, preferably a non-replicative and non-infectious
structure
resembling a virus particle, preferably a capsid of a virus. The term "non-
replicative", as used
herein, refers to being incapable of replicating the genome comprised by the
VLP. The term
"non-infectious", as used herein, refers to being incapable of entering the
host cell. Preferably
a virus-like particle in accordance with the invention is non-replicative
and/or non-infectious
since it lacks all or part of the viral genome or genome function due to
physical, chemical
inactivation or due to genetic manipulation. Typically and preferably a virus-
like particle
lacks all or part of the replicative and infectious components of the viral
genome. A virus-like
particle in accordance with the invention may contain nucleic acid distinct
from their genome.
A typical and preferred embodiment of a virus-like particle in accordance with
the present
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invention is a viral capsid such as the viral capsid of the corresponding
virus, bacteriophage,
preferably RNA-bacteriophage. The terms "viral capsid" or "capsid", refer to a
macromolecular assembly composed of viral protein subunits. Typically, there
are 60, 120,
180, 240, 300, 360 and more than 360 viral protein subunits. Typically and
preferably, the
interactions of these subunits lead to the formation of viral capsid or viral-
capsid like structure
with an inherent repetitive organization, wherein said structure is,
typically, spherical or
tubular. The term "capsid-like structure" as used herein, refers to a
macromolecular assembly
composed of viral protein subunits resembling the capsid morphology in the
above defined
sense but deviating from the typical symmetrical assembly while maintaining a
sufficient
degree of order and repetitiveness.
[0030] One common feature of virus particle and virus-like particle is its
highly
ordered and repetitive arrangement of its subunits.
[0031] Virus-like particle of an RNA-bacteriophage: As used herein, the term
"virus-
like particle of an RNA-bacteriophage" refers to a virus-like particle
resembles the structure
of an RNA-bacteriophage, being non replicative or non-infectious, and
typically and
preferably being non replicative and non-infectious. Typically and preferably,
the term "virus-
like particle of an RNA-bacteriophage" should furthermore refer to a virus-
like particle of an
RNA-bacteriophage which lacks at least one of the genes, preferably all of the
genes,
encoding for the replication machinery of the RNA-bacteriophage, and typically
and further
preferably even at least one of the genes, preferably all of the genes,
encoding the protein or
proteins responsible for viral attachment to or entry into the host. This
definition should,
however, also encompass virus-like particles of RNA-bacteriophages, in which
the
aforementioned gene or genes are still present but inactive, and, therefore,
also leading to non-
replicative and/or noninfectious virus-like particles of an RNA-bacteriophage.
Moreover, the
term "virus-like particle of an RNA-bacteriophage" should therefore also
encompass in its
broadest definition a virus particle of an RNA-bacteriophage, the genome of
which has been
inactivated by physical or chemical or genetic methods so that the virus
particle is not capable
of infecting and/or replicating. Preferred VLPs derived from RNA-
bacteriophages exhibit
icosahedral symmetry and consist of 180 subunits. Within this present
disclosure the term
"subunit" and "monomer" are interexchangeably and equivalently used within
this context.
[0032] One, a, or an: when the terms "one", "a", or "an" are used in this
disclosure,
they mean "at least one" or "one or more" unless otherwise indicated.
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[0033] Within this application, antibodies are defined to be specifically
binding if they
bind to the antigen with a binding affinity (Ka) of 106 M-1 or greater,
preferably 107 M-1 or
greater, more preferably 108 M-1 or greater, and most preferably 109 M-1 or
greater. The
affinity of an antibody can be readily determined by one of ordinary skill in
the art (for
example, by Scatchard analysis.)
[0034] The amino acid sequence identity of polypeptides can be determined
conventionally using known computer programs such as the Bestfit program. When
using
Bestfit or any other sequence alignment program, preferably using Bestfit, to
determine
whether a particular sequence is, for instance, 95% identical to a reference
amino acid
sequence, the parameters are set such that the percentage of identity is
calculated over the full
length of the reference amino acid sequence and that gaps in homology of up to
5% of the
total number of amino acid residues in the reference sequence are allowed.
This
aforementioned method in determining the percentage of identity between
polypeptides is
applicable to all proteins, polypeptides or a fragment thereof disclosed in
this invention.
[0035] Conservative amino acid substitutions, as understood by a skilled
person in the
art, include isosteric substitutions, substitutions where the charged, polar,
aromatic, aliphatic
or hydrophobic nature of the amino acid is maintained. Typical conservative
amino acid
substitutions are substitutions between amino acids within one of the
following groups: Gly,
Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr, Cys; Lys, Arg; and Phe and
Tyr.
[0036] In one aspect, the invention provides a composition comprising: (a) a
virus-like
particle with at least one first attachment site; (b) at least one antigenic
peptide with at least
one second attachment site, wherein said antigenic peptide comprises: (i) CCR5
PNt domain
comprising an amino acid sequence KINVK (SEQ ID NO:25); (ii) a first amino
acid
comprising a first reactive group and wherein said first amino acid is located
at the N-terminal
of KINVK; (iii) a second amino acid comprising a second reactive group and
wherein said
second amino acid is located at the C-terminal of KINVK, wherein said first
and said second
amino acid does not comprise said second attachment site linking said first
attachment site;
wherein said first reactive group binds to said second reactive group by at
least one covalent
bond so that the peptide starting from said first amino acid and ending with
said second amino
acid is looped; and wherein said virus-like particle and said at least one
antigenic peptide are
linked through said first and said second attachment site.
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[0037] Any virus known in the art having an ordered and repetitive structure
may be
selected as a VLP of the invention. Illustrative DNA or RNA viruses, the coat
or capsid
protein of which can be used for the preparation of VLPs have been disclosed
in WO
2004/009124 on page 25, line 10-21, on page 26, line 11-28, and on page 28,
line 4 to page
31, line 4. These disclosures are incorporated herein by way of reference.
[0038] Virus or virus-like particle can be produced and purified from virus-
infected
cell culture. The resulting virus or virus-like particle for vaccine purpose
needs to be devoid
of virulence. Besides genetic engineering, physical or chemical methods can be
employed to
inactivate the viral genome function, such as UV irradiation, formaldehyde
treatment.
[0039] In one preferred embodiment, the VLP is a recombinant VLP. Almost all
commonly known viruses have been sequenced and are readily available to the
public. The
gene encoding the coat protein can be easily identified by a skilled artisan.
The preparation of
VLPs by recombinantly expressing the coat protein in a host is within the
common knowledge
of a skilled artisan.
[0040] In one preferred embodiment, the virus-like particle comprises, or
alternatively
consists of, recombinant proteins or preferably coat proteins, mutants or
fragments thereof, of
a virus selected form the group consisting of: a) RNA-bacteriophage s; b)
bacteriophage; c)
Hepatitis B virus, preferably its capsid protein (Ulrich, et al., Virus Res.
50:141-182 (1998))
or its surface protein (WO 92/11291); d) measles virus (Warnes, et al., Gene
160:173-178
(1995)); e) Sindbis virus; f) rotavirus (US 5,071,651 and US 5,374,426); g)
foot-and-mouth-
disease virus (Twomey, et al., Vaccine 13:1603 1610, (1995)); h) Norwalk virus
(Jiang, X., et
al., Science 250:1580 1583 (1990); Matsui, S.M., et al., J. Clin. Invest.
87:1456 1461 (1991));
i) Alphavirus; j) retrovirus, preferably its GAG protein (WO 96/30523); k)
retrotransposon
Ty, preferably the protein pl; 1) human Papilloma virus (WO 98/15631); m)
Polyoma virus;
n) Tobacco mosaic virus; and o) Flock House Virus p) Cowpea Chlorotic Mottle
Virus; q) a
Cowpea Mosaic Virus; and r) an Alfalfa Mosaic Virus.
[0041] Assembly of the fragment or mutant of recombinant protein or coat
protein
into a VLP may be tested, as one skilled in the art would appreciate by
expressing the protein
in E.coli, optionally purifying the capsids by gel filtration from cell
lysate, and analysing the
capsid formation in an immunodiffusion assay (Ouchterlony test) or by Electron
Microscopy
(EM) (Kozlovska, T. M.. et al., Gene 137:133-37 (1993)). Immunodiffusion
assays and EM
may be directly performed on cell lysate.
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12
[0042] In one preferred embodiment, the VLP comprises, or consists of, more
than
one amino acid sequence, preferably two amino acid sequences, of the
recombinant proteins,
mutants or fragments thereof. VLP comprises or consists of more than one amino
acid
sequence is referred, in this application, as mosaic VLP.
[0043] The term "fragment of a recombinant protein" or the term "fragment of a
coat
protein", as used herein, is defined as a polypeptide, which is of at least
70%, preferably at
least 80%, more preferably at least 90%, even more preferably at least 95% the
length of the
wild-type recombinant protein, or coat protein, respectively and which
preferably retains the
capability of forming VLP. Preferably the fragment is obtained by at least one
internal
deletion, at least one truncation or at least one combination thereof. The
term "fragment of a
recombinant protein" or "fragment of a coat protein" shall further encompass
polypeptide,
which has at least 80%, preferably 90%, even more preferably 95% amino acid
sequence
identity with the "fragment of a recombinant protein" or "fragment of a coat
protein",
respectively, as defined above and which is preferably capable of assembling
into a virus-like
particle.
[0044] The term "mutant recombinant protein" or the term "mutant of a
recombinant
protein" as interchangeably used in this invention, or the term "mutant coat
protein" or the
term "mutant of a coat protein", as interchangeably used in this invention,
refers to a
polypeptide having an amino acid sequence derived from the wild type
recombinant protein,
or coat protein, respectively, wherein the amino acid sequence is at least
80%, preferably at
least 85%, 90%, 95%, 97%, or 99% identical to the wild type sequence and
preferably retains
the ability to assemble into a VLP.
