Note: Descriptions are shown in the official language in which they were submitted.
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NEW HBV SEQUENCES
FIELD OF THE INVENTION
The present invention relates to the field of Hepatitis B virus (HBV)
diagnostics and
therapeutics. The present invention provides sequences of a new HBV genotype G
and a new mechanism for HBeAg modulation for use in diagnosis, prevention and
treatment of HBV infections.
BACKGROUND OF THE INVENTION
Human Hepatitis B virus (HBV), which is the prototype member of the family
Hepadnaviridae, is a circular, partially double-stranded DNA virus of
approximately
3200 nucleotides (Magnius and Norder, 1995). This highly compact genome
contains
the four major open reading frames (ORFs) encoding the envelope (preSl, preS2
and
surface antigen HBsAg), core (preCore precursor protein, HBeAg and HBcAg),
polymerase (HBpol) and X (HBX) proteins, respectively.
By using subtype-specific antibodies against HBsAg, nine different serological
subtypes were defined, reflecting the genetic variability of HBV. Of the
defined
determinants, one is common to all subtypes (a determinant), but also .two
pairs of
mutually exclusive subdeterminants (d or y, and w or r) were commonly found.
By
using this tool in epidemiological studies, nine serological subtypes have
been
identified: aywl, ayw2, ayw3, ayw4, ayr, adw2, adw4, adrq+ and adrq- (Swenson
et
al., 1991).
Genotypically, HBV genomes have been classified into six groups, designated A-
F,
based on an intergroup divergence of 8% or more in the complete nucleotide
sequence
(Okamoto et al., 1988; Norder et al., 1992; Magnius and Norder, 1995). These
six
different genotypes show a characteristic geographical distribution: genotype
A is
pandemic, but most prevalent in north-west Europe, North America, and Central
Africa; genotype B is mostly found in Indonesia, China and Vietnam; genotype C
is
found in East Asia, Korea, China, Japan, Polynesia and Vietnam; genotype D is
also
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more or less pandemic, but is predominant in the Mediterranean area and the
Middle
East extending to India; genotype E is endemic in Africa; and genotype F is
found in
American natives and in Polynesia (Van Geyt et al., 1998; Magnius & Norder,
1995).
Some studies have shown that, in certain populations where HBV is endemic, a
higher
variability of HBV might be expected (Bowyer et al., 1997; Carman et al.,
1997). In
the present prevalence study of the HBV genotypes in France and the USA, HBV
strains were identified that did not allow unique type recognition.
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AIMS OF THE INVENTION
It is an aim of the present invention to provide new HBV polynucleic acid
sequences
that are unique to a heretofore unidentified HBV genotype, genotype G, which
is
phylogenetically different from HBV genotypes A, B, C, D, E and F.
It is another aim of the invention to provide fragments of the HBV polynucleic
acids
as described above, that are unique to the heretofore unidentified HBV
genotype G
and that contain at least one nucleotide or a combination of nucleotides
different from
known nucleic acid sequences of HBV genotypes A, B, C, D, E or F, or the
complement of said fragments.
It is another aim of the present invention to provide fragments of the HBV
polynucleic acids as described above that are universal to genotypes A, B, C,
D, E, F
and G.
It is another aim of the invention to provide a method for the detection in a
biological
sample of the presence of an HBV genotype G specific polynucleic acid.
It is another aim of the invention to provide a universal method for the
detection of
HBV genotypes A, B, C, D, E, F and G.
It is another aim of the invention to provide an oligonucleotide primer that
is able to
act as primer for specifically sequencing or specifically amplifying a nucleic
acid or
part of a nucleic acid characteristic to HBV genotype G.
It is another aim of the invention to provide a universal primer that is able
to act as a
primer for sequencing or amplifying nucleic acids from HBV genotypes A, B, C,
D,
E, F and G.
It is another aim of the invention to provide an oligonucleotide probe that is
able to
act as a hybridization probe for specific detection of a nucleic acid or part
of a nucleic
acid characteristic to HBV genotype G.
It is another aim of the invention to provide a universal probe that is able
to act as a
hybridization probe for the universal detection of nucleic acids from HBV
genotypes
A, B, C, D, E, F and G.
It is another aim of the invention to provide a method for HBV genotyping.
It is another aim of the invention to provide a diagnostic kit for the
detection of a
nucleic acid or part of a nucleic acid characteristic to HBV genotype G.
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It is another aim of the invention to provide a diagnostic kit for the
universal detection
of HBV genotypes A, B, C, D, E, F and G.
It is another aim of the present invention to provide a HBV genotype G
specific
polypeptide.
S It is another aim of the present invention to provide a universal peptide
characteristic
to genotypes A, B, C, D, E, F, and G.
It is another aim of the present invention to provide a method for the
production of a
polypeptide of the invention.
It is another aim of the present invention to provide an expression vector
that allows
the expression of a peptide of the invention.
It is another aim of the present invention to provide a host cell transformed
with an
expression vector of the invention.
It is another aim of the present invention to provide a method for the
detection of
antibodies that are specific to the heretofore unidentified HBV genotype or
antibodies
that recognize HBV genotypes A, B, C, D, E, F and G.
It is another aim of the present invention to provide a diagnostic kit for the
detection
of antibodies that are specific to the heretofore unidentified HBV genotype or
for the
detection of antibodies that recognize HBV genotypes A, B, C, D, E, F and G.
It is another aim of the present invention to provide an antibody or a ligand
that
specifically binds a peptide of the invention.
It is another aim of the present invention to provide an antibody or a ligand
that
specifically recognizes the HBV polymerase, X protein, preCore, HBcAg, HBeAg,
preSl, preS2 or HBsAg protein of genotype G.
It is another aim of the present invention to provide an antibody or a ligand
that
recognizes the HBV polymerase, preCore, HBcAg, HBeAg, preSl, preS2 or HBsAg
protein of genotypes A, B, C, D, E, F and G.
It is another aim of the present invention to provide a method for the
detection of a
polypeptide of the invention.
It is another aim of the present invention to provide a diagnostic kit that
allows the
detection of a polypeptide of the invention.
It is another aim of the present invention to provide a monoclonal antibody
for the
prevention or treatment of HBV infections.
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It is another aim of the present invention to provide a vaccine or a
pharmaceutical
composition for the prevention or treatment of HBV infections.
It is another aim of the present invention to provide a method to identify
compounds
or agents that can be used for the prevention or treatment of HBV infections.
5 It is another aim of the present invention to provide an HBV genotype G
infected
clone for use in a drug-screening assay.
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FIGURE LEGENDS
Figure 1. Complete nucleotide sequence of FRl with amino acid translation of
the
four major ORFs. Target sequences for HBV genotype G specific probes and/or
primers are indicated with an horizontal line. Nucleotides and nucleotide
combinations that are unique to HBV genotype G are shown in bold and with au ,
respectively.
Figure 2. Complete coding sequence of the HBV preC/C gene as described by Tran
et
al. (1991).
Figure 3. Complete coding sequence of the HBV preS/S gene as described by Tran
et
al. (1991).
Figure 4. The genome organization and open reading frames of HBV genotype G
virus compared to other genotypes. For each genotype, only one representative
genome is included (genotype A: X70185; B: D00331; C: X01587; D: X72702; E:
X75664; F: X75663; G: FR1). Positions in the viral genome where extensive
variability is observed between the genotypes, are indicated as a black zone.
Nucleotide and amino acid numbering is indicated. (1): Most isolates of
genotype G
contain translational stop codons, influencing the length of the preCore
region. xxx:
stands for the amino acid numbering for the M-residue in the conserved YMDD
motif
of the different genotypes. The positions and orientation of the primers shown
in table
1 are indicated with arrows.
Figure 5. Amino acid sequence alignment of the preCore and Core regions of the
different HBV genotypes. For each genotype, only one representative genome is
included (genotype A: X70185; B: D00331; C: X01587; D: X72702; E: X75664; F:
X75663; G: FR1). The amino acid sequence was derived from the nucleotide
sequence. X: translational stop.
Figure 6. Amino acid sequence alignment of the preS 1, preS2 and HBsAg open
reading frame of the different HBV genotypes. For each genotype, only one
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7
representative genome is included (genotype A: X70185; B: D00331; C: X01587;
D:
X72702; E: X75664; F: X75663; G: FR1). Top: preSl; middle: preS2; bottom:
HBsAg. The amino acid sequence was derived from the nucleotide sequence.
Figure 7. Alignment of preCore/Core sequences of a HBV genotype A strain
(HBVXCPS) and of 7 HBV strains that belong to the newly identified genotype G
(FR1, FR2, US1, US3, USS, US6, US7, US10). N indicates the absence of a
nucleotide at this position.
Figure 8. Alignment of preSl/preS2/HBsAg sequences of a HBV genotype A strain
(HBVXCPS) and of 7 HBV strains that belong to the newly identified genotype G
(FR1, FR2, US1, US3, US6, US7, US9, US10). N indicates the absence of a
nucleotide at this position.
Figure 9. Phylogenetic distances, obtained by the program DNADIST, between the
different HBV genotypes. In total 36 complete genomes (accession numbers are
given
in Fig. 10) were compared with the FR1 (genotype G) sequence. The mean for
each
group is indicated (A: ~; B: ~; C: ~; D: X; E: ~k; F:~; G: -f-), including the
standard
error of the mean; the latter gives a 95% confidence interval around these
distances.
Figure 10. Phylogenetic trees of the HBV genotypes. Virus isolates are
indicated by
their GenBank accession number. (a) Complete genomes. (b) Open reading frame
of
the surface gene (including preSl/preS2/HBsAg).
Figure 11. Result obtained with some representative samples of genotypes A, B,
C,
D, E, F and G after hybridization of their PCR product to the genotyping LiPA
as
designed in example 4. Strip G: incubated with PCR fragments of FR1. The right
strip
shows the positions of all probes applied. The numbering of the probes is
described in
Van Geyt et al. (1998).
Conj.contr: conjugate control; Ampl.contr: amplification control (probe
reacting with
all genotypes, located in conserved area). The probe lines with "target A" and
"target
B" contain probes recognizing the target A and target B region as indicated in
Figure
1. Probes 193 and 77 recognize genotype A only. Probe 78 recognizes genotype B
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only. Probes 153, 154 and 204 recognize genotype C only. Probes 165 and 208
recognize genotype D only. Probes 172, 177 and 213 recognize genotype E only.
Probes 186, 216, and 219 recognize genotype F only. Probe 140 recognizes
genotypes
A and G. Probe 148 recognizes genotypes A, B and G. Probe 80 recognizes
genotypes
C, D, E and G. Probe 239 recognizes genotypes B, E and G.
Figure 12. Complete coding sequence of the HBV preC/C gene as described by
Bhat
et al. ( 1990).
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TABLES
O
Z
A .~ N M ~ v7 ~D I~ 00 Q\ O ~ N M ~t V~
N N N N N N N N N M M M M M M M
a
w
00 00 ~O00 O O .~ M O V7~ O
i~ ~ oo ~ l~ N N I~ l~ N v1 '~i'N l~ M t~
M
O o0 0o N a1 .~ ,~ l~ ~O t~ Ov o0 00N ~ v1
M
N N M .~ M M ~ .~ .-~l~ ~ ,~,~ N .~
d'
vi O l~ ~Oo0 ~ ~ V7 N ~ V1 W O V7 ~ Ov
O ~n ~ N ~ O O ~n ~n O l~ N O ~n O
p.io0 0o N O~ -~ ~ ~ ~O l~ l~ 00 0oN ~ ~n
N N M .-.M M ,-~.~ ,-~~ .~ N
O O ~ ~ ~ ~' '_' b0
O O O O d
O
A ~ ~ " ~ ~ ~ ~ x ~ ~ ~ x o
x x
0
C~
'' U U
U U Cd.7 ~ H d
M ~ ~ H U d d
E-~ CH,7 U H U C7 ~ F-' U
in ~ d ~ d U C7 U U
a C~7U~U~dH~HE'U''HUUCH.7CU.7U
H ~ ~ E'' U U U E'' E-~-, ~ ~ E"' ~ d C7
HUUCU.7UUUHH~C7UC7dL7 U
~. E-~ d U d U d ~ U U ~ C7 F-~ U ~ ~ U
U
HU~HCU.7UEH-~~~ UU~Cd.7Cd,7
U~~~7Cd7UHd ~UHUL7dH
U ~dd U d ~ ~ ~ ~ ~ H ~ ~ U C7 C7 Ed..,,
H~UUC.~7~C7~HdH HCd.7UC7
C7 ~ U H C~.7 H ~ U ~ C7 H U H U C.U7
C7 C7 U U H U U d E-~ C7 H U U U H
00 01O .~ M
v~ ~ M V'1~O I~ d' O O ~ ~ -~
M
i, .~ N M l~ .~ M I~ 00 00 01 ~ ~ --~.--r.~
.--i
~. it 1-W, Y. i.r~, ~. ~, F, i-~i-W. i..iy.
P-~ G, C~..~P-iP..~P-~P-~~r P..~P-~P-~P-iP-~P-~4r
P~
P-~
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z
A t~ oo a, o --~ N
M M M ~ ~ ~t
a
w
N Ov M o0
b N ~ I~ 00 N o0
O N ~n N ~n M o0
~' 00 N ~-~ l~ N N
~ i i
v~ M M V7 00 M 00
O O O~ v7 M O ~O
N ~ ~ N N
d d
C~~ O O
O
x
x
A
x x
..,
a ~ d
°
c~
c
C7 d
C7~ C.~7U H
~ C~7H H
U
H d V
d d7 C7 C7
,
d7d C7
d c.~7d d.77
7 ~
d U C.U7d ~ d
C7 U U ~ ~ d
v H H d U
U U C7 Ed-C7 U
0
U M ~ O
L 7 D 0
'~ ~ OMM ~' ~ ~ f,"
t- W..ni.~1..4, s.,
H w x ~ x x
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Table 2. Summary of the genotyping result obtained from 121 individuals
infected
with HBV.
Genotype A B C D E F G Total
France 18 0 2 16 1 0 2 39
Georgia, 48 4 12 7 0 0 11 82
U.S.
Total 66(54%) 4(3%) 14(12%) 23(19%) 1(1%) 0(0%) 13(11%) 121(100%)
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DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to an isolated and/or purified HBV polynucleic
acid,
having a nucleotide sequence which is unique to a heretofore unidentified HBV
genotype and which is phylogenetically different from HBV genotypes A, B, C,
D, E
and F characterized by:
(a) a sequence as defined in Figure 1 (SEQ ID NO 1); or
(b) a sequence with at least 90%, more preferably 91 %, most preferably
92% identity to the sequence as defined in Figure 1; or
(c) a sequence which is degenerate as a result of the genetic code to the
sequence as defined in (a) and (b).
An "individual genetic group" or "phylogenetically different genotype" of HBV
can
be defined if a certain viral strain differs by more than 8% from all other
HBV
genomes (Okamoto et al., 1988; Norder et al., 1994). Thus, virus strains
differing
from other isolates by more than 8% over their entire genome (not focussing on
possible deletions or insertions) are classified as new genotypes. Based on
results
obtained from several epidemiological studies (Blitz et al., 1998; Norder et
al., 1994;
Magnius and Norder, 1995; Telenta et al., 1997; Alvarado-Esquivel et al.,
1998), six
major genetic groups of HBV (A, B, C, D, E and F) were recognized. Apart from
the
detection of these commonly found genotypes, in a prevalence study on samples
from
chronic HBV carriers from Atlanta, USA and Lyon, France, the present inventors
have isolated a new viral HBV strain with at least 11.7% (new genotype versus
genotype E) and at most 15.3% (new genotype versus genotype F) divergence over
the complete genome. Accordingly, this new viral HBV strain has been
classified as a
new HBV genotype G. Surprisingly, the prevalence of this viral variant in the
US
exceeded 11% of all infections, although it had not been identified in all of
the
previous molecular epidemiology studies. In addition to the prominent
prevalence of
this new genotype G virus in the USA samples, it was also found in samples
originating from France.
The term "isolated" refers to material that is free from components that
normally
accompany it as found in its naturally occurring environment. However, it
should be
clear that the isolated nucleic acid of the present invention might comprise
heterologous cell components or a label and the like.
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The terms "nucleic acid" or "polynucleic acid" are used interchangeable
throughout
the present application and refer to a deoxyribonucleotide or ribonucleotide
polymer
in either single- or double stranded form, which may encompass known analogues
of
natural nucleotides that hybridize to nucleic acids in a manner similar to
naturally
occurring nucleotides. The term specifically refers to the DNA sequence as
defined in
Figure 1 and given by SEQ ID NO 1 or the complementary strand thereof. Also
within the scope of the present invention are all HBV nucleic acids that
belong to the
new HBV genotype G and that thus differ 10%, more preferably 9%, most
preferably
8% or less from the nucleotide sequence as depicted in Figure 1 or that are at
least
90%, more preferably 91%, most preferably 92% identical to the sequence as
depicted
in Figure 1.
A nucleic acid sequence that "differs 10%, 9%, 8% or less from another nucleic
acid
sequence" is a nucleic acid sequence of which respectively 10%, 9%, 8% or less
of its
total nucleotides are different from the nucleotides of the nucleic acid
sequence it is
compared with.
A nucleic acid sequence that "is at least 90%, 91%, or 92% identical to
another
nucleic acid sequence" is a nucleic acid sequence of which respectively at
least 90%,
91%, or 92% of its total nucleotides are identical to the nucleotides of the
nucleic acid
sequence it is compared with.
As a result of the genetic code, also nucleic acid sequences that show less
than 90%,
more preferably 91 %, most preferably 92% identity to the sequence as depicted
in
Figure 1 can encode the same proteins as is encoded by the nucleotide
sequences of
HBV strains belonging to the new genotype G. Therefore also these nucleic acid
sequences, that are degenerate as a result of the genetic code, fall within
the scope of
the present invention.
In contrast to the known HBV genotypes, the complete genome of the HBV
sequence
of the invention, belonging to the newly identified genotype G, was found to
be 3248
by long (Fig. 4). The preCore region has translational stops at codon 2 (TAA
instead
of CAA) and codon 28 (TAG instead of TGG) (Fig. 5). The Core region is 585
nucleotides long, encoding a Core protein of 195 amino acids, and has a
nucleotide
insert of 36 bp, located after the fifth nucleotide following the Core
translation
initiation point (A at position 1901). Similar to all other genotypes except
genotype A,
the HBV sequence belonging to genotype G showed a 6 nucleotide deletion in the
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carboxy-terminal part of the HBcAg ORF (Figs. 4 and 5). The preS 1 region
contains
354 by (118 amino acids), the presS2 region 165 by (55 amino acids) and the
HBsAg
region 678 by (226 amino acids) (Fig. 6). Like genotype E, the HBV sequence
belonging to genotype G showed a 3 nucleotide deletion (1 amino acid at
position 11;
S Fig. 6) in the amino-terminal part of preS 1.
In accordance, the present invention specifically relates to an HBV
polynucleic acid
as described above, further characterized that its nucleotide sequence differs
from
HBV genotype A, B, C, D, E or F in at least one of the following
characteristics:
(a) it has a length of 3248 basepairs;
(b) it encodes a translational stop codon at amino acids 2 and 28 of the
preCore region;
(c) it has a 36 nucleotide insert in the N-terminal part of the Core region;
(d) it has a 6 nucleotide deletion in the C-terminal part of the HBcAg ORF;
(e) it has a 3 nucleotide deletion in the N-terminal part of the preS 1 ORF.
In this specific embodiment, the HBV polynucleic acid sequence of the
invention thus
has 1, 2, 3, 4 or 5 of the above mentioned characteristics.
In a more specific embodiment, the present invention relates to an HBV
polynucleic
acid as described above, comprising at least one of the following sequences
(Figs. 7
and 8): SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO
48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 53, SEQ ID NO 54,
SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59,
SEQ ID NO 60.