[0045] In one preferred embodiment, the virus-like particle of the invention
is a virus-
like particle of a Hepatitis B virus. The preparation of Hepatitis B virus-
like particles have
been disclosed, inter alia, in WO 00/32227, WO 01/85208 and in WO 01/056905.
All three
documents are explicitly incorporated herein by way of reference. Other
variants of HBcAg
suitable for use in the practice of the present invention have been disclosed
in page 34-39 WO
01/056905.
[0046] In one further preferred embodiments of the invention, a lysine residue
is
introduced into the HBcAg polypeptide, to mediate the linking of the antigenic
peptide of the
invention to the VLP of HBcAg. In preferred embodiments, VLPs and compositions
of the
invention are prepared using a HBcAg comprising, or alternatively consisting
of, amino acids
1-144, or 1-149, 1-185 of SEQ ID NO:20, which is modified so that the amino
acids at
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13
positions 79 and 80 are replaced with a peptide having the amino acid sequence
of Gly-Gly-
Lys-Gly-Gly. This modification changes the SEQ ID NO:20 to SEQ ID NO:21. In
further
preferred embodiments, the cysteine residues at positions 48 and 110 of SEQ ID
NO:21, or its
corresponding fragments, preferably 1-144 or 1-149, are mutated to serine. The
invention
further includes compositions comprising Hepatitis B core protein mutants
having above
noted corresponding amino acid alterations. The invention further includes
compositions and
vaccines, respectively, comprising HBcAg polypeptides which comprise, or
alternatively
consist of, amino acid sequences which are at least 80%, 85%, 90%, 95%, 97% or
99%
identical to SEQ ID NO:21.
[0047] In one preferred embodiment, the virus-like particle is of a Cowpea
Chlorotic
Mottle Virus, a Cowpea Mosaic Virus or an Alfalfa Mosaic Virus. Methods to
produce VLP
of these viruses have been described in US 2005/0260758 and in WO05067478.
[0048] In one preferred embodiment, the virus-like particle of the invention
is a virus-
like particle of an RNA-bacteriophage, wherein preferably said RNA-
bacteriophage is Q(3,
AP205, GA or fr, further preferably said RNA-bacteriophage is Q(3.
[0049] In one preferred embodiment of the invention, the virus-like particle
of an
RNA-bacteriophage comprises, consists essentially of, or alternatively
consists of,
recombinant coat proteins, mutants or fragments thereof, of an RNA-
bacteriophage.
Preferably, the RNA-bacteriophage is selected from the group consisting of a)
bacteriophage
Q(3; b) bacteriophage R17; c) bacteriophage fr; d) bacteriophage GA; e)
bacteriophage SP; f)
bacteriophage MS2; g) bacteriophage Ml l; h) bacteriophage MX 1; i)
bacteriophage NL95; k)
bacteriophage f2; 1) bacteriophage PP7 and m) bacteriophage AP205.
[0050] In one further preferred embodiment, the virus-like particle of an RNA-
bacteriophage comprises, consists essentially of, or alternatively consists
of, recombinant coat
proteins, of an RNA-bacteriophage. Preferably, the RNA-bacteriophage is
selected from the
group consisting of a) bacteriophage Q(3; b) bacteriophage R17; c)
bacteriophage fr; d)
bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2; g) bacteriophage
M11; h)
bacteriophage MXl; i) bacteriophage NL95; k) bacteriophage f2; 1)
bacteriophage PP7 and m)
bacteriophage AP205.
[0051] In one preferred embodiment of the invention, the composition comprises
coat
protein, mutants or fragments thereof, of RNA-bacteriophages, wherein the coat
protein has
amino acid sequence selected from the group consisting of: (a) SEQ ID NO:l;
referring to Q(3
CP; (b) a mixture of SEQ ID NO:l and SEQ ID NO:2 (referring to Q(3 Al
protein); (c) SEQ
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14
ID NO:3; (d) SEQ ID NO:4; (e) SEQ ID NO:5; (f) SEQ ID NO:6, (g) a mixture of
SEQ ID
NO:6 and SEQ ID NO:7; (h) SEQ ID NO:8; (i) SEQ ID NO:9; (j) SEQ ID NO:10; (k)
SEQ
ID NO:l l; (1) SEQ ID NO:12; (m) SEQ ID NO:13; and (n) SEQ ID NO:14.
[0052] In one preferred embodiment of the invention, the VLP is a mosaic VLP
comprising or alternatively consisting of more than one amino acid sequence,
preferably two
amino acid sequences, of coat proteins, mutants or fragments thereof, of an
RNA-
bacteriophage .
[0053] In one very preferred embodiment, the VLP comprises or alternatively
consists
of two different coat proteins of an RNA-bacteriophage, said two coat proteins
have an amino
acid sequence of SEQ ID NO: 1 and SEQ ID NO:2, or of SEQ ID NO:6 and SEQ ID
NO:7.
[0054] In preferred embodiments of the present invention, the virus-like
particle of the
invention comprises, or alternatively consists essentially of, or
alternatively consists of
recombinant coat proteins, mutants or fragments thereof, of the RNA-
bacteriophage Q(3, fr,
AP205 or GA.
[0055] In one preferred embodiment, the VLP of the invention is a VLP of RNA-
bacteriophage Q(3. The capsid or virus-like particle of Q(3 showed an
icosahedral phage-like
capsid structure with a diameter of 25 nm and T=3 quasi symmetry. The capsid
contains 180
copies of the coat protein, which are linked in covalent pentamers and
hexamers by disulfide
bridges (Golmohammadi, R. et al., Structure 4:543-5554 (1996)), leading to a
remarkable
stability of the Q(3 capsid. Capsids or VLPs made from recombinant Q(3 coat
protein may
contain, however, subunits not linked via disulfide bonds to other subunits
within the capsid,
or incompletely linked. The capsid or VLP of Q(3 shows unusual resistance to
organic
solvents and denaturing agents. Surprisingly, we have observed that DMSO and
acetonitrile
concentrations as high as 30%, and guanidinium concentrations as high as 1 M
do not affect
the stability of the capsid. The high stability of the capsid or VLP of Q(3 is
an advantageous
feature, in particular, for its use in immunization and vaccination of mammals
and humans in
accordance of the present invention.
[0056] Further preferred virus-like particles of RNA-bacteriophages, in
particular of
Q(3 and fr in accordance of this invention are disclosed in WO 02/056905, the
disclosure of
which is herewith incorporated by reference in its entirety. Particular
example 18 of WO
02/056905 gave detailed description of preparation of VLP particles from Q(3.
[0057] In another preferred embodiment, the VLP of the invention is a VLP of
RNA-
bacteriophage AP205. Assembly-competent mutant forms of AP205 VLPs, including
AP205
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WO 2008/074895 PCT/EP2007/064522
coat protein with the substitution of proline at amino acid 5 to threonine or
AP205 coat
protein with the substitution of asparigine at amino acid 14 to aspartic acid,
may also be used
in the practice of the invention and leads to other preferred embodiments of
the invention.
WO 2004/007538 describes, in particular in Example 1 and Example 2, how to
obtain VLP
comprising AP205 coat proteins, and hereby in particular the expression and
the purification
thereto. WO 2004/007538 is incorporated herein by way of reference. AP205 VLPs
are highly
immunogenic, and can be linked with antigen to typically and preferably
generate vaccine
constructs displaying the antigen in oriented in a repetitive manner. High
antibody titer is
elicited against the so displayed antigens showing that linked antigens are
accessible for
interacting with antibody molecules and are immunogenic.
[0058] In one preferred embodiment, the VLP of the invention comprises or
consists
of a mutant coat protein of a virus, preferably an RNA-bacteriophage, wherein
the mutant
coat protein has been modified by removal of at least one lysine residue by
way of
substitution and/or by way of deletion. In another preferred embodiment, the
VLP of the
invention comprises or consists of a mutant coat protein of a virus,
preferably an RNA-
bacteriophage , wherein the mutant coat protein has been modified by addition
of at least one
lysine residue by way of substitution and/or by way of insertion. The
deletion, substitution or
addition of at least one lysine residue allows varying the degree of coupling,
i.e. the amount
of antigen per subunits of the VLP of a virus, preferably of an RNA-
bacteriophage, in
particular, to match and tailor the requirements of the vaccine.
[0059] In one preferred embodiment, the compositions and vaccines of the
invention
have an antigen density being from 0.5 to 4Ø The term "antigen density", as
used herein,
refers to the average number of antigen which is linked per subunit,
preferably per coat
protein, of the VLP, and hereby preferably of the VLP of an RNA-bacteriophage.
Thus, this
value is calculated as an average over all the subunits or monomers of the
VLP, preferably of
the VLP of the RNA-bacteriophage, in the composition or vaccines of the
invention.
[0060] VLPs or capsids of Q(3 coat protein display a defined number of lysine
residues
on their surface, with a defined topology with three lysine residues pointing
towards the
interior of the capsid and interacting with the RNA, and four other lysine
residues exposed to
the exterior of the capsid. Preferably, the at least one first attachment site
is a lysine residue,
pointing to or being on the exterior of the VLP.
[0061] Q(3 mutants, of which exposed lysine residues are replaced by arginines
can be
used for the present invention. Thus, in another preferred embodiment of the
present
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16
invention, the virus-like particle comprises, consists essentially of or
alternatively consists of
mutant Q(3 coat proteins. Preferably these mutant coat proteins comprise or
alternatively
consist of an amino acid sequence selected from the group of a) Q(3-240 (SEQ
ID NO:15,
Lysl3-Arg of SEQ ID NO: 1) b) Q(3-243 (SEQ ID NO:16, AsnlO-Lys of SEQ ID
NO:l); c)
Q(3-250 (SEQ ID NO:17, Lys2-Arg of SEQ ID NO: 1) d) Q(3-251 (SEQ ID NO:18,
Lysl6-Arg
of SEQ ID NO:l); and e) Q(3-259" (SEQ ID NO:19, Lys2-Arg, Lysl6-Arg of SEQ ID
NO:l).