The present invention further relates to a fragment of a polynucleic acid as
described
above characterized that said fragment is unique to the heretofore
unidentified HBV
genotype G and that said fragment contains at least one nucleotide or a
combination
of nucleotides different from known HBV genotypes A, B, C, D, E or F
nucleotide
sequences, or the complement of said fragment, provided that said fragment is
not
equal to the preC/C gene sequence as depicted in Figure 2 (SEQ ID NO 7) or the
preS/S gene sequence as depicted in Figure 3 (SEQ ID NO 8).
Tran et al. (1991) have described the preS/S and preC/C sequences of an HBV
recombinant strain which accidentally differ less than 8% from the preS/S and
preC/C
sequences of the strains of the present invention. In contrast to the newly
identified
HBV genotype G of the present invention, however, this atypical HBV strain was
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described in the context of an event of emergence of and take over by HBV,
with
rearrangements in the preS/S and preC/C genes (Trap et al., 1991). This
atypical HBV
strain was never recognized as a new genotype. In order to study the genotype
G
sequence with respect to such recombination events (as described by Tran et
al. 1991 ),
5 the present inventors have inspected the complete FR1 genome for such events
on the
nucleotide level, and phylogenetically for co-segregation with other known
genotypes.
Evidence for recombination was not found. The absence of recombination events
in
the newly isolated strain and the high prevalence of this new viral variant
indicate that
it concerns a new HBV genotype. The accidentally homologous HBV sequences
10 described by Tran et al. (1991) which have emerged only as a result of a
recombination event, are disclaimed from the present invention. In accordance,
the
HBV preC/C gene sequence as shown in Figure 2 (SEQ ID NO 7; Accession number
M74501; Tran et al. 1991) and the preS/S gene sequence as shown in Figure 3
(SEQ
ID NO 8; Accession number M74499; Tran et al. 1991) do not form part of the
1 S present invention. In the same respect, the HBV preC/C gene sequence as
shown in
Figure 12 (SEQ ID NO 169; Bhat et al. 1990) does not form part of the present
invention and is therefore disclaimed. The HBV preC/C sequence by Bhat et al
was
disclosed and discussed in the context of a chronic Garner who lacked anti-HBc
but
carned exceedingly high levels of HBV DNA in serum. As for Tran et al (1991),
the
link with the presence of a new genotype was never made.
The present invention also relates to a fragment of a polynucleic acid as
described
above characterized that said fragment is a universal fragment which is
comprised in
nucleic acid sequences of HBV genotypes A, B, C, D, E, F and G. Since the
present
invention provides nucleic acid alignments of 7 genotypes, the invention
allows for
the first time to delineate universal HBV nucleic acids sequences,
characteristic for
HBV genotypes A, B, C, D, E, F and G.
The term "universal" as used in the present application means that it is
present in,
corresponds to, or is characteristic to HBV genotypes A, B, C, D, E, F, and G.
The fragment of the invention can be single stranded or double stranded. It
can be
from S to 3247 by long. More preferably this fragment is from 5 to 2600 by
long,
from 5 to 1300 by long, from 5 to 700 by long, from 5 to 500 by long, from 5
to 300
by long and most preferably from 5 to 200 by long. The fragment can be used
for
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16
cloning and expression or the fragment can be used as primer or a probe in an
HBV
typing method.
The present invention therefore also relates to an oligonucleotide primer
comprising a
fragment as described above, with said primer being a HBV genotype G specific
primer, able to act as primer for specifically sequencing or specifically
amplifying a
nucleic acid or part of a nucleic acid characteristic to HBV genotype G. The
present
invention also relates to an oligonucleotide primer comprising a fragment as
described
above, with said primer being a universal primer, able to act as primer for
sequencing
or amplifying nucleic acids from HBV genotypes A, B, C, D, E, F and G. The
term
"primer" refers to a single stranded oligonucleotide sequence capable of
acting as a
point of initiation for synthesis of a primer extension product that is
complementary to
the nucleic acid strand to be copied. The length and the sequence of the
primer must
be such that they allow to prime the synthesis of the extension products.
Preferably,
the length of the primer is about 5-50 nucleotides. More preferably, the
length of the
primer is about 10-30 nucleotides. Most preferably, the length of the primers
is about
20-25 nucleotides. Specific length and sequence will depend on the complexity
of the
required DNA or RNA target, as well as on the conditions at which the primer
is used,
such as temperature and ionic strength.
The present invention also relates to an oligonucleotide probe comprising a
fragment
as described above, with said probe being a HBV genotype G specific probe,
able to
act as a hybridization probe for the specific detection of a nucleic acid or
part of a
nucleic acid characteristic to HBV genotype G. The present invention also
relates to
an oligonucleotide probe comprising a fragment as described above, with said
probe
being a universal probe, able to act as a hybridization probe for nucleic
acids from
HBV genotypes A, B, C, D, E, F and G. The term "probe" refers to a single
stranded
sequence-specific oligonucleotide that has a sequence that is complementary to
the
target sequence in the HBV genome. Preferably, these probes are about 5 to 50
nucleotides long, more preferably from about 10 to 25 nucleotides.
Particularly preferred
lengths of probes include 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24 or 25
nucleotides. The nucleotides used in the probes of the present invention may
be
ribonucleotides, deoxyribonucleotides and modified nucleotides such as
inosine, or
nucleotides containing modified groups that do not essentially alter their
hybridization
characteristics.
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The probes and/or primers according to the invention can be prepared by
cloning
recombinant plasmids containing inserts including the corresponding nucleotide
sequence, if needed followed by excision of the latter from the cloned
plasmids by use
of the adequate nucleases and recovery of the primers and/or probes, e.g. by
fractionation according to molecular weight. The primers and/or probes
according to
the present invention can also be synthesized chemically, for instance by the
conventional phospho-triester method. They can also be part of a branched DNA
capture or detection probe (Sanchez-Pescador et al., 1988; Urdea et al., 1991)
The primers and/or probes of the invention may be labeled. Labeling may be
carried
out by any method known to the person skilled in the art. The nature of the
label may
be isotopic (32P, 3sS, etc.) or non-isotopic (biotin, digoxigenin, etc.) or
may reside on a
branched DNA probe.
The oligonucleotides used as primers and/or probes may also contain or consist
of
nucleotide analogues such as phosphorothiates (Matsukura et al., 1987),
alkylphosphorothiates (Miller et al., 1979) or peptide nucleic acids (Nielsen
et al., 1991;
Nielsen et al., 1993) or may contain intercalating agents (Asseline et al.,
1984). The
introduction of these modifications may be advantageous in order to positively
influence
characteristics such as hybridization kinetics, reversibility of the hybrid-
formation,
biological stability of the oligonucleotide molecules, etc.
As most other variations or modifications introduced into the original DNA
sequences
of primers and/or probes, these variations will necessitate adaptations with
respect to
the conditions under which the oligonucleotide should be used to obtain the
required
specificity and sensitivity. The eventual results of the priming or
hybridization with
these modified oligonucleotides, however, should be essentially the same as
those
obtained with the unmodified oligonucleotides.
In a preferred embodiment of the invention, the probe and/or primer recognizes
one of
the target sequences as indicated in Figure 1.
The term "target sequence" of a probe and/or primer, according to the present
invention, is a sequence within the genome of the newly identified genotype G
that
comprises one or more nucleotides that are unique on their own or as a
combination to
the newly identified genotype G and to which the probe or primer is
complementary
or partially complementary (i.e. with up to 20%, more preferably 15%, more
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18
preferably 10% or most preferably 5% mismatches). It is to be understood that
the
complement of said target sequence is also a suitable target sequence in some
cases.
It should be understood that probes that are designed to specifically
hybridize to a
target sequence of a nucleic acid, may fall within said target sequence or may
to a
large extent overlap with said target sequence (i.e. form a duplex with
nucleotides
outside as well as within said target sequence).
In a very specific embodiment of the invention the target sequence is chosen
within.
the 36 nucleotide insert in the N-terminal part of the Core region of HBV
genotype G:
TAGAACAACTTTGCCATATGGCCTTTTTGGCTTAGA (SEQ ID NO 61)
More specifically, the primer and/or probe of the invention recognizes one or
more of
the following sequences:
TAGAA (SEQ ID NO 62);
AGAAC (SEQ ID NO 63);
GAACA (SEQ ID NO 64);
AACAA (SEQ ID NO 65);
ACAAC (SEQ ID NO 66);
CAACT (SEQ ID NO 67);
AACTT (SEQ ID NO 68);
ACTTT (SEQ ID NO 69);
CTTTG (SEQ ID NO 70);
TTTGC (SEQ ID NO 71 );
TTGCC (SEQ ID NO 72);
TGCCA (SEQ ID NO 73);
GCCAT (SEQ ID NO 74);
CCATA (SEQ ID NO 75);
CATAT (SEQ ID NO '76);
ATATG (SEQ ID NO 77);
TATGG (SEQ ID NO 78);
ATGGC (SEQ ID NO 79);
TGGCC (SEQ ID NO 80);
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GGCCT (SEQ ID NO 81);
GCCTT (SEQ ID NO 82);
CCTTT (SEQ ID NO 83);
CTTTT (SEQ ID NO 84);
TTTTT (SEQ ID NO 85);
TTTTG (SEQ ID NO 86);
TTTGG (SEQ ID NO 87);
TTGGC (SEQ ID NO 88);
TGGCT (SEQ ID NO 89);
GGCTT (SEQ ID NO 90);
GCTTA (SEQ ID NO 91 );
CTTAG (SEQ ID NO 92);
TTAGA (SEQ ID NO 93);
TAGAAC (SEQ ID NO 94);
AGAACA (SEQ ID NO 95);
GAACAA (SEQ ID NO 96);
AACAAC (SEQ ID NO 97);
ACAACT (SEQ ID NO 98);
CAACTT (SEQ ID NO 99);
AACTTT (SEQ ID NO 100);
ACTTTG (SEQ ID NO 1 O l );
CTTTGC (SEQ ID NO 102);
TTTGCC (SEQ ID NO 103);
TTGCCA (SEQ ID NO 104);
TGCCAT (SEQ ID NO 105);
GCCATA (SEQ ID NO 106);
CCATAT (SEQ ID NO 107);
CATATG (SEQ ID NO 108);
ATATGG (SEQ ID NO 109);
TATGGC (SEQ ID NO 110);
ATGGCC (SEQ ID NO 111);
TGGCCT (SEQ ID NO 112);
GGCCTT (SEQ ID NO 113);
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GCCTTT (SEQ ID NO 114);
CCTTTT (SEQ ID NO 115);
CTTTTT (SEQ ID NO 116);
TTTTTG (SEQ ID NO 117);
S TTTTGG (SEQ ID NO 118);
TTTGGC (SEQ ID NO 119);
TTGGCT (SEQ ID NO 120);
TGGCTT (SEQ ID NO 121);
GGCTTA (SEQ ID NO 122);
10 GCTTAG (SEQ ID NO 123);
CTTAGA (SEQ ID NO 124);
TAGAACA (SEQ ID NO 125);
AGAACAA (SEQ ID NO 126);
GAACAAC (SEQ ID NO 127);
1 S AACAACT (SEQ ID NO 128);
ACAACTT (SEQ ID NO 129);
CAACTTT (SEQ ID NO 130);
AACTTTG (SEQ ID NO 131);
ACTTTGC (SEQ ID NO 132);
20 CTTTGCC (SEQ ID NO 133);
TTTGCCA (SEQ ID NO 134);
TTGCCAT (SEQ ID NO 135);
TGCCATA (SEQ ID NO 136);
GCCATAT (SEQ ID NO 137);
CCATATG (SEQ ID NO 138);
CATATGG (SEQ ID NO 139);
ATATGGC (SEQ ID NO 140);
TATGGCC (SEQ ID NO 141);
ATGGCCT (SEQ ID NO 142);
TGGCCTT (SEQ ID NO 143);
GGCCTTT (SEQ ID NO 144);
GCCTTTT (SEQ ID NO 145);
CCTTTTT (SEQ ID NO 146);
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CTTTTTG (SEQ ID NO 147);
TTTTTGG (SEQ ID NO 148);
TTTTGGC (SEQ ID NO 149);
TTTGGCT (SEQ ID NO 150);
TTGGCTT (SEQ ID NO 151);
TGGCTTA (SEQ ID NO 152);
GGCTTAG (SEQ ID NO 153);
GCTTAGA (SEQ ID NO 154);
The invention also relates to the use of a HBV genotype specific primer and/or
a
probe of the invention in a method to detect, in a biological sample, the
presence of
the new HBV genotype G.