The construction, expression and purification of the above indicated Q(3
mutant coat proteins,
mutant Q(3 coat protein VLPs and capsids, respectively, are described in WO
02/056905. In
particular is hereby referred to Example 18 of above mentioned application.
[0062] In another preferred embodiment of the present invention, the virus-
like
particle comprises, or alternatively consists essentially of, or alternatively
consists of mutant
coat protein of Q(3, or mutants or fragments thereof, and the corresponding Al
protein. In a
further preferred embodiment, the virus-like particle comprises, or
alternatively consists
essentially of, or alternatively consists of mutant coat protein with amino
acid sequence SEQ
ID NO:15, 16, 17, 18, or 19 and the corresponding Al protein.
[0063] Further RNA-bacteriophage coat proteins have also been shown to self-
assemble upon expression in a bacterial host (Kastelein, RA. et al., Gene
23:245-254 (1983),
Kozlovskaya, TM. et al., Dokl. Akad. Nauk SSSR 287:452-455 (1986), Adhin, MR.
et al.,
Virology 170:238-242 (1989), Priano, C. et al., J. Mol. Biol. 249:283-297
(1995)). In
particular the biological and biochemical properties of GA (Ni, CZ., et al.,
Protein Sci.
5:2485-2493 (1996), Tars, K et al., J. Mol.Biol. 271:759-773(1997)) and of fr
(Pushko P. et
al., Prot. Eng. 6:883-891 (1993), Liljas, L et al. J Mol. Biol. 244:279-290,
(1994)) have been
disclosed. The crystal structure of several RNA bacteriophages has been
determined
(Golmohammadi, R. et al., Structure 4:543-554 (1996)). Using such information,
surface
exposed residues can be identified and, thus, RNA-bacteriophage coat proteins
can be
modified such that one or more reactive amino acid residues can be inserted by
way of
insertion or substitution. Another advantage of the VLPs derived from RNA-
bacteriophages is
their high expression yield in bacteria that allows production of large
quantities of material at
affordable cost.
[0064] In one embodiment, the first amino acid is at the very N-terminus of
the
antigenic peptide of the invention.
[0065] In one embodiment, the first amino acid is located at the N-terminal of
KINVK
and not more than 16 amino acids, preferably not more than 15, 14, 13, 12, 11,
10, 9, 8, 7, 6,
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17
5, 4, 3, 2, or 1 amino acid away from the first amino acid K of KINVK. In one
preferred
embodiment, the first amino acid is not more than 16 amino acids, preferably
not more than
10, more preferably not more than 5 amino acids away from the first amino acid
K of
KINVK.
[0066] In one preferred embodiment, the first amino acid is at -16, -15 or -5
position
relative to the first K (position 0) of KINVK. In one further preferred
embodiment, the first
amino acid is a cysteine residue. Typically and preferably the cysteine is
generated by
insertion, or preferably by substitution of the naturally occurring amino acid
residue, normally
serine, at that position into cysteine, wherein further preferably the
naturally occurring
cysteine within the PNt domain will be deleted or preferably substituted,
preferably by a
serine or an alanine substitution.
[0067] In one very preferred embodiment, the first amino acid is at -2
position relative
to the first K (position 0) of KINVK. In one further preferred embodiment, the
first amino
acid corresponds to, or preferably is, the cysteine residue within SEQ ID
NO:22.
[0068] In one embodiment, the second amino acid is located at the C-terminal
of
KINVK and not more than 14, 12, preferably not more than 10 amino acids,
preferably not
more than 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid away from V of KINVK,
further preferably not
more than 8 amino acids away from amino acid V of KINVK. In one preferred
embodiment,
the second amino acid is at least 4, preferably at least 5, more preferably at
least 6 amino
acids away from V of KINVK. In one preferred embodiment, the second amino acid
is at least
and not more than 10 amino acids away from V of KINVK.
[0069] In one preferred embodiment, the second amino acid is located at +2,
+3, +4,
+5, +6, or +7 position relative to amino acid V (position 0) of KINVK. Further
preferably the
second amino acid is located at +7 position relative to amino acid V (position
0) of KINVK.
[0070] In one embodiment, the second amino acid is located at +2 position
relative to
amino acid V (position 0) of KINVK, further preferably the second amino acid
is a cysteine.
Typically and preferably the cysteine is generated by insertion, or preferably
by substitution
of the naturally occurring amino acid residue, normally glutamine, at that
position into
cysteine.
[0071] In one embodiment, the first reactive group comprises or is a
sulfhydryl group,
preferably a sulfhydryl group of a cysteine. In one embodiment, the second
reactive group
comprises or is a sulfhydryl group, preferably a sulfhydryl group of a
cysteine.
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[0072] In one preferred embodiment, the first reactive group and the second
reactive
group is a sulfhydryl group, preferably a sulfhydryl group of a cysteine
residue. In one
preferred embodiment, the first amino acid corresponds to, or preferably is,
the cysteine
residue of SEQ ID NO:22. In one preferred embodiment, the second amino acid is
a cysteine
residue located at the C-terminal of KINVK and not more than 10 amino acids
away from
amino acid V of KINVK. The introduction of the cysteine at any one of the +2
to +6 positions
relative to amino acid V (position 0) of KINVK can be achieved by insertion or
preferably by
substitution of any amino acid of QIAAR within SEQ ID NO:22. In one preferred
embodiment, the second amino acid is a cysteine residue located at any one of
+7, +8, +9 and
+10 position relative to amino acid amino acid V (position 0) of KINVK. The
cysteine is
introduced by insertion. Additional spacing amino acids may be added,
preferably alanine and
glycine.
[0073] In one preferred embodiment, the first amino acid corresponds to, or
preferably
is, the cysteine within SEQ ID NO:22 and the second amino acid is a cysteine
located at any
one of the +7, +8, +9 and +10 position relative to amino acid amino acid V
(position 0) of
KINVK. In one further preferred embodiment, the CCR5 PNt domain comprises,
consists
essentially of, or preferably consists of SEQ ID NO:22.
[0074] In one preferred embodiment, the first amino acid corresponds to, or
preferably
is, the cysteine within SEQ ID NO:22 and the second amino acid is a cysteine
located at +7
position relative to amino acid amino acid V (position 0) of KINVK. In one
still further
preferred embodiment, the antigenic peptide of the invention comprises,
consists essentially
of, or preferably consists of an amino acid sequence of SEQ ID NO:23.
[0075] In one preferred embodiment, the first amino acid corresponds to, or
preferably
is, the cysteine within SEQ ID NO:22 and the second amino acid is a cysteine
by substituting
amino acid Q of QIAAR within SEQ ID NO:22. In one further preferred
embodiment, the
antigenic peptide of the invention comprises, consists essentially of, or
preferably consists of
an amino acid sequence of SEQ ID NO:24.
[0076] The side chain of an amino acid which may serve as the first or the
second
reactive group include thiol (C), amine (K), amido (QQ, arginine (R),
carboxylic acid (DE),
alcohol (ST), thioether (M), imidazol (K), phenyl (F), phenol (Y), indole (W),
and aliphatic
(AVILP).
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[0077] In one preferred embodiment, the first reactive group binds to the
second
reactive group through at least one intermediate molecule, preferably two,
more preferably
only one intermediate molecule.
[0078] In one preferred embodiment, one of the two reactive groups is
derivatized by
at least one, preferably only one, intermediate molecule so that it receives a
new functionality
which is readily reactive to the other reactive group. For example, the amino
group of a lysine
may be derivatized by an intermediate molecule, eg, SMPH, so that after
derivatization it
becomes readily reactive to a sulfhydryl group.
[0079] In one preferred embodiment, both of the two reactive groups are
derivatized
by at least one, preferably only one, intermediate molecule, respectively, so
that after
derivatization both are readily to react with each other.
[0080] In one preferred embodiment, the first and the second amino acid are
identical.
In one preferred embodiment, the first reactive group and the second reactive
group are
identical. In one alternative embodiment, the first amino acid and the second
amino acid,
either after derivatization of one of the two reactive groups or after
derivatization of both
reactive groups, have the same functionality.
[0081] Thus in one preferred embodiment, the second reactive group is an amino
group of a lysine. In one further preferred embodiment, said lysine
corresponds to or
preferably is the 26th lysine within SEQ ID NO:22. In one further preferred
embodiment, the
first reactive group is a sulfhydryl group, preferably a sulfhydryl group of a
cysteine residue.
In one further preferred embodiment, the first amino acid corresponds to or
preferably is the
cysteine within SEQ ID NO:22 and the second amino acid corresponds to or
preferably is the
26'h lysine within SEQ ID NO:22.
[0082] In one preferred embodiment, the first reactive group binds to the
second
reactive group by exclusively non-peptide covalent bond.
[0083] In one preferred embodiment, the first and the second reactive group is
a
sulfhydryl group, preferably a sulfhydryl group of a cysteine. In one
preferred embodiment,
the first reactive group binds to the second reactive group by at least one
covalent bond,
wherein said covalent bond is a disulfide bond. Methods of forming disulfide
bond between
two sulfhydryl groups are disclosed (see e.g. US8929758; US4518711; US5169833;
W09109051; W09108759). Preferably the second attachment site is then a -NH-NH2
group
and one way of coupling of the antigenic peptide via -NH-NH2 to the VLP is
described in
EXAMPLE 4.
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[0084] In one embodiment, the intermediate molecule comprises at least two
functional reactive sites and wherein the two functional reactive site binds
to the two reactive
group respectively. In one preferred embodiment, at least one. Preferably both
of the bounds
between the functional reactive site and the reactive group comprises a
thioether bond.