The invention also relates to the use of a universal primer and/or a probe of
the
invention in a universal method to detect, in a biological sample, the
presence of
HBV.
The present invention further relates to a method for the detection in a
biological
sample of the presence of a polynucleic acid characteristic to HBV genotype G.
The present invention further relates to a method for HBV genotyping using a
primer
and/or probe of the present invention.
More specifically, the present invention relates to a method as described
above,
further characterized that it comprises the following step:
(i) possibly extracting the nucleic acid present in the biological sample;
(ii) amplifying the nucleic acid characteristic to HBV genotype G;
(iii) detecting the amplified nucleic acid characteristic to HBV genotype G.
Primers used for amplification may be generic primers or genotype G specific
primers. Generic primers allow the amplification of DNA from strains that
belong to
genotype G as well as DNA from other strains belonging to other HBV genotypes.
Genotype G specific primers, in contrast, will allow only the amplification of
the
nucleic acids from strains that belong to genotype G. These genotype G
specific
primers also fall within the scope of the present invention.
The present invention further relates to a method for the universal detection
in a
biological sample of the presence of HBV.
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More specifically, the present invention relates to a universal method as
described
above, further characterized that it comprises the following step:
(i) possibly extracting the nucleic acid present in the biological sample;
(ii) amplifying the nucleic acid;
(iii) detecting the amplified nucleic acid.
Primers used for amplification in said universal method are universal primers
that
allow the amplification of DNA from all genotypes A, B, C, D, E, F and G.
The term "biological sample" as used in the present invention refers to any
biological
material (tissue or fluid) taken either directly from the infected human
being, or after
culturing (enrichment) and containing HBV nucleic acid sequences. Biological
material
may be e.g. expectoration's of any kind, broncheolavages, blood, skin tissue,
biopsies,
sperm, lymphocyte blood culture material, colonies, liquid cultures, fecal
samples, urine,
hepatocytes, etc. More particularly "biological sample" refers to blood serum
or plasma
samples.
The nucleic acids are released, concentrated and/or isolated from the
biological
sample by any method known in the art. Currently, various commercial kits are
available such as the QIAamp Blood Kit from Qiagen (Hilden, Germany) for the
isolation of nucleic acids from blood samples and the 'High pure PCR Template
Preparation Kit' (Roche Diagnostics, Brussels, Belgium). Other well-known
procedures for isolation of DNA or RNA from a biological sample are also
available
(Sambrook et al., 1989).
The nucleic acids of the invention can be amplified by polymerase chain
reaction
(PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al., 1988;
Wu and
Wallace, 1989; Barany, 1991), nucleic acid sequence-based amplification
(NASBA;
Guatelli et al., 1990; Compton, 1991), transcription-based amplification
system (TAS;
Kwoh et al., 1989), strand displacement amplification (SDA; Duck, 1990) or
amplification by means of Q13 replicase (Lomeli et al., 1989) or by any other
suitable
method known in the art, that allows the amplification of nucleic acid
molecules. Also
TMA (Guatelli et al., 1990) or bDNA (Sanchez-Pescador et al., 1988; Urdea et
al.,
1991) techniques can be used in the method of the present invention.
The products of this amplification step can subsequently be used for detection
of the
presence, and/ or for typing of the nucleic acids present in the sample.
Analysis of the
presence and typing of these nucleic acids can be done by any method known in
the
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23
art, such as duplex analysis of the PCR products (Clay et al., 1994), single-
stranded
conformational polymorphism analysis of the PCR product (PCR-SSCP; Yoshida et
al., 1992), sequence-based typing (SBT; Santamaria et al., 1992 and 1993), the
use of
sequence specific primers in PCR reaction (PCR-SSP; Olerup and Zetterquist,
1991),
the use of PCR in combination with sequence-specific oligonucleotide probing
(PCR-
SSOP; Saiki et al., 1986), TMA or bDNA techniques. Sequencing can be performed
via any known sequencing method such as the enzymatic dideoxy method of Sanger
et al (1977) or the chemical method of Maxam and Gilbert (1977, 1980). Kits
and/or
tools for thermal-cycle sequencing, solid-phase sequencing and automated
sequencing
are commercially available. Recent sequencing techniques provide for
simultaneous
sequencing in the 5' and 3' directions. Some examples of automated sequencers
are
the MicroGene ClipperTM 2 Dye and the MicroGene BlasterTM from the OpenGeneTM
system (Visible Genetics Inc., Toronto, Ontario, Canada) and the ABI PRISM~
system (Perkin Elmer Inc., PE Biosystems, PE Applied Biosystems, Foster City,
California, USA). Assay methods that rely on the formation of a hybrid between
the
nucleic acids in the biological sample and the oligonucleotide probe include
Southern
blot, Northern blot or dot blot format (Saiki et al., 1989), the unlabelled
amplified
sample being bound to a membrane, the membrane being incorporated with at
least one
labeled probe under suitable hybridization and wash conditions, and the
presence of
bound probe being monitored. An alternative is a "reverse" format, in which
the
amplified sequence contains a label. In this format, the selected probes are
immobilized
to certain locations on a solid support and the amplified polynucleic acids
are labeled
in order to enable the detection of the hybrids formed. The term "solid
support" can
refer to any substrate to which an oligonucleotide probe can be coupled,
provided that
it retains its hybridization characteristics and provided that the background
level of
hybridization remains low. Usually the solid substrate will be a microtiter
plate (e.g.
in the DEIA technique), a membrane (e.g. nylon or nitrocellulose) or a
microsphere
(bead) or a chip. Prior to application to the membrane or fixation it may be
convenient
to modify the nucleic acid probe in order to facilitate fixation or improve
the
hybridization efficiency. Such modifications may encompass homopolymer
tailing,
coupling with different reactive groups such as aliphatic groups, NHz groups,
SH
groups, carboxylic groups, or coupling with biotin, haptens or proteins.
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A preferred embodiment of the present invention thus relates to a method as
described
above, further characterized that it comprises the following step:
(i) possibly extracting the nucleic acid present in the biological sample;
(ii) amplifying the nucleic acid;
(iii) hybridizing the nucleic acid, possibly under denatured conditions, at
appropriate conditions with one or more probes, with said probes being
possibly attached to a solid substrate;
(iv) possibly washing at appropriate conditions;
(v) detecting the hybrids formed.
In order to obtain fast and easy results, if a multitude of probes is
involved, a reverse
hybridization format may be convenient. In this preferred embodiment the
selected
probes are immobilized to certain locations on a solid support and the
amplified
polynucleic acids are labeled in order to enable the detection of the hybrids
formed.
The present invention further relates to a diagnostic kit for use in a method
as
described above, comprising the following components:
(i) when appropriate, a means for releasing, isolating or concentrating the
nucleic acids present in the sample;
(ii) a primer set or a primer mix, possibly according to the invention;
(iii) a means for detection or typing of the nucleic acid, present in the
sample.
The present invention specifically relates to a diagnostic kit as described
above, said
kit comprising at least one primer and/or at least one probe of the invention.
A
specific and very user-friendly diagnostic kit is the line probe assay
(Stuyver et al.,
1996), comprising the following components:
(i) when appropriate, a means for releasing, isolating or concentrating the
nucleic acids present in said sample;
(ii) a primer pair or a primer mix, possibly according to the invention;
(iii) at least one probe that specifically hybridizes with a nucleic acid
characteristic to HBV genotype G, or at least one universal probe, fixed
to a solid support;
(iv) a hybridization buffer, or components necessary for producing said
buffer;
(v) a wash solution, or components necessary for producing said solution;
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(vi) when appropriate, a means for detecting the hybrids resulting from the
preceding hybridization.
In this embodiment, the selected set of probes is immobilized to a membrane
strip in a
line fashion. Said probes may be immobilized individually or as mixtures to
the
5 delineated locations. The amplified HBV polynucleic acids can be labeled
with biotin,
and the hybrid can then, via a biotin-streptavidine coupling, be detected with
a non-
radioactive color developing system.