[0085] In one preferred embodiment, the first reactive group and the second
reactive
group comprises or preferably is a sulfhydryl group, preferably a sulfhydryl
group of a
cysteine, wherein said first reactive group binds to said second reactive
group through at least
one, preferably only one, intermediate molecule. Preferably the first reactive
group and the
second reactive group binds to the only one intermediate molecule,
respectively. Further
preferably at least one of the bounds between the reactive group and the
intermediate
molecule comprises a thioether bond.
[0086] In one preferred embodiment, the intermediate molecule comprises a
halogenoalkane. Halogenoalkanes (also known as haloalkanes or alkyl halides)
are
compounds containing a halogen atom (fluorine, chlorine, bromine or iodine)
joined to one or
more carbon atoms in a chain. Provided herein are dihalo-intermediate
molecules comprising
two halogen atoms, and tri- and tetrahalo-intermediate molecules.
[0087] In one preferred embodiment, the intermediate molecule comprises at
least
two, preferably two halogen atoms, wherein preferably said two halogen atoms
are two Cl
atoms, more preferably one Cl atom and one Br atom, even more preferably two
Br atoms.
[0088] In one preferred embodiment, the intermediate molecule comprises an
aromatic compound, wherein preferably said aromatic compound comprises at
least two
benzylic halogen substituents. Preferably, the aromatic compound comprises at
least two
benzylic halogen substituents, like for instance halomethyl groups. Suitable
examples include,
but are not limited, to di(halomethyl)benzene, tri(halomethyl)benzene or
tetra(halomethyl)benzene and derivatives thereof. In one further preferred
embodiment, the
intermediate molecule is a di(halomethyl)benzene (T2), preferably wherein the
di(halomethyl)benzene is 1,3-bis(bromomethyl)benzene.
[0089] In one preferred embodiment, the intermediate molecule is a
tri(halomethyl)benzene or a derivative thereof (T3). Further preferably the
tri(halomethyl)benzene is 2,4,6-tris(bromomethyl)mesitylene. In one preferred
embodiment,
antigenic peptide with two cysteines may be looped through the two cysteines
via reaction
with T3. The so-looped T3 peptide may react with excessive amount of DTT,
which provides
a -SH group serving as the second attachment site.
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21
[0090] In one preferred embodiment, the intermediate molecule comprises an
allylic
system. In an allylic system, there are three carbon atoms, two of which are
connected
through a carbon-carbon double bond. In a preferred embodiment, the formation
of a bond
between the intermediate molecule and the reactive group substitution occurs
via an allylic
substitution reaction. In one further preferred embodiment, the intermediate
molecule
comprises at least one carbon-oxygen double bond (carbonyl group). For
example, a scaffold
comprises two or more reactive groups comprising the structure - C(O)-CH2-
halogen.
[0091] The intermediate molecule may comprise polycyclic aromatic compounds
with
smaller or larger ring structures. Furthermore, the intermediate molecule may
comprise a
cyclic molecule with at least one atom other than carbon in the ring
structure. A preferred
intermediate molecule is meta-dibromo-pyridine. Still furthermore, the
intermediate molecule
may comprise multiple ring aromatic structure, such as fused-ring aromatic
compounds. Still
furthermore, the intermediate molecule may comprise multiple aromatic
conjugated systems
wherein the systems do not share a pair of carbon atoms, e.g. benzene rings
are connected
directly via a carbon-carbon bond. Further intermediate molecules which can be
used for the
present invention have been disclosed in W02004/077062 and this descriptions
are
incorporated herein by way of reference.
[0092] In one preferred embodiment, the intermediate molecule is selected from
a
group consisting of: bis-; tris-; or tetra(halomethyl)benzene; bis-; tr.is-;
or
tetra(halomethyl)pyridine; bis-; tris-; or tetra (halomethyl)pyridazine; bis-;
tria-; or
tetra(halomethyl)pyrimidine; bis-; tris-; or tetra(halomethyl)pyrazine; bis-;
tris-; or
tetra(halomethyl)-1,2,3-triazine; bis-; tris-; or tetra(halomethyl)-1,2,4-
triazine; bis-; tris-; or
tetra(halomethyl)pyrrole,-furan, -thiophene; bis-; tris-; or
tetra(halomethyl)imidazole, -
oxazole, -thiazol; bis-; tris-; or tetra(halomethyl)-8H- pyrazole, -
isooxazole, -isothiazol; bis-;
tris-; or tetra(halomethyl)biphenylene; bis-; tris-; or
tetra(halomethyl)terphenylene; 1,8-
bis(halomethyl)naphthalene; bis-; tris-; or tetra(halomethyl)anthracene; bis-;
tris-; or tetra(2-
halomethylphenyl)methane; or, if applicable, another regioisomer thereof. For
example,
provide6 is 1,2-bis(halomethyl)benzene; 3,4-bis(halomethyl)pyridine; 3,4-
bis(halomethyl)pyridazine; 4,5-bis(halomethyl)pyrimidine; 4,5-
bis(halomethyl)pyrazine; 4,5-
bis(halomethyl)-1,2, 3-triazine; 5 , 6-bis(halomethyl)-1,2,4-triazine; 3,4-
bis(halomethyl)pyrrole,-furan, -thiophene and other regioisomers, 4,5-
bis(halomethyl)imilazole, -oxazole, -thiazol; 4,5-bis(halomethyl)-8H-pyxazole,
- isooxazole, -
isothiazol; 2,2'-bis(halomethyl)biphenylene; 2,2"- bis(halomethyl)
terphenylene; 1, 8-
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22
bis(halomethyl) naphthalene 1, 1 0-bis(halomethyl)anthracene; bis(2-
halomethylphenyl)methane; 1,2,8-tris(halomethyl)benzene; 2,3,4-
tris(halomethyl)pyridine;
2,8,4- tris(halomethyl)pyrid.azine; 3,4,5-tris(halomethyl)pyrimidine; 4,5,6-
tris(halomethyl)-
1,2,8-triazine; 2,8,4-tris(halomethyl)pyrrole, -furan, -thiophene; 2,4,5-
bis(halomethyl)imidazole, -oxazole, -thiazol; 8,4,5-bis(halomethyl)-1H-
pyrazole, -
isooxazole, -isothiazol; 2,4,2'-tris(halomethyl)biphenylene; 2,3',2"-
tris(halomethyl)terphenylene; 1,8,8-tris(halomethyl)naphthalene 1,8, 10-
tris(halomethyl)
anthracene; bis(2-halomethylphenyl) methane; 1,2,4,5-tetra(halomethyl)benzene;
1,2,4,5-
tetra(halomethyl)pyridine; 2,4,5,6- tetra(halomethyl)pyrimidine; 2,3,4,5-
tetra(halomethyl)pyrrole; -furan; -thiophene; 2,2',6,6'-
tetra(halomethyl)biphenylene;
2,2",6,6"-tetra(halomethyl) terphenylene 2,8,5,6-tetra(halomethyl)naphthalene
and. 2,8,7,8-
tetra(halomethyl) anthracene; Bis(2,4-bis(halomethyl)phenyl)methane.
[0093] In one preferred embodiment, the CCR5 PNt domain comprises, consists
essentially of, or consists of an amino acid sequence of SEQ ID NO:22, in
which at most
three, preferably two, more preferably at most one amino acid has been
deleted, inserted or
substituted, preferably by conservative substitution. In one preferred
embodiment, CCR5 PNt
domain comprises, consists essentially of, or preferably consists of an amino
acid sequence of
SEQ ID NO:22.
[0094] In one preferred embodiment of the invention, the VLP with at least one
first
attachment site is linked to the antigenic peptide with at least one second
attachment site via
at least one peptide bond, preferably via exclusively peptide bond, wherein
the antigen
peptide comprises (i) CCR5 PNt domain comprising an amino acid sequence KINVK
(SEQ
ID NO:25); (ii) wherein the first amino acid is a cysteine located at the N-
terminal of KINVK
and wherein the second amino acid is a cysteine located at the C-terminal of
KINVK, and
wherein the two cysteines binds to each other by a disulfide bond.
[0095] In one preferred embodiment, the first cysteine corresponds to, or
preferably is,
the cysteine within SEQ ID NO:22. In one preferred embodiment, the second
cysteine is
located at any one of the +2 to +10 position relative to amino acid V
(position 0) of KINVK.
Further preferably the second cysteine is located at +7 position relative to
amino acid V
(position 0) of KINVK.
[0096] In one preferred embodiment, the CCR5 PNt domain comprises, consists
essentially of, or consists of an amino acid sequence of SEQ ID NO:22. In one
preferred
embodiment, the antigenic peptide of the invention comprises or consists
essentially of an
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23
amino acid sequence of SEQ ID NO:23, wherein the first cysteine and the second
cysteine are
linked by a disulfide bond and wherein the first attachment site and the
second attachment site
are linked by one peptide bond, preferably through the second cysteine (at
position 32 of SEQ
ID NO:23) to the coat protein of a virus-like particle, preferably to the N-
terminus of the coat
protein of bacteriophage AP205.
[0097] Gene encoding antigenic peptide is in-frame ligated, either internally
or
preferably to the N- or the C-terminus to the gene encoding the coat protein
of the VLP.
Fusion may also be effected by fusing sequences of the antigenic peptide into
a mutant of a
coat protein where part of the coat protein sequence has been deleted, that
are further referred
to as truncation mutants. Truncation mutants may have N- or C-terminal, or
internal deletions
of part of the sequence of the coat protein. For example for the specific VLP
HBcAg, amino
acids 79-80 are replaced with a foreign epitope. The fusion protein shall
retain the ability of
assembly into a VLP upon expression which can be examined by
electromicroscopy.