The term "hybridization buffer" means a buffer allowing a hybridization
reaction
between the probes and the polynucleic acids present in the sample, or the
amplified
10 products, under the appropriate stringency conditions.
The term "wash solution" means a solution enabling washing of the hybrids
formed
under the appropriate stringency conditions.
The invention also relates to a peptide or a polypeptide having an amino acid
15 sequence encoded by a polynucleic acid of the invention. The terms
"peptide" and
"polypeptide" are used interchangeable throughout the present application and
refer to
a polymer of amino acids (aa) and do not refer to a specific length of the
product.
Thus, oligopeptides, polypeptides, peptides, proteins and precursors are
included
within this definition. More particularly, the polypeptide of the present
invention is a
20 HBV genotype G specific polypeptide such as the preSl, preS2, surface
antigen
HBsAg, preCore precursor protein, HBeAg, HBcAg, the polymerase or the X
protein
of the HBV strains belonging to the newly identified genotype G, or any part
thereof
or any combination thereof, provided that said polypeptide differs in at least
one
amino acid from known polypeptides from HBV strains belonging to genotypes A,
B,
25 C, D, E or F. In a very specific embodiment, the present invention relates
to the
HBcAg or HBeAg of the HBV genotype G or any part thereof. The insert in the
core
region, encoding twelve additional amino acids RTTLPYGLFGLD (SEQ ID NO 155)
may be responsible for a novel mechanism of expression of HBcAg and HBeAg, and
core and a antigens with a unique structure and immunological properties may
exist.
Therefore, the present invention specifically relates to a peptide derived
from the
HBcAg or HBeAg of HBV genotype G comprising one of the following sequences:
RTTLPY (SEQ ID NO 156);
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26
TTLPYG (SEQ ID NO 157);
TLPYGL (SEQ ID NO 158);
LPYGLF (SEQ ID NO 159);
PYGLFG (SEQ ID NO 160);
S YGLFGL (SEQ ID NO 161);
GLFGLD (SEQ ID NO 162);
RTTLPYG (SEQ ID NO 163);
TTLPYGL (SEQ ID NO 164);
TLPYGLF (SEQ ID NO 165);
LPYGLFG (SEQ ID NO 166);
PYGLFGL (SEQ ID NO 167);
YGLFGLD (SEQ ID NO 168);
The present invention further relates to a universal peptide, characterized
that said
peptide is comprised in peptide sequences of HBV genotypes A, B, C, D, E, F
and G.
Since the present invention provides amino acid sequence alignments of 7
genotypes,
the invention allows for the first time to delineate universal HBV amino acid
sequences, characteristic for HBV genotypes A, B, C, D, E, F and G.
Also included within the present invention are post-translational
modifications such as
glycosylation, acetylation, phosphorylation, modifications with fatty acids
and the
like. Also included within the definition are, for example, polypeptides
containing one
or more analogues of an as (including unnatural amino acids), polypeptides
with
substituted linkages, mutated versions or natural sequence variations of the
polypeptides, polypeptides containing disulfide bounds between cysteine
residues,
biotinylated polypeptides as well as other modifications known in the art.
The polypeptides of the invention can be produced by any method known in the
art
such as classical chemical synthesis as described by Houbenweyl (1974) and
Atherton
and Shepard (1989) or by means of recombinant DNA techniques as described by
Sambrook et al. (1989).
In accordance, the present invention also relates to a polypeptide as
described above
which is recombinantly expressed. The term "recombinantly expressed" refers to
the
fact that the peptide is produced by recombinant expression methods be it in
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27
prokaryotes, or lower or higher eukaryotes as discussed below. The present
invention
thus also relates to a method for the production of a recombinant polypeptide
of the
invention comprising the following steps:
(i) transformation of an appropriate cellular host with an expression vector;
(ii) culturing said transformed cellular host under conditions enabling the
expression of the polynucleic acid inserted in the expression vector; and
(iii) harvesting said polypeptide.
Therefore, also comprised within the scope of the present invention is an
expression
vector, comprising a polynucleic acid or part thereof of the invention,
operably linked
to prokaryotic, eukaryotic or viral transcription and translation control
elements. The
term "expression vector" refers to a vector sequence of the type plasmid,
cosmid,
phage or viral DNA wherein a nucleic acid of the invention is inserted, and
which
contains the necessary elements to promote the transcription and translation
of the
latter polypeptide(s) by a host cell.
The present invention also relates to a host cell transformed with an
expression vector
as defined above. The term "host cell" denotes micro-organisms or higher
eukaryotic
cell lines cultured as unicellular entities and refers to cells which can be
or have been
used as recipients for a recombinant vector. The prokaryotes used as host cell
can be
chosen from bacteria such as Escherichia coli, Lactobacillus, Lactococcus,
Salmonella, Streptococcus, Bacillus or Streptomyces. Preferred lower
eukaryotic host
cells are yeasts, particularly species within Saccharomyces,
Schizosaccharomyces,
Kluyveromyces, Pichia (e.g. Pichia pastoris), Hansenula (e.g. Hansenula
polymorpha), Yarowia, Schwanniomyces, Zygosaccharomyces and the like.
Saccharomyces cerevisiae, Saccharomyces carlsbergensis and K. lactis are the
most
commonly used yeast hosts. Fungal host cells include Neurospora crassa. Host
cells
from higher eukaryotes include those from higher animals such as mammals,
reptiles,
insects, and the like. Presently, preferred higher eukaryotic host cells are
derived from
Chinese hamster (e.g. CHO), monkey (e.g. COS and Vero cells), baby hamster
kidney
(BHK), pig kidney (PK15), rabbit kidney 13 cells (RK13), the human
osteosarcoma
cell line 143 B, the human cell line HeLa and human hepatoma cell lines like
Hep G2,
and insect cell lines (e.g. Spodoptera frugiperda). The host cells may be
provided in
suspension as flask cultures, tissue cultures, organ cultures and the like.
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Any of the known purification methods for recombinant peptides can be used for
the
production of the recombinant peptides of the present invention.
The present invention also relates to the use of a HBV genotype G specific
polypeptide of the invention in a method for detecting antibodies specific to
the
heretofore unidentified HBV genotype G, preferably specific to the HBcAg
and/or
HBeAg of the heretofore unidentified HBV genotype G present in a biological
sample.
The present invention also relates to a method for detecting antibodies
specific to the
heretofore unidentified HBV genotype G, preferably specific to the HBcAg
and/or
HBeAg of the heretofore unidentified HBV genotype G present in a biological
sample, comprising the following steps:
(i) contacting the biological sample with a HBV genotype G specific
polypeptide according to the invention;
(ii) detecting the immunological complex formed between said antibodies
and said polypeptide.
The present invention also relates to a universal method for detecting
antibodies
specific to genotypes A, B, C, D, E, F and G present in a biological sample,
comprising the following steps:
(i) contacting the biological sample with a universal polypeptide according
to the invention;
(ii) detecting the immunological complex formed between said antibodies
and said polypeptide.
The present invention also relates to a universal method for HBV genotyping of
a
biological sample, comprising the following steps:
(i) contacting the biological sample with at least one polypeptide according
to the invention;
(ii) detecting the immunological complex formed between said antibodies
and said polypeptide.
Design of the immunoassay is subject to a great deal of variation, and many
formats
are known in the art. Protocols may, for example, use solid supports, or
immunoprecipitation. Most assays involve the use of labeled polypeptides. The
label
may be, for example, enzymatic, fluorescent, chemiluminescent, radioactive, or
dye
molecules. Assays that amplify the signals from the immune complex include the
use
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29
of biotin and avidin or streptavidin, and enzyme-labeled and mediated
immunoassays,
such as ELISA assays.
The immunoassay may be, without limitation, in a heterogeneous or in a
homogeneous format, and of a standard or competitive type. In a heterogeneous
format, the polypeptide is typically bound to a solid matrix or support to
facilitate
separation of the sample from the polypeptide after incubation. Examples of
solid
supports that can be used are nitrocellulose (e.g., in membrane or microtiter
well
form), polyvinyl chloride (e.g., in sheets or microtiter wells), polystyrene
latex (e.g.,
in beads or microtiter plates, polyvinylidene fluoride (known as ImmunolonTM),
diazotized paper, nylon membranes, activated beads, and Protein A beads. For
example, Dynatech ImmunolonTM 1 or ImmunolonTM 2 microtiter plates or 0.25-
inch
polystyrene beads (Precision Plastic Ball) can be used in the heterogeneous
format.
The solid support containing the antigenic peptides is typically washed after
separating it from the biological sample, and prior to detection of the bound
1 S antibodies. Both standard and competitive formats are known in the art.
In a homogeneous format, the test sample is incubated with the combination of
antigens in solution. For example, it may be under conditions that will
precipitate any
antigen-antibody that is formed. Both standard and competitive formats for
these
assays are known in the art.
The present invention also relates to a diagnostic kit for use in a method as
described
above, said kit comprising at least one polypeptide according to the
invention, with
said polypeptide possibly bound to a solid support. In a preferred embodiment
said kit
may comprise a range of said polypeptides, wherein said polypeptides are
attached to
specific locations on a solid substrate. More preferably, said solid support
is a
membrane strip and said polypeptides are coupled to the membrane in the form
of
parallel lines.
The present invention further relates to the polypeptides of the invention for
use as an
active substance in a vaccine or a medicament. Alternatively, anti-idiotypic
antibodies
that specifically bind an antibody of the present invention can be used as
immunogens.
The present invention further relates to the use of a polypeptide or an anti-
idiotypic
antibody as described above for the preparation of a vaccine for the
prevention or
treatment of HBV infections.
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The present invention also relates to a vaccine composition comprising as an
active
substance a polypeptide or an anti-idiotypic antibody of the invention that
can be used
as an inoculum to vaccinate humans (or non human mammals) against infection
with
HBV or to therapeutically vaccinate human (or non human mammal) carriers of
HBV.
S The terms "vaccine" or "vaccine composition" relate to an immunogenic
composition
capable of eliciting protection against HBV, whether partial or complete. The
term "as
an active substance" relates to the component of the vaccine composition that
elicits
protection against HBV. An active substance (i.e. the polypeptides of the
present
invention) can be used as such, in a biotinylated form (as explained in WO
93/18054)
10 and/or complexed to Neutralite Avidin according to the manufacturer's
instruction
sheet (Molecular Probes Inc., Eugene, OR). It should also be noted that a
"vaccine" or
a "vaccine composition" comprises, in addition to an active substance, a
suitable
excipient, diluent, carrier and/or adjuvant that, by them, do not induce the
production
of antibodies harmful to the individual receiving the composition nor do they
elicit
15 protection. Suitable Garners are typically large slowly metabolized
macromolecules
such as proteins, polysaccharides, polylactic acids, polyglycolic acids,
polymeric
amino acids, amino acid copolymers and inactive virus particles. Such Garners
are
well known to those skilled in the art. Preferred adjuvants to enhance
effectiveness of
the composition include, but are not limited to: aluminium hydroxide,
aluminium in
20 combination with 3-0-deacylated monophosphoryl lipid A as described in WO
93/19780, aluminium phosphate as described in WO 93/24148, N-acetyl-muramyl-L-
threonyl-D-isoglutamine as described in U.S. Patent N° 4,606,918, N-
acetyl-
normuramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-
alanine2(1'2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamine and
RIBI
25 (ImmunoChem Research Inc., Hamilton, MT) which contains monophosphoryl
lipid
A, detoxified endotoxin, trehalose-6,6-dimycolate, and cell wall skeleton (MPL
+
TDM + CWS) in a 2% squalene/Tween 80 emulsion. Any of the three components
MPL, TDM or CWS may also be used alone or combined 2 by 2. Additionally,
adjuvants such as Stimulon (Cambridge Bioscience, Worcester, MA) or SAF-1
30 (Syntex), RC259 (Coryxa), SBAS2, SBAS3, SBAS4, SBASS, SBAS6, SBAS7,
SBAS8 (Smith Kline Beecham, Rixensart, Belgium) may be used. Furthermore,
Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA) may be
used for non-human applications and research purposes. A "vaccine composition"
will
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31
further contain excipients and diluents, which are inherently non-toxic and
non-
therapeutic, such as water, saline, glycerol, ethanol, wetting or emulsifying
agents, pH
buffering substances, preservatives, and the like. Typically, a vaccine
composition is
prepared as an injectable, either as a liquid solution or suspension. Solid
forms,
suitable for solution on, or suspension in, liquid vehicles prior to injection
may also be
prepared. The preparation may also be emulsified or encapsulated in liposomes
for
enhancing adjuvant effect. The polypeptides may also be incorporated into
Immune
Stimulating Complexes together with saponins, for example Quil A (ISCOMS).
Vaccine compositions comprise an immunologically effective amount of the
polypeptides of the present invention, as well as any other of the above-
mentioned
components. "Immunologically effective amount" means that the administration
of
that amount to an individual, either in a single dosis or as part of a series,
is effective
for prevention or treatment. This amount varies depending upon the health and
physical condition of the individual to be treated, the taxonomic group of the
1 S individual to be treated (e.g. nonhuman primate, primate, etc.), the
capacity of the
individual's immune system to mount an effective immune response, the degree
of
protection desired, the formulation of the vaccine, the treating's doctor
assessment, the
strain of the infecting HBV, and other relevant factors. It is expected that
the amount
will fall in a relatively broad range that can be determined through routine
trials.
Usually, the amount will vary from 0.01 to 1000 ~g/dose, more particularly
from 0.1
to 100 pg/dose. The vaccine compositions are conventionally administered
parenterally, typically by injection, for example, subcutaneously or
intramuscularly.
Additional formulations suitable for other methods of administration include
oral
formulations and suppositories. Dosage treatment may be a single dose schedule
or a
multiple dose schedule. The vaccine may be administered in conjunction with
other
immunoregulatory agents. It should be noted that a vaccine might also be
useful for
treatment of an individual, in which case it is called a "therapeutic
vaccine".
The present invention also relates to a HBV genotype G specific ligand or to a
HBV
genotype G specific antibody specifically raised upon immunization with at
least one
HBV genotype G specific polypeptide of the invention, with said antibody being
specifically reactive with said polypeptide and not reactive with a peptide
characteristic for HBV genotype A, B, C, D, E and/or F. In a preferred
embodiment,
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32
the antibody specifically recognizes the HBV polymerise, X protein, preCore,
HBcAg, HBeAg, preS 1, preS2 or HBsAg of HBV genotype G.
The present invention also relates to a universal ligand or to a universal
antibody
specifically raised upon immunization with a universal polypeptide of the
invention,
with said antibody being reactive with said universal polypeptide. In a
preferred
embodiment, the antibody specifically recognizes the HBV polymerise, preCore,
HBcAg, HBeAg, preSl, preS2 or HBsAg protein of genotypes A, B, C, D, E, F and
G.
The antibody may be polyclonal or monoclonal. Also fragments derived from
these
monoclonal antibodies such as Fab, F(ab)'2, scFv ("single chain variable
fragment") and
other antibody-like constructs that retain the variable region of the
antibody, providing
they have retained the original binding properties, fall within the scope of
the present
invention and can be used in a method of the present invention. Such fragments
are
commonly generated by, for instance, enzymatic digestion of the antibodies
with papain,
pepsin, or other proteases. It is well known to the person skilled in the art
that
monoclonal antibodies, or fragments thereof, can be modified for various uses.
Also
mini-antibodies and multivalent antibodies such as diabodies, triabodies,
tetravalent
antibodies and peptabodies can be used in a method of the invention. The
preparation
and use of these fragments and multivalent antibodies has been described
extensively in
International Patent Application WO 98/29442. The monoclonal antibodies used
in a
method of the invention may be humanized versions of the mouse monoclonal
antibodies made by means of recombinant DNA technology, departing from the
mouse
and/or human genomic DNA sequences coding for H and L chains or from cDNA
clones
coding for H and L chains. Alternatively the monoclonal antibodies used in a
method of
the invention may be human monoclonal antibodies. The term "humanized
antibody"
means that at least a portion of the framework regions of an immunoglobulin is
derived
from human immunoglobulin sequences. The antibodies used in a method of the
present
invention may be labeled by an appropriate label of the enzymatic,
fluorescent, or
radioactive type.
The present invention further relates to the use of a HBV genotype G specific
antibody or ligand of the invention in a method for detecting a HBV genotype G
specific polypeptide of the invention, preferably for detecting the HBcAg
and/or
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33
HBeAg of the heretofore unidentified HBV genotype G, present in a biological
sample.
The present invention also relates to a method for detecting a HBV genotype G
specific polypeptide of the invention, preferably for detecting the HBcAg
and/or
HBeAg of the heretofore unidentified HBV genotype G present in a biological
sample, comprising the following steps:
(i) bringing said biological sample into contact with a HBV genotype G
specific antibody or ligand of the invention recognizing said HBV
genotype G specific polypeptide, under conditions being suitable for
producing an antigen-antibody or an antigen-ligand complex; and
(ii) detecting the immunological binding of said antibody or ligand to said
polypeptide.
The present invention further relates to the use of a universal antibody or
ligand of the
invention in a method for detecting a universal polypeptide of the invention,
present
in a biological sample.
The present invention also relates to a method for detecting a universal
polypeptide of
the invention, present in a biological sample, comprising the following steps:
(i) bringing said biological sample into contact with a universal antibody or
ligand of the invention recognizing said universal polypeptide, under
conditions being suitable for producing an antigen-antibody or an
antigen-ligand complex; and
(ii) detecting the immunological binding of said antibody or ligand to said
polypeptide.
The process for the detection of the immunological binding can then be carried
out by
bringing together said polypeptide-antibody complex formed by the polypeptide
of
the invention and the antibody recognizing said polypeptide with:
(i) a secondary antibody (or detector antibody) recognizing a specific
epitope of the polypeptide-antibody complex but not recognizing the
primary antibody alone;
(ii) a marker either for specific tagging or coupling with said secondary
antibody, with said marker being any possible marker known to the
person skilled in the art;
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(iii) appropriate buffer solutions for carrying out the immunological reaction
between the secondary antibody and the polypeptide-primary antibody
complex and/or the bound secondary antibody and the marker on the
other hand.
Advantageously, the antibodies used in the invention are in an immobilized
state on a
suitable support. The secondary antibody itself may carry a marker or a group
for
direct or indirect coupling with a marker. Alternatively, the present process
may be
put into practice by using any other immunoassay format known to the person
skilled
in the art.
The term "epitope" refers to that portion of the polypeptide-antibody complex
that is
specifically bound by an antibody-combining site. Epitopes may be determined
by
any of the techniques known in the art or may be predicted by a variety of
computer
prediction models known in the art.
The expression "recognizing", "reacting with", "immunological binding" or
"producing a polypeptide-antibody complex" as used in the present invention is
to be
interpreted that binding between the polypeptide and the antibody occurs under
all
conditions that respect the immunological properties of the antibody and the
polypeptide of the invention.
The terms "specifically recognizing", "specifically binding to", "specifically
reactive
with" or the like as used in the present invention in respect of antibodies
and ligands
are to be interpreted that the antibody or the ligand binds to a polypeptide
that is
unique to the heretofore unidentified HBV genotype G (and not to any
polypeptide of
the HBV genotypes A to F) or that the antibody or the ligand binds to a
polypeptide
that is universal to HBV genotypes A, B, C, D, E, F and G but not to any other
polypeptide.
The present invention also relates to a diagnostic kit for use in the
detection of a
heretofore unidentified HBV genotype G present in a biological sample, said
kit
comprising at least one HBV genotype G specific antibody according to the
invention.
In a specific embodiment the present invention relates to a diagnostic kit for
use in the
detection of a heretofore unidentified HBV genotype G present in a biological
sample,
said kit comprising:
(r) at least a primary antibody recognizing a HBV genotype G specific
polypeptide of the invention (or capturing antibody);
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(ii) possibly, a secondary antibody (or detector antibody) forming an
immunological complex with an epitope of the polypeptide-primary
antibody complex but not with the primary antibody alone;
(iii) possibly, a marker either for specific tagging or coupling with said
5 secondary antibody;
(iv) possibly, appropriate buffer solutions for carrying out the immunological
reaction between the primary antibody and the polypeptide of the
invention, between the secondary antibody and the polypeptide-primary
antibody complex and/or between the bound secondary antibody and the
10 marker;
(v) possibly, for standardization purposes, a purified or synthetic peptide
that
is specifically recognized by the antibodies of the kit.