[0098] Spacer of short amino acid stretch may be added to increase the
distance
between the coat protein and the antigenice peptide. Glycine and serine
residues are
particularly favored amino acids to be used in the spacer sequences. Such a
spacer confers
additional flexibility, which may diminish the potential destabilizing effect
of fusing a foreign
sequence into the sequence of a VLP subunit and diminish the interference with
the assembly
by the presence of the foreign epitope.
[0099] The antigenic peptide can be fused to a number of viral coat proteins,
by way
of examples, to the C-terminus of a truncated form of the Al protein of Q(3
(Kozlovska, T.
M., et al., Intervirology 39:9-15 (1996)), or being inserted between position
72 and 73 of the
CP extension. For example, Kozlovska et al., (Intervirology, 39: 9-15 (1996))
describe Q(3A1
protein fusions where the epitope is fused at the C-terminus of the Q(3CP
extension truncated
at position 19. As another example, the antigenic peptide can be inserted
between amino acid
2 and 3 of the fr CP (Pushko P. et al., Prot. Eng. 6:883-891 (1993)).
Furthermore, the
antigenic peptide of the invention can be fused to the N-terminal protuberant
(3-hairpin of the
coat protein of RNA-bacteriophage MS-2 (WO 92/13081). Alternatively, the
antigenic
peptide can be fused to a capsid protein of papillomavirus, preferably to the
major capsid
protein Ll of bovine papillomavirus type 1(BPV-l) (Chackerian, B. et al.,
Proc. Natl. Acad.
Sci.USA 96:2373-2378 (1999), WO 00/23955). Substitution of amino acids 130-136
of BPV-
1 Ll with the antigenic peptide is also an embodiment of the invention.
Further embodiments
o fusing antigenic peptide to coat protein, mutants or fragements thereof, to
a coat protein of a
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24
virus have been disclosed in WO 2004/009124 page 62 line 20 to page 68 line 17
and herein
are incorporated by way of reference.
[00100] In another preferred embodiment, the antigenic peptide is fused to
either the N-
or the C-terminus of a coat protein, mutants or fragments thereof, of RNA-
bacteriophage
AP205. In one further preferred embodiment, the fusion protein further
comprises a spacer,
wherein said spacer is positioned between the coat protein, fragments or
mutants thereof, of
AP205 and the antigenic peptide. Preferably said spacer composed of less than
20, even more
preferably less than 15, more preferably less than 12, 10, still more
preferably less than 8, less
than 5 amino acids (see W02006/032674 for detail).
[00101] In one preferred embodiment, the antigenic peptide is fused at the N-
terminus
of the coat protein of fr. In one further preferred embodiment, the CCR5 PNt
domain has an
amino acid sequence of SEQ ID NO:22, wherein the first cysteine is the
cysteine within SEQ
ID NO:22 and wherein the second cysteine is added at the C-terminal of CCR5
PNt domain.
[00102] In one preferred embodiment, the antigenic peptide of the invention is
fused to
the N-terminus of the coat protein of AP205. In one further preferred
embodiment, the CCR5
PNt domain has an amino acid sequence of SEQ ID NO:22, wherein the first
cysteine is the
cysteine within SEQ ID NO:22 and wherein the second cysteine is added at the C-
terminal of
CCR5 PNt domain.
[00103] In one preferred embodiment, the antigenic peptide is fused to the N-
terminus
of coat protein of AP205 or fr through a spacer comprising at least one amino
acid, preferably
comprises less than 20, even more preferably less than 15, more preferably
less than 12, 10,
still more preferably less than 8, less than 7, 6, 5 or 4 amino acids.
[00104] In one preferred embodiment, the composition comprises or
alternatively
consists essentially of a virus-like particle with at least one first
attachment site linked to at
least one antigenic peptide of the invention with at least one second
attachment site via at
least one non-peptide bond, preferably the non-peptide bond is a covalent
bond. Preferably
the first attachment site does not comprise or is not sulfhydryl group of
cysteine. Further
preferably the first attachment site does not comprise or is not sulfhydryl
group. In one
preferred embodiment, the first attachment site comprises, or preferably is,
an amino group,
preferably the amino group of a lysine residue. In another preferred
embodiment, the second
attachment site comprises, or preferably is, a sulfhydryl group, preferably a
sulfhydryl group
of a cysteine.
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[00105] In a very preferred embodiment, the at least one first attachment site
is an
amino group, preferably an amino group of a lysine and the at least one second
attachment
site is a sulfhydryl group, preferably a sulfhydryl group of a cysteine.
[00106] In one preferred embodiment, the antigenic peptide of the invention
further
comprises an amino acid linker, wherein said amino acid linker comprises at
least one second
attachment site. The linker is associated to the antigenic peptide by way of
at least one
covalent bond, preferably, by at least one, typically one peptide bond. In a
further preferred
embodiment, the linker comprises a sulfhydryl group, preferably of a cysteine
residue. In
another preferred embodiment, the amino acid linker is a cysteine residue.
[00107] The selection of linkers has been disclosed in W02005/108425A1, page
32-33,
which is incorporated herein by way of reference. In one preferred embodiment,
the amino
acid linker is selected from the group consisting of: (a) C; (b) GC; (c) GGC;
(d) GSC; (e)
GGC-CONH2; (f) GC-CONH2; (g) C-CONH2; and (h) GSC-CONH2.
[00108] In one preferred embodiment, the second attachment site comprises or
is a
sulfhydryl group, preferably a sulfhydryl group of a cysteine. In one
preferred embodiment,
the amino acid linker is fused to the C-terminal of the PNt domain. In one
further preferred
embodiment, the second attachment site is located at the C-terminal of the
second amino acid
of the invention.
[00109] In one preferred embodiment, the at least one antigenic peptide with
at least
one second attachment site of the invention comprises or consists essentially
of or consists of
a peptide having an amino acid sequence selected from the group consisting of:
(a) MDYQVSSPIYDINYYTSEPCQKINVKQIAARCC (SEQ ID NO:27) ; (b)
MDYQVSSPIYDINYYTSEPC QKINVKQIAARCGSC (SEQ ID NO:26); (c)
MDYQVSSPIYDINYYTSEPCQKINVKCC (SEQ ID NO:29); and (d) MDYQVSSPIY
DINYYTSEPC QKINVKCSGGSC (SEQ ID NO:28). In one very preferred embodiment, the
at least one antigenic peptide with at least one second attachment site of the
invention
comprises or consists essentially of or consists of MDYQVSSPIYDINYYTSEPC
QKINVKQIAARCGSC (SEQ ID NO:26).
If the antigenic peptide with at least one second attachment site comprises at
least
three, preferably consists of three, cysteine residues, the one cysteine,
which serves as the
second attachment site and which is preferably located at the very C-terminus
of the antigenic
peptide, shall be protected by commonly known method in the art, for example
by
acetamidomethyl group, before the peptide reacts with the intermediate
molecule.
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26
[00110] In one preferred embodiment of the invention, the antigenic peptide of
the
invention is linked to the VLP by way of chemical cross-linking, typically and
preferably by
using a heterobifunctional cross-linker. In preferred embodiments, the hetero-
bifunctional
cross-linker contains a functional group which can react with the preferred
first attachment
sites, preferably with the amino group, more preferably with the amino groups
of lysine
residue(s) of the VLP, and a further functional group which can react with the
preferred
second attachment site, i.e. a sulfhydryl group, preferably of cysteine
artificially added to,
preferably at the C-terminal of, the antigenic peptide, and optionally also
made available for
reaction by reduction. Several hetero-bifunctional cross-linkers are known to
the art. These
include the preferred cross-linkers SMPH (Pierce), Sulfo-MBS, Sulfo-EMCS,
Sulfo-GMBS,
Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIA and other cross-linkers
available for
example from the Pierce Chemical Company, and having one functional group
reactive
towards amino groups and one functional group reactive towards sulfhydryl
groups. The
above mentioned cross-linkers all lead to formation of an amide bond after
reaction with the
amino group and a thioether linkage with the sulfhydryl groups. Another class
of cross-linkers
suitable in the practice of the invention is characterized by the introduction
of a disulfide
linkage between the antigenic peptide and the VLP upon coupling. Preferred
cross-linkers
belonging to this class include, for example, SPDP and Sulfo-LC-SPDP (Pierce).
[00111] Linking of the antigenic peptide of the invention to the VLP by using
a hetero-
bifunctional cross-linker according to the preferred methods described above,
allows coupling
of the antigenic peptide to the VLP in an oriented fashion. Other methods of
linking the
antigenic peptide to the VLP include methods wherein the antigenic peptide is
cross-linked to
the VLP, using the carbodiimide EDC, and NHS. The antigenic peptide may also
be first
thiolated through reaction, for example with SATA, SATP or iminothiolane. The
antigenic
peptide, after deprotection if required, may then be coupled to the VLP as
follows. After
separation of the excess thiolation reagent, the antigenic peptide is reacted
with the VLP,
previously activated with a hetero-bifunctional cross-linker comprising a
cysteine reactive
moiety, and therefore displaying at least one or several functional groups
reactive towards
cysteine residues, to which the antigenic peptide can react, such as described
above.
Optionally, low amounts of a reducing agent are included in the reaction
mixture. In further
methods, the antigenic peptide is attached to the VLP, using a homo-
bifunctional cross-linker
such as glutaraldehyde, DSG, BM[PEO]4, BS3, (Pierce) or other known homo-
bifunctional
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27
cross-linkers with functional groups reactive towards amine groups or carboxyl
groups of the
VLP.
[00112] In other embodiments of the present invention, the composition
comprises or
alternatively consists essentially of a virus-like particle linked to
antigenic peptide via
chemical interactions, wherein at least one of these interactions is not a
covalent bond. Such
interactions include but not limited to antigen-antibody interaction, receptor-
ligand
interaction. Linking of the VLP to the antigenic peptide can be effected by
biotinylating the
VLP and expressing the antigenic peptide as a streptavidin-fusion protein.