The present invention also relates to a diagnostic kit for use in the
universal detection
of HBV present in a biological sample, said kit comprising at least one
universal
15 antibody according to the invention.
In a specific embodiment the present invention relates to a diagnostic kit for
use in the
universal detection of HBV present in a biological sample, said kit
comprising:
(i) at least a primary antibody recognizing a universal polypeptide of the
invention (or capturing antibody);
20 (ii) possibly, a secondary antibody (or detector antibody) forming an
immunological complex with an epitope of the polypeptide-primary
antibody complex but not with the primary antibody alone;
(iii) possibly, a marker either for specific tagging or coupling with said
secondary antibody;
25 (iv) possibly, appropriate buffer solutions for carrying out the
immunological
reaction between the primary antibody and the polypeptide of the
invention, between the secondary antibody and the polypeptide-primary
antibody complex and/or between the bound secondary antibody and the
marker;
30 (v) possibly, for standardization purposes, a purified or synthetic peptide
that
is specifically recognized by the antibodies of the kit.
The present invention further relates to polypeptide, an antibody or a ligand
of the
invention for use as a medicament.
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36
The present invention further relates to the use of a peptide, an antibody or
a ligand of
the present invention for the preparation of a medicament to prevent humans of
HBV
infection or to treat humans with HBV infection.
The present invention also relates to a pharmaceutical composition or
medicament
comprising as an active substance a peptide, an antibody or a ligand of the
present
invention that can be used for the treatment of HBV infection.
The term "medicament" or "pharmaceutical composition" (both terms can be used
interchangeable) refers to a composition comprising an antibody according to
the
present invention, possibly in the presence of suitable excipients known to
the skilled
man such as saline, Ringer's solution, dextrose solution, Hank's solution,
fixed oils,
ethyl oleate, S% dextrose in saline, substances that enhance isotonicity and
chemical
stability, buffers and preservatives. The "medicament" may be administered by
any
suitable method within the knowledge of the skilled man. The preferred route
of
administration is parenterally. In parental administration, the medicament of
this
invention will be formulated in a unit dosage injectable form such as a
solution,
suspension or emulsion, in association with the pharmaceutically acceptable
excipients as defined above. However, the dosage and mode of administration
will
depend on the individual. Generally, the medicament is administered so that
the
antibody is given at a dose between 1 ~g/kg and 10 mg/kg, more preferably
between
10 ~g/kg and 5 mg/kg, most preferably between 0.1 and 2 mg/kg. Preferably, it
is
given as a bolus dose. Continuous infusion may also be used. If so, the
medicament
may be infused at a dose between 5 and 20 ~g/kg/minute, more preferably
between 7
and 15 pg/kg/minute. Dosage forms suitable for oral administration include but
are
not limited to tablets, pills, capsules, caplets, granules, powders, elixirs,
syrups,
solutions and aqueous or oil suspensions. A suitable daily dose of the active
compound for administration to human beings is generally from about 1 mg to
about
5000 mg, more usually from about 5 mg to about 1000 mg, given in a single dose
or
in divided doses at one or more times during the day.
It should also be clear that the pharmaceutical composition of the present
invention
might comprise a functionally equivalent variant of the antibody of the
invention. The
latter terms refer to a molecule which contains the antibody of the invention,
to which
certain modifications have been applied, and which retains all or part of its
biological
properties. Such modifications include but are not limited to the addition of
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37
polysaccharide chains, the addition of certain chemical groups, the addition
of lipid
moieties, the fusion with other peptide or protein sequences and the formation
of
intramolecular cross-links.
The present invention also provides methods for identifying compounds or
agents
(herein referred to as "test compound") that can be used to treat disorders
characterized by (or associated with) HBV infection. These methods are also
referred
to herein as "drug screening assays" or "bioassays."
In one embodiment, the drug screening assay may include the step of screening
a test
compound for the ability to interact with (e.g., bind to) an HBV genotype G
protein,
or any functionally equivalent part thereof, to modulate the interaction of an
HBV
protein and a target molecule. Typically, the assay is a cell-free assay
including the
following steps:
(i) combining the HBV genotype G protein of the invention or an
immunogenic fragments thereof, and a test compound under conditions
which allow for interaction of (e.g., binding of) the test compound to the
HBV genotype G protein or a portion thereof to form a complex;
(ii) detecting the formation of a complex.
The ability of the test compound to interact with (e.g. bind to) the HBV
genotype G
protein or portion thereof is indicated by the presence of the test compound
in the
complex. Formation of complexes between the HBV genotype G protein and the
test
compound can be quantitated, for example, using a standard immunoassay. The
HBV
genotype G protein or its immunogenic fragment employed in such a test, may be
free
in solution, affixed to a solid support, borne on a cell surface, or located
intracellularly.
In another embodiment, the invention provides a screening assay to identify
test
compounds which modulate (e.g., stimulate or inhibit) the interaction (and
most likely
HBV genotype G protein activity as well) between an HBV genotype G protein
and:
- a molecule (target molecule) to which the HBV genotype G protein
normally interacts; or
- antibodies which specifically recognize the HBV genotype G protein.
Target molecules include proteins in the same signaling path as the HBV
genotype G
protein, e.g., proteins which may function upstream (including both
stimulators and
inhibitors of activity) or downstream of the HBV protein signaling pathway [Zn-
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38
fingers, protease activity, regulators of cysteine redox status]. Typically,
the assay is a
cell-free assays, which includes the following steps:
(i) combining an HBV genotype G protein or a immunogenic fragment
thereof, an HBV protein target molecule (e.g., an HBV protein ligand) or
a specific antibody and a test compound under conditions which allow for
interaction of (e.g., binding of) the HBV genotype G protein or a portion
thereof with the target molecule or the antibody;
(ii) detecting the formation of a complex which includes the HBV genotype G
protein and the target molecule or the antibody, or detecting the
interaction/reaction of the HBV genotype G protein and the target
molecule or antibody.
To perform the above-described drug-screening assay, it is feasible to
immobilize
either the HBV genotype G protein or its target molecule to facilitate
separation and
to accommodate automation of the assay. Detection of complex formation can
include
direct quantitation of the complex by, for example, measuring inductive
effects of the
HBV genotype G protein. A statistically significant change, such as a
decrease, in the
interaction of the HBV genotype G protein and the target molecule (e.g., in
the
formation of a complex between the HBV genotype G protein and the target
molecule) in the presence of a test compound (relative to what is detected in
the
absence of the test compound) is indicative of a modulation (e.g., stimulation
or
inhibition) of the interaction between the HBV genotype G protein and the
target
molecule. Modulation of the formation of complexes between the HBV genotype G
protein and the target molecule can be quantitated using, for example, an
immunoassay.
It should be clear that a modulator of the interaction between a HBV genotype
G
protein and a target molecule, when identified by any of the herein-described
methods, is contemplated in the invention.
Another technique for drug screening which provides for high throughput
screening
of compounds having suitable binding affinity to the HBV genotype G protein is
described in detail in "Determination of Amino Acid Sequence Antigenicity" by
Geysen HN, WO Application 84/03564, published on 13/09/84, and incorporated
herein by reference. In summary, large numbers of different small peptide test
compounds are synthesized on a solid substrate, such as plastic pins or some
other
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39
surface. The protein test compounds are reacted with fragments of HBV genotype
G
protein and washed. Bound HBV genotype G protein is then detected by methods
well
known in the art.
This invention also contemplates the use of a competitive drug screening
assays in
which neutralizing antibodies capable of binding a HBV genotype G protein
specifically compete with a test compound for binding the HBV genotype G
protein.
In this manner, the antibodies can be used to detect the presence of any
protein that
shares one or more antigenic determinants with the HBV genotype G protein.
In yet another embodiment, the invention provides a method for identifying a
compound capable of modulating HBV nucleic acid expression and/or HBV protein
activity. Methods for assaying the ability of a test compound to modulate the
expression of a HBV nucleic acid or activity of a HBV genotype G protein are
typically cell-based assays. However, HBV genotype G infected animals are also
contemplated herein. HBV genotype G infected or transfected cells which
transduce
signals via a pathway involving a HBV genotype G protein can be induced to
overexpress an HBV protein in the presence and absence of a test compound.
These
HBV genotype G infected cells also form part of the present invention. Said
HBV
genotype G infected cells and animals are further also referred to as HBV
genotype G
infected clones.
Throughout this specification and the claims which follow, unless the context
requires
otherwise, the word "comprise", and variations such as "comprises" and
"comprising", will be understood to imply the inclusion of a stated integer or
step or
group of stated integers or steps but not to the exclusion of any other
integer or step or
group of integers or steps. The disclosures of the various patent
applications, patents
and/or publications that are cited, as well as the references cited in these
publications,
are incorporated by reference herein. This, however, does not imply that the
content
of all of these disclosures is to be seen as part of common general knowledge.
The present invention claims priority under 35 USC 119 from the US patent
application No. 60/169,287, which was filed on 7 December 1999. The present
invention further claims priority under PCT Article 8 from US patent
application No.
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60/169,287, which was filed on 7 December 1999 and from EP 99870252.6, which
was filed on 3 December 1999.
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41
EXAMPLES
Example 1: Prevalence of the different HBV genotypes
1.1 Sample collection
A total of 121 HBV-positive plasma samples were collected in France (n=39) and
the
United States (n=82). The samples were divided into aliquots and stored at -
20°C until
use. Samples were taken from chronic HBV carriers and were randomly selected
as
they became available.
1.2 HBV DNA extraction and amplification
HBV DNA was extracted from 100 ~1 serum sample using the High Pure PCR
Template Preparation kit (Roche Diagnostics, Brussels, Belgium) essentially as
previously described (Stuyver et al., 1999). A PCR fragment was generated
covering
the preS 1, preS2 and HBsAg region by use of primers HBPrl and HBPr135 (outer
PCR) followed by a nested reaction using HBPr2 and HBPr94 (Fig. 4; Table 1).
Outer
PCR amplified the viral DNA over 40 cycles, with denaturation at 94°C
for 30 s,
annealing at 50°C for 30 s, and elongation at 72 °C for 30 s.
Samples negative in first-
round PCR were further amplified with nested PCR primers for 35 cycles with
the
same thermal profile.
1.3 HBV genotyping
The 121 serum samples were further genotyped by using a research version of
the
HBV genotyping Line Probe Assay (LiPA) (Van Geyt et al., 1998). The results
are
summarized in Table 2. A very typical but previously unrecognized reactivity
pattern
was obtained for 2 samples from Europe and 11 samples from the United States:
hybridization reactions were observed on probe 140 (specifically designed for
genotype A), probe 148 (designed for genotypes A and B), probe 80 (designed
for
genotypes C, D and E) and probe 239 (designed for genotypes B and E) (Van Geyt
et
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42
al. 1998). Because this mixed hybridization pattern did not allow unique type
recognition, further characterization was done via sequence analysis.
Example 2: Characterization of the samples that showed a mixed hybridization
pattern
2.1 HBV DNA extraction and amplification
HBV DNA was extracted from 100 ~1 serum sample using the High Pure PCR
Template Preparation kit (Roche ' Diagnostics, Brussels, Belgium) essentially
as
previously described (Stuyver et al., 1999). The complete genome of HBV was
amplified using the Expand High Fidelity PCR system (Roche Diagnostics,
Brussels,
Belgium). The amplification was performed on 5 ~1 of the extracted DNA with
the
primers HBPr108 and HBPr109 (Table 1). A 45 ~1 reaction mix was made,
containing
5 ~1 10 x Expand High Fidelity PCR system buffer, 2.6 U Expand High Fidelity
PCR
system enzyme mix, 200 ~,M dNTPs, 300 nM of each primer and sterile HzO.
Amplification was performed with denaturation at 94 °C for 40 s,
annealing (after
shifting to 60 °C in 50 s) for 1 min and elongation (after shifting to
72 °C in 15 s) for
4 min, with an increment of 5 s/cycle (Giinther et al., 1998). Two shorter PCR
fragments were also generated: (i) the first amplicon covered the preSl,
preS2, and
HBsAg region and was amplified using primers HBPrl and HBPr135 (outer PCR)
followed by a nested reaction using HBPr2 and HBPr94 (Table 1 ); and (ii) the
second
amplicon covered the preCore/Core region and was amplified by means of a hemi-
nested set of PCR primers (HBPr86 and HBPr303, followed by HBPr87 and
HBPr303). Outer PCR amplified the viral DNA over 40 cycles, with denaturation
at
94 °C for 30 s, annealing at 50 °C for 30 s and elongation at 72
°C for 30 s. Samples
negative in first-round PCR were further amplified with nested PCR primers for
35
cycles with the same thermal profile.
2.2 Sequencing
Sequencing was performed on an automated DNA sequencer ABI 377 (PE Applied
Biosystems, Foster City, CA, USA), using fluorescence-labeled
dideoxynucleotide
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chain terminators (ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction
Kit with AmpliTaq DNA polymerase FS; PE Applied Biosystems). The primers used
for sequencing the complete genome are summarized in Table 1. The primers used
to
sequence the shorter PCR fragments are the same as the amplification primers.
S One European virus isolate (FR1) was selected for whole genome sequencing
(Fig. 1;
SEQ ID NO 1), whereas the preCore/Core and preS/S genes were sequenced from
another seven samples (Figs. 7 and 8; SEQ ID NO 43-56).
2.3 Homology percentages with other genotypes
In order to compare the genetic relatedness of the FR1 strain with 36 other
complete
HBV genomes, homology percentages were calculated. The FR1 sequence showed an
average homology of 87.1 % (min. 86.2%, max. 87.7%) with genotype A (five
sequences); 86.6% (min. 86.5%, max. 86.6%) with genotype B (four sequences);
86.5% (min. 86.0%, max. 87.0%) with genotype C (14 sequences); 86.9% (min.
86.3%, max. 87.3%) with genotype D (eight sequences); 88.3% (min. 88.2%, max.
88.4%) with genotype E (two sequences); and 84.7% (min. 84.5%, max. 84.8%)
with
genotype F (three sequences).
2.4 Phylogenetic distance with other genotypes
In order to compare the genetic relatedness of the FR1 strain with 36 other
complete
HBV genomes, also the phylogenetic distances were calculated. Phylogenetic
distances between the six recognized HBV genotypes (36 sequences) were
compared
to each other and to the FR1 strain (Fig. 9). A clear difference emerged
between the
phylogenetic distances (i) within one genotype (distance range of 0.01 - 0.06)
and (ii)
between different genotypes (distance range 0.08 - 0.17). Using a t-test for
the mean
and a distance of 0.08 as border value between genotypes, FR1 was found to be
significantly different from genotypes A - F (range 0.11 - 0.17; P < 0.019).
Phylogenetic trees were created and analyzed by distance matrix comparison
using
DNADIST, Neighbor and Drawgram software programs of PHYLIP version 3.5c
(Felsenstein, 1993). Statistical analysis (t-test for the mean) was performed
using
MedCalc Version 4.05-Windows 95 (Medcalc Software, Mariakerke, Belgium). A P
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44
value of < 0.05 was considered to be statistically significant. Complete
genome
sequences representing the different genotypes were retrieved from the
GenBank;
their accession numbers are indicated in Figure 10. Phylogenetic trees of the
complete
genome sequences (Fig. 10a), as well as of the individual ORF [Fig. lOb for
the
S surface region (preSl, preS2, HBsAg); data not shown for the other ORFs]
were
constructed, illustrating that FR1 is indeed located on a separate branch.
Fig. lOb
further illustrates that the HBV samples from the USA and France, are closely
related
to each other. Based on these calculations and illustrated by means of
phylogenetic
trees, FR1 and the other virus strains shown belong to a new HBV genotype,
called
genotype G.
Example 3: Characterization of the genome structure and open reading frames
in HBV strains belonging to genotype G
The complete genome structure of FR1 was similar to that described for the
known
HBV genotypes, but was found to be 3248 by long (Fig. 4).
The preCore region has translational stops at codon 2 (TAA instead of CAA) and
codon 28 (TAG instead of TGG) in all eight isolates sequenced. Based on the
presence of this dual stop codon, the presence of HBeAg is generally not
expected.
Paradoxically, sample FR2 showed the presence of HBeAg in the plasma. The Core
region is 585 nucleotides long and encodes a Core protein of 195 amino acids
(Fig. 5).
In contrast to the other genotypes, the Core region had a nucleotide insert of
36 bp,
located after the fifth nucleotide following the Core translation initiation
point (A at
position 1901). Therefore, the insert in the core region encoding twelve
additional
amino acids RTTLPYGLFGLD (SEQ ID NO 155) may be responsible for a novel
mechanism of expression of HBcAg and HBeAg, and core and a antigens with
unique
structures and immunological properties may exist. Genotype G, as well as all
other
genotypes except A, showed a 6 nucleotide deletion in the carboxy-terminal
part of
the HBcAg ORF (Figs. 4 and 5).
The preSl region contains 354 by (118 amino acids), the preS2 region 165 by
(55
amino acids), and the HBsAg region 678 by (226 amino acids) (Fig. 6). Like
genotype
E, genotype G strains showed a 3 nucleotide deletion (1 amino acid at position
11;
Fig. 6) in the amino-terminal part of preS 1. Based on the presence of a
lysine (K) at
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HBsAg position 122, a lysine (K) at position 160 and a proline (P) at position
127, the
serological subtype of this genotype G strain was predicted to be adw2.
The polymerise region of this genotype contains 2526 by (842 amino acids). The
deletion in the carboxy-terminal part of HBcAg, as well as the deletion at the
amino
5 terminus of preS 1 affects the numbering of the polymerise protein. The
methionine
residue, which is prone to changes during lamivudine therapy, is located at
position
549 in the highly conserved YMDD motif (Bartholomeusz et al., 1998). Figure 6
also
shows the exact numbering for this methionine residue in the other genotypes.
10 Example 4: Method for the detection of HBV genotype G
4.1 HBV DNA extraction and amplification
HBV DNA was extracted from serum samples with the High Pure PCR Template
15 Preparation Kit (Roche Diagnostics, Brussels, Belgium). A nested set of PCR
primers
was designed, allowing the amplification of the HBsAg region and generating a
341
by fragment (outer sense primer HBPr 134; outer antisense primer HBPr 135;
nested
sense primer HBPr 75; nested antisense primer HBPr 94; table 1). All primers
were
tagged with a biotin group at the 5' end. Outer PCR amplified the DNA over 40
20 cycles (30 s at 95°C; 30 s at 45°C; 30 s at 72°C).
Samples negative in first round PCR
were further amplified with nested PCR primers for 35 cycles with the same
thermal
profile.
4.2 Design of a LiPA for HBV genotyping
A total of 20 genotype-specific probes are designed. Probes designed for the
identification of genotypes A, B, C, D, E and F are described in Van Geyt et
al.
(1998). By way of example, probes specifically selected for the identification
of HBV
genotype G nucleic acid sequences recognize the regions indicated as target A
or
target B in Figure 1. After enzymatic addition of a poly d(T) tail, the
selected
oligonucleotide probes are applied as horizontal lines on nitrocellulose
membranes.
One line of biotinylated DNA is applied alongside as a positive control.
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4.3 Testing of the LiPA with a cloned reference panel and with representative
clinical samples
The assay is essentially carned out as described for the HCV line probe assay
(Stuyver et al., 1996). The reactivity of the different probes on the 1TBV
genotyping
LiPA is assessed with a cloned reference panel and with clinical samples.
Figure 11
shows the result for some representative samples after hybridization of their
PCR
product to the probes on the LiPA. PCR fragments from HBV strain FR1 hybridize
with the generic probes 140, 148, 80 and 239 and with the genotype G specific
probes
selected to recognize target A or target B as indicated in Figure 1.
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