[00113] One or several antigen molecules, i.e. antigenic peptide, can be
attached to one
subunit of the VLP, preferably of RNA-bacteriophage, preferably through the
exposed lysine
residues, if sterically allowable. A specific feature of the VLPs of RNA-
bacteriophage and in
particular of the VLP of RNA-bacteriophage Q(3 is thus the possibility to
couple several
antigenic peptides per subunit. This allows for the generation of a dense
antigen array.
[00114] In a very preferred embodiment of the invention, the antigenic peptide
is
linked via a cysteine residue, having been added to the C-terminus of the
antigenic peptide, to
lysine residues on the surface of the VLPs of RNA-bacteriophages, and in
particular to the
VLP of RNA-bacteriophages Q.
[00115] As described above, four lysine residues are exposed on the surface of
the VLP
of Q(3 coat protein. Typically and preferably these residues are derivatized
upon reaction with
a cross-linker molecule. In the instance where not all of the exposed lysine
residues can be
coupled to an antigen, the lysine residues which have reacted with the cross-
linker are left
with a cross-linker molecule attached to the c-amino group after the
derivatization step. This
leads to disappearance of one or several positive charges, which may be
detrimental to the
solubility and stability of the VLP. By replacing some of the lysine residues
with arginines, as
in the disclosed Q(3coat protein mutants, we prevent the excessive
disappearance of positive
charges since the arginine residues do not react with the preferred cross-
linkers. Moreover,
replacement of lysine residues by arginine residues may lead to more defined
antigen arrays,
as fewer sites are available for reaction to the antigen.
[00116] Accordingly, exposed lysine residues were replaced by arginines in the
following Q(3coat protein mutants: Q(3-240 (Lysl3-Arg; SEQ ID NO:15), Q(3-250
(Lys 2-Arg,
Lysl3-Arg; SEQ ID NO:17), Q(3-259 (Lys 2-Arg, Lysl6-Arg; SEQ ID NO:19) and Q(3-
251;
(Lysl6-Arg, SEQ ID NO: 18). In a further embodiment, we disclose a Q(3 mutant
coat protein
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28
with one additional lysine residue Q(3-243 (Asn l0-Lys; SEQ ID NO:16),
suitable for
obtaining even higher density arrays of antigens.
[00117] In one aspect, the invention provide an antigenic peptide comprising:
(i) CCR5
PNt domain comprising an amino acid sequence KINVK (SEQ ID NO:25); (ii) a
first amino
acid comprising a first reactive group and wherein said first amino acid is
located at the N-
terminal of KINVK; (iii) a second amino acid comprising a second reactive
group and
wherein said second amino acid is located at the C-terminal of KINVK, wherein
said first
reactive group binds to said second reactive group by at least one covalent
bond so that the
peptide starting from said first amino acid and ending with said second amino
acid is looped.
[00118] The preferred nature and location of the first and the second amino
acid and
the preferred CCR5 PNt domain have been described throughout this application.
[00119] In one preferred embodiment, the antigenic peptide of the invention
comprises,
consists essentially of, or consists of an amino acid sequence of SEQ ID
NO:22.
[00120] In one preferred embodiment, the first amino acid is a cysteine. In
one
preferred embodiment, the second amino acid is a cysteine. In one preferred
embodiment, the
first and the second amino acid is a cysteine.
[00121] In one preferred embodiment, the first amino acid is cysteine, wherein
preferably said first amino acid corresponds to, or preferably is, the
cysteine within SEQ ID
NO:22. In one preferred embodiment, the second amino acid is a cysteine
residue located at
any one of +5, +6, +7, +8, +9 and +10 position relative to amino acid amino
acid V (position
0) of KINVK. In one further preferred embodiment, second amino acid is
cysteine, wherein
preferably second amino acid is located +7 position relative to amino acid V
(position 0) of
KINVK.
[00122] In one preferred embodiment, the antigenic peptide of the invention
comprises,
consists essentially of, or consists of an amino acid sequence selected from
the group
consisting of: (i) SEQ ID NO:23 and (ii) SEQ ID NO:24. In one very preferred
embodiment,
the antigenic peptide comprises or preferably consists of amino acid sequence
of SEQ ID
NO:23.
[00123] In one preferred embodiment, the antigenic peptide further comprising
at least
one, preferably one, intermediate molecule, wherein said intermediate molecule
comprises at
least two, preferably two, functional reactive sites, wherein said at least
two, preferably two,
functional reactive sites bind to said first reactive and said second reactive
group respectively,
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29
wherein preferably at least one of the bounds between the functional reactive
site and reactive
group comprises a thioether bond.
[00124] In one preferred embodiment, the intermediate molecule comprises a
halogenoalkane, wherein preferably said intermediate molecule comprises at
least two,
preferably two halogen atoms, wherein preferably said two halogen atoms are
two Cl atoms,
more preferably one Cl atom and one Br atom, even more preferably two Br
atoms.
Preferably the intermediate molecule comprises an aromatic compound, wherein
preferably
said aromatic compound comprises at least two benzylic halogen substituents.
[00125] In one preferred embodiment, the intermediate molecule is a
halomethylarene,
preferably selected from the group consisting of bis(bromomethyl)benzene,
tris(bromomethyl)benzene and tetra(bromomethyl)benzene, or a derivative
thereof. Further
preferably the intermediate molecule is a di(halomethyl)benzene, preferably
wherein said
di(halomethyl)benzene is 1,3-bis(bromomethyl)benzene.
[00126] The utility of the antigenic peptide lies preferably in that it mimics
the tertiary
structure of the CCR5 PNt domain. Thus the antigenic peptide of the invention
may be used
as antigenic site to raise antibodies or as a bait for screening useful drugs
binding to it.
Furthermore the antigenic peptide may be useful in the treatment or prevention
of HIV-
infection.
[00127] In one aspect, the invention provides a vaccine composition comprising
the
composition of the invention.
[00128] In one preferred embodiment, the vaccine composition further comprises
at
least one, preferably one, adjuvant. The administration of the at least one
adjuvant may
hereby occur prior to, contemporaneously or after the administration of the
inventive
composition. The term "adjuvant" as used herein refers to non-specific
stimulators of the
immune response or substances that allow generation of a depot in the host
which when
combined with the vaccine and pharmaceutical composition, respectively, of the
present
invention may provide for an even more enhanced immune response. Examples of
the at least
one adjuvant include and preferably consist of complete and incomplete
Freund's adjuvant,
aluminum hydroxide, aluminium salts, and modified muramyldipeptide. Further
adjuvants are
mineral gels such as aluminum hydroxide, surface active substances such as
lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG (bacille
Calmette-Guerin)
and Corynebacterium parvum. Such adjuvants are also well known in the art.
Further
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adjuvants that can be administered with the compositions of the invention
include, but are not
limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,
CRL1005, Aluminum salts (Alum), MF-59, OM-174, OM-197, OM-294, and Virosomal
adjuvant technology. Still further adjuvant include immunostimulatory nucleic
acid,
preferably the immunostimulatory nucleic acid contains one or more
modifications in the
backbone, preferably phosphorothioate modifications. The modification is to
stabilize the
nucleic acid against degradation.
[00129] In another preferred embodiment, the vaccine composition is devoid of
adjuvant.
[00130] An advantageous feature of the present invention is the high
immunogenicity
of the composition, even in the absence of adjuvants. Thus, the administration
of the vaccine
of the invention to a patient will preferably occur without administering at
least one adjuvant
to the same patient prior to, contemporaneously or after the administration of
the vaccine.
VLP has been generally described as an adjuvant. However, the term "adjuvant",
as used
within the context of this application, refers to an adjuvant not being the
VLP used for the
inventive compositions, rather in addition to said VLP.
[00131] The invention further discloses a method of immunization comprising
administering the vaccine of the present invention to an animal or a human,
preferably to a
human. The vaccine may be administered by various methods known in the art,
but will
normally be administered by injection, infusion, inhalation, oral
administration, or other
suitable physical methods. The conjugates may alternatively be administered
intramuscularly,
intravenously, transmucosally, transdermally, intranasally, intraperitoneally
or
subcutaneously. Components of conjugates for administration include sterile
aqueous (e.g.,
physiological saline) or non-aqueous solutions and suspensions. Examples of
non-aqueous
solvents are propylene glycol, polyethylene glycol, vegetable oils such as
olive oil, and
injectable organic esters such as ethyl oleate. Carriers or occlusive
dressings can be used to
increase skin permeability and enhance antigen absorption.
[00132] Vaccines of the invention are said to be "pharmacologically
acceptable" if their
administration can be tolerated by a recipient individual. Further, the
vaccines of the
invention will be administered in a "therapeutically effective amount" (i.e.,
an amount that
produces a desired physiological effect).
[00133] In one aspect, the invention provides a pharmaceutical composition
comprising
the composition as taught in the present invention and an acceptable
pharmaceutical carrier.
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31
When vaccine of the invention is administered to an individual, it may be in a
form which
contains salts, buffers, adjuvants, or other substances which are desirable
for improving the
efficacy of the conjugate. Examples of materials suitable for use in
preparation of
pharmaceutical compositions are provided in numerous sources including
REMivGTON'S
PHARMACEUTICAL SCIENCES (Osol, A, ed., Mack Publishing Co., (1990)).
[00134] The invention teaches a process for producing the composition of the
invention
comprising the steps of: (a) providing a VLP with at least one first
attachment site; (b)
providing at least one antigenic peptide with at least one second attachment
site, and wherein
said antigenic peptide comprises: (i) CCR5 PNt domain comprising an amino acid
sequence
KINVK (SEQ ID NO:25); (ii) a first amino acid comprising a first reactive
group and wherein
said first amino acid is located at the N-terminal of KINVK; (iii) a second
amino acid
comprising a second reactive group and wherein said second amino acid is
located at the C-
terminal of KINVK, wherein said first and said second amino acid does not
comprise said
second attachment site linking said first attachment site; wherein said first
reactive group
binds to said second reactive group by at least one covalent bond so that the
peptide starting
from said first amino acid and ending with said second amino acid is looped;
(c) linking said
VLP and said at least one antigenic peptide to produce said composition,
wherein said at least
one antigenic peptide and said VLP are linked through said at least one first
and said at least
one second attachment site.
[00135] In one aspect, the present invention provides a method of preventing
and/or
treating, preferably treating, HIV infection, wherein the method comprises
administering the
inventive composition or the inventive vaccine composition, respectively, to a
human.
[00136] In another aspect, the invention provides for the use of the
composition for the
manufacture of a medicament for prevention and/or treatment of HIV infection
in human.
EXAMPLES
EXAMPLE 1
Synthesis of peptides P36 and P37
[00137] Peptides P36 and P37 were synthesized by solid-phase peptide synthesis
using
a 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy (RinkAmide) resin
(BACHEM) on
a Syro-synthesizer (MultiSynTech). All Fmoc-amino acids were purchased from
Orpegen
Pharma or Senn Chemicals with side-chain functionalities protected as N-t-Boc
(KW), O-t-Bu
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32
(DESTY), N-Trt (HNQ), S-Trt (C), S(Acm) (C), or N-Pbf (R) groups. A coupling
protocol
using a 6.5-fold excess of HBTU/HOBt/amino acid/DIPEA (1:1:1:2) in NMP with a
30
minute activation time using double couplings was employed. Acetylation of
peptides was
performed by reacting the resin with NMP/Ac20/DIEA (10:1:0.1, v/v/v) for 30
min at room
temperature. Acylated peptides were cleaved from the resin by reaction with
TFA (15 ml/g
resin) containing 13.3% (w) phenol, 5% (v) thioanisole, 2.5% (v) 1,2-
ethanedithiol, and 5%
(v) milliQ-H20 for 2-4 hrs at RT. The crude peptides were purified by reversed-
phase high
performance liquid chromatography (RP-HPLC), either on a "DeltaPack" (25x100
or 40x210
mm inner diameter, 15 um particle size, 100 A pore size; Waters) or on a
"XTERRA" (19 x
100 mm inner diameter, 5 um particle size (Waters) RP-18 preparative C18
column with a
lineair AB gradient of 1-2% B/min. where solvent A was 0.05% TFA in water and
solvent B
was 0.05% TFA in ACN. The correct primary ion molecular weights of the
peptides was
confirmed by electron-spray ionization mass spectrometry on a Micromass ZQ
(Micromass)
or a VG Quattro II (VG Organic) mass spectrometer.
[00138] The peptides synthesized are the following:
P36: Ac-MDYQVSSPIYDINYYTSEPC(SH) QKINVKC(SH) SGGSC(Acm)-CONHz (SEQ
ID NO:28)
P37: Ac-MDYQVSSPIYDINYYTSEPC(SH)QKINVKQIAARC(SH)GSC(Acm)-CONH2
(SEQ ID NO:26)
[00139] P36 and P37 peptides without amino-terminal acetyl-group are
synthesized as
they may better mimic the PNt domain with N-terminal free methoinine.
EXAMPLE 2
Synthesis of T2-P36-SH and T2-P37-SH
[00140] Peptides P36 and P37 were cyclized onto a T2-scaffold via reaction
with 1.05
equivalent of 1,3- (bisbromomethyl)benzene in 25% ACN/75% ammonium bicarbonate
(20
mM, pH 7.8) for 3 hours at room temperature. The solutions were freeze-dried
and the crude
peptides were purified by RP-HPLC (for conditions see above) and freeze-dried.
For removal
of the Acm protective group the peptide constructs were treated with excess
(10 equiv.) of 12
in a mixture of MeOH/DMSO (9:1, v/v) at 1 mM (final concentration) for 15 min.
at room
temperature, followed by destruction of excess of Iz with vitC (200 mM). The
reaction
mixtures were then diluted with 9 volumes of H20 and filtered over a RP C18-
cartridge (Sep-
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33
Pak Vac 3cc for HPLC-extraction, Waters Corporation). The peptide-constructs
were then
collected by elution with ACN/H20 (6 mL, 1:1 v/v) followed by removal of the
solvent by
freeze-drying. Subsequently, the peptide-constructs T2-P36-SH and T2-P37-SH
were purified
by RP-HPLC and freeze-dried (3x) from ACN/milliQ-H20 solution in order to
ensure
complete removal of traces of TFA and/or ammonium bicarbonate. 1,3-
(bisbromomethyl)benzene (T2) was purchased from Sigma-Aldrich. The final
products are
the following:
T2
T2-P36-SH: Ac-MDYQVSSPIYDINYYTSEPCQKINVKCSGGSC(SH)-CONH2
F_'T2:'7
T2-P37-SH: Ac-MDYQVSSPIYDINYYTSEPCQKINVKQIAARCGSC(SH)-CONHz
EXAMPLE 3
Coupling of antigenic peptide P36, P37 to the virus-like particle of Q13
[00141] 2 g/l virus-like particle of Q(3 were derivatised with 1.14 mM SMPH
(Pierce)
for 30 minutes at 25 C and then dialysed against 20 mM phosphate pH7.4. 0.284
mM peptide
P36 (from 5 mM stock in DMSO) and 1 g/l derivatised Q(3 VLPs were incubated
for two
hours at 25 C. The coupling products were analysed by SDS-page.
[00142] 2 g/l virus-like particle of Q(3 were derivatised with 1.29 mM SMPH
(Pierce)
for 30 minutes at 25 C and then dialysed against 20 mM MES pH6. 0.178 mM
peptide P37
(from 5 mM stock in DMSO) and 1 g/l derivatised Q(3 VLPs were incubated for
two hours at
25 C. The coupling products were analysed by SDS-PAGE as shown in FIG. 1.
EXAMPLE 4
Coupling of peptide R8, R9 to virus-like particle of Q13
[00143] Peptide R8 and R9 were chemically synthesized. The two cysteines
within the
peptide were linked by one disulfide bond. The -NH-NH2 group at the very C-
terminus of the
peptide was used as the second attachment site for coupling to VLP.
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34
R8
MDYQVSSPIYDINYYTSEPCQKINVKCG-NH-NH2 (S-S disulfide bridged) (SEQ ID
NO:30)
R9
MDYQVSSPIYDINYYTSEPCQKINVKQIAARCG-NH-NH2 (S-S disulfide bridged) (SEQ
ID NO:31)
[00144] 0.7 g/l virus-like particle of Q(3 were derivatised with 0.5 mM SFB
(Succinimidyl 4-formylbenzoate dissolved in DMSO, Novabiochem) in 0.1 M
phosphate
pH7.4, 0.15 M NaC1 for 150 minutes at 25 C and then dialysed against 10 mM
Hepes pH8.
0.24 mM peptide R8 (from 5 mM stock in DMSO) and 0.66 g/l derivatised Q(3 VLPs
were
incubated over night at 25 C. The coupling products were analysed by SDS-page.
[00145] 0.7 g/l virus-like particle of Q(3 were derivatised with 0.5 mM SFB in
0.1 M
phosphate pH7.4, 0.15 M NaC1 for 150 minutes at 25 C and then dialysed against
10 mM
Hepes pH8. 0.188 mM peptide R9 (from 5 mM stock in DMSO) and 0.66 g/l
derivatised Q(3
VLPs were incubated over night at 25 C. The coupling products were analysed by
SDS-
PAGE.
EXAMPLE 5
Fusing antigenic peptide to the N-terminus of AP205 coat protein
[00146] The construction of Construct 378-2 (with a short GSGG spacer at the N-
terminus of the AP205 coat protein) and Construct 382-2 (with a long
GSGTAGGGSGS
spacer at the N-terminus of AP205) has been described in detail in EXAMPLE 1
of
W02006/032674.
[00147] The antigenic peptide with sequence of SEQ ID NO:23 is fused to the
coat
protein of AP205 via either the short spacer or the longer spacer.
[00148] E.coli JM109 cells are transformed with the corresponding AP205 fusion
protein
plasmids. For expression, the overnight culture is diluted at 1:50 in M9
medium supplemented
with casaminoacids (Difco) and containing 20 mg/l Ampicillin and growth of the
culture
carried out at 37 C with vigorous aeration for 14-20 hours.
[00149] Cells are lysed by ultrasonication in lysis buffer (50 mM Tris, 5 mM
EDTA 0.1 %
Tween 20, pH 8.0) supplemented with 5 g/ml PMSF. The lysate is clarified by
centrifugation, and the pellet washed with lysis buffer containing 1 M urea.
The pooled
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supematants are purified over a Sepharose CL-4B column in NET buffer. Eluted
fractions
containing the VLPs are pooled, concentrated using an Amicon centrifugal
filter unit, and
purified over a Sepharose 6B column in NET buffer. The fractions containing
VLPs are
pooled, concentrated with a centrifugal filter unit and dialyzed against 10 mM
Hepes pH 7.5,
Particle assembly and display of the antigenic peptide is demonstrated by
analysis of purified
VLPs by SDS-PAGE and EM.
EXAMPLE 6
Mouse immunisation with QB-P16, QB-P36 and QB-P37
[00150] BalbC mice were primed with 50 g Q(3 VLP-P36 on day 0 subcutaneously
in 0.2
m120 mM phosphate pH 7.4. Mice were further boosted with the same vaccine on
day 14 and
day 28. BalbC mice injected with Q(3 VLP only were used as control. The a-QB
and the a-
P36 antibody titers were checked by ELISA at day 21 and day 35. QB-P16
immunization
followed the same regimen.
[00151] The serum IgG titers against coated RNAse-P36 were between 2'500 -
8'000 at
days 21 and 35.
[00152] BalbC mice were primed with 50 g Q(3 VLP-P37 on day 0 subcutaneously
in 0.2
ml 20 mM MES pH7.4. Mice were further boosted with the same vaccine on day 14
and day
58 BalbC mice injected with Q(3 VLP only were used as control. The a-Q13 and
the a-P37
antibody titers were checked by ELISA at day 65.
[00153] The serum IgG titers against coated RNAse-P37 peptide were between
20'000 -
100' 000 at day 65.
EXAMPLE 7
Rabbit immunisation with Q13-P16, Q13-P36 and Q13-P37
[00154] New Zealand White rabbits were primed with 100 g Q(3-P36 peptide on
day 0
intradermic in 0.2 ml PBS. After boosting with the same vaccine on days 0, 21,
42, 70, the a-
Q13 and the a-CCR5 antibody titers were checked by ELISA at days 0, 21, 49,
63, 80.
[00155] The serum IgG titers against coated RNAse-P36 peptide were around 1500
at day
86.
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36
EXAMPLE 8
Purification of polyclonal mouse and rabbit IgG
[00156] Serum from five immunised mice obtained from EXAMPLE 6 or 9 ml of
rabbit
serum obtained from EXAMPLE 7 were centrifuged for five minutes at 14'000 rpm.
The
supematant was loaded on a column of 3.3 ml prewashed protein G and protein A
sefarose.
The column was washed with PBS and eluted with 100 mM glycine pH2.8. 1 ml
fractions
were collected in tubes previously provided with 120 1 1 M Tris pH8. Peak
fractions
absorbing at 280 nm were pooled. The eluted antibodies were dialysed against
PBS buffer.
EXAMPLE 9
FACS staining of cellular CCR5 with polyclonal IgG
[00157] In order to check whether the antigenic peptide used for immunisation
can induce
antibodies that bind to CCR5, we have performed a FACS (fluorescence activated
cell
sorting) staining of cell-surface exposed CCR5. CEM.NKR-CCR5 is a CCR5-
expressing
variant of the CEM.NKR, a human cell line that naturally expresses CD4 (Trkola
et al., J.
Virol., 1999, page 8966).
[00158] CEM.NKR-CCR5 cells were grown in RPMI 1640 culture medium (with 10%
FCS, glutamine, and antibiotics). Cells were pelleted and resuspended in
phosphate-buffered
saline (PBS) containing 1% fetal calf serum (FCS). 1 g/l human y-globulin
(Jackson Immuno
Research) was added as a blocking agent and incubated for 20 minutes. 0.1 ml
cells in the
concentration of 2.3x105 cells/well were plated and then pelleted in a V-
bottom 96-well plate.
The cells were then resuspended with 0.1 ml a-CCR5 polyclonal antibodies
obtained from
EXAMPLE 8 (1 mg/l purified IgG or 1/50 diluted serum; dilutions with 1% FCS
/PBS). After
20 minutes on ice, cells were washed twice in 1% FCS/PBS and stained for 20
minutes on ice
in 1% FCS/PBS with either 0.4 mg/l PE-goat-a-mouse-immunoglobulin (Pharmingen)
or
with 1/800 diluted PE-donkey-a-rabbit-IgG (Jackson Immuno Research). After two
washes in
1% FCS/PBS, cells were resuspended in 0.5 mg/l propidium iodide, 1% FCS/PBS.
[00159] 5'000 - 10'000 live cells (propidium iodide negative) were analysed by
flow
cytometry for binding of PE labelled antibodies. The geometric mean of each
staining was
determined using the "FlowJo" flow cytometry software.
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37
[00160] Sera of days 21 and 35 from three out of five mice immunized with Q(3
VLP-P36
positively stained CEM.NKR cells, whereas a-Q(3 sera did not stain CEM.NKR
cells. Sera of
day 65 from 7 out of 9 mice immunized with Q(3 VLP-P37 positively stained
CEM.NKR
cells. Monoclonal antibodies 2D7 (Pharmingen) or 45531 (R&D systems) were used
as
positive controls. The data were shown in FIG. 2. Similar positive data of
FACS staining were
also obtained for sera of 21 days of mice immunized with P 16 (data not
shown).
EXAMPLE 10
Stimulated primary CD8 depleted PBMC (for HIV neutralisation assays)
[00161] Buffy coats obtained from three healthy blood donors were depleted of
CD8+ T
cells using Rosette Sep cocktail (StemCell Technologies Inc.) and PBMC
isolated by Ficoll-
Hypaque centrifugation. Cells were adjusted to 4x106 per ml in culture medium
(RPMI 1640,
10% FCS, 10 U/ml IL-2, glutamine and antibiotics), divided into three parts
and stimulated
with either 5 g/ml phytohemagglutinin (PHA), 0.5 g/ml PHA or anti-CD3 MAb
OKT3 as
described (Rusert P. et al, Virology 326: 113-129). After 72 h, cells from all
three
stimulations were combined (referred to as 3-way stimulated PBMC) and used as
source of
stimulated CD4+ T cells for infection and virus isolation experiments.
EXAMPLE 11
HIV-Neutralisation assay
[00162] A HIV neutralisation assay with stimulated CD8-depleted PBMC was
carried out
essentially as described (Trkola et al., 1998, J. Virol. 72, 396).
[00163] The sources of the R5 viruses (CCR5 co-receptor specific strains: JR-
FL and
SF162) have been described (O'Brien et al., Nature 1990, 348, page 69; and
Shioda et al.,
Nature 1991, 349, page 167). The HIV-1 inoculums were adjusted to contain
approximately
1,000 to 4,000 TCID50/ml in assay medium (TCID50: 50% tissue culture infective
dose).
Aliquots (60 l) were incubated with serial dilutions of purified polyclonal
rabbit IgG or
control antibody 2D7 (25 ng/ml - 25 g/ml; Pharmingen) for 1 h at 37 C.
[00164] Briefly, cells were incubated with serial dilutions of purified
polyclonal rabbit IgG
or control antibody 2D7 (25 g/ml - 25 ng/ml; Pharmingen) in 96-well culture
plates for lh at
37 C. Then virus inoculum (100 TCID50; 50% tissue culture infective dose;
Trkola et al., J.
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38
Virol., 1999, page 8966) was added and plates cultured for 4-14 days. Either
JR-FL or SF162
R5 virus was used for infection. The total infection volume was 200 1. On day
6 post
infection, the supernatant medium was assayed for the HIV-1 p24 antigen
production by using
an immunoassay, as described (Moore et al., 1990. Science 250, page 1139). The
calculated
inhibitory doses refer to the concentration of antibodies used. p24 antigen
production in the
absence of testing antibodies was designated as 100%, and the p24 antigen
production in
antibody-containing cultures was calculated relative to this. The antibody
concentrations
causing 50%, 70% and 90% reduction in p24 antigen production were determined
by linear
regression analysis. If the appropriate degree of inhibition was not achieved
at the highest or
lowest antibody concentration, a value of > or < was recorded and these upper
or lower limits
were used for statistical analysis.
[00165] Alternatively R5 tropic virus stocks of RHPA Env pseudotyped virus
carrying a
luciferase reporter gene were prepared by transfecting 10 cm dishes seeded
with 293-T cells
with 15 g of the backbone plasmid (pNLluc-AM) and 5 g of the envelope clone
and 40 g
of PEI (linear, 25 kDa, Polysciences, Inc.). Virus stocks were titrated on TZM-
bl cells as
described (Huber M et al, (2006) PLoS Medicine 3: e441). Neutralization
activity of MAbs
against pseudotyped virus carrying the patient derived and in vitro selected
envelope genes
was evaluated on CD8 depleted activated PBMC obtained from EXAMPLE 10 by
preincubating the cells for lh with serial dilutions of purified antibodies
obtained from
EXAMPLE 8. Then, 10000 - 20000 TCID50/ml of the virus in assay medium
(RPMI1640,
10% FCS, 100 U IL-2, 2 g/ml polybrene) was added and incubated for 48h. The
antibody
concentration causing 50% (IC50), 70% (IC70) and 90% (IC90) reduction in
luciferase
reporter gene production after 48h was determined by regression analysis. The
monoclonal
antibody PA14 was used the positive control. The result is shown in FIG. 3.
While 90%
neutralization was not achieved with the concentration 100ug/ml of Q(3
antibodies, the highest
concentration measured, antibodies raised against peptide P16, P36 and P37
neutralized 90%
virus infection within a concentration comparable to the positive control.
EXAMPLE 12
Synthesize peptide with T3/DTT
[00166] Antigenic peptide with two cysteines, such as:
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39
XXXXC(SH)XXXXC(SH)XXXX (X represents any amino acid except cysteine) may also
be
looped through the two cysteines by T3as the following:
[00167] After synthesize, antigenic peptide was reacted with -3 equivalent of
2,4,6-
tris(bromomethyl)mesitylene in 50% ACN/50% ammonium bicarbonate (20 mM, pH
7.8) for
1-5 min. at room temperature, followed by reaction with excess (20-50 equiv.)
of 1,4-
dithiothreitol (DTT). The solvents were removed by freeze-drying and the crude
T3/DTT
peptide was purified by RP-HPLC (for conditions see above) followed by removal
of solvent
via freeze-drying. 2,4,6-tris(bromomethyl)mesitylene (T3) and 1,4-
dithiothreitol (DTT) were
purchased from Sigma-Aldrich. DTT provides an additional -SH group which
serves as the
second attachment site for coupling to VLP.
[00168] FIG. 4 showed P16 (SEQ ID NO:32) peptide, which was synthesized
according to
the above described method.
$
