U.S. patent application number 10/320786 was filed with the patent office on 2003-09-25 for hiv-1 group o antigens and uses thereof.
This patent application is currently assigned to INNOGENETICS N.V.. Invention is credited to Delaporte, Eric, Peeters, Martine, Saman, Eric, Vanden Haesevelde, Marleen.
Application Number | 20030180759 10/320786 |
Document ID | / |
Family ID | 8231024 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030180759 |
Kind Code |
A1 |
Delaporte, Eric ; et
al. |
September 25, 2003 |
HIV-1 group O antigens and uses thereof
Abstract
The current invention relates to new HIV-1 group O antigens,
nucleic acids encoding them, and the use of said antigens and/or
nucleic acids as reagents in the diagnosis and prophylaxis of AIDS.
It also relates to new HIV-1 group O strains comprising these
antigens.
Inventors: |
Delaporte, Eric; (Saint Jean
de Cuculles, FR) ; Peeters, Martine; (Saint Jean de
Cuculles, FR) ; Saman, Eric; (Bornem, BE) ;
Vanden Haesevelde, Marleen; (Oudenaarde, BE) |
Correspondence
Address: |
Matthew L. Madsen
HOWREY SIMON ARNOLD & WHITE, LLP
750 Bering Drive
Houston
TX
77057-2198
US
|
Assignee: |
INNOGENETICS N.V.
|
Family ID: |
8231024 |
Appl. No.: |
10/320786 |
Filed: |
December 16, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10320786 |
Dec 16, 2002 |
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09462917 |
Apr 3, 2000 |
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6511801 |
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09462917 |
Apr 3, 2000 |
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PCT/EP98/04522 |
Jul 20, 1998 |
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Current U.S.
Class: |
435/5 ; 435/69.1;
435/7.1; 435/974; 435/975; 436/536 |
Current CPC
Class: |
Y10S 530/826 20130101;
C07K 14/005 20130101; C12N 2740/15022 20130101; A61K 2039/51
20130101; C12N 2740/16122 20130101; C12N 7/00 20130101; A61K 39/00
20130101; C12N 2740/16021 20130101; Y10S 435/974 20130101; Y10S
435/975 20130101 |
Class at
Publication: |
435/6 ; 435/7.1;
435/974; 435/975; 436/536; 435/5; 435/69.1 |
International
Class: |
C12Q 001/70; C12Q
001/68; G01N 033/53; C12P 021/06; G01N 033/536; G01N 033/564 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 18, 1997 |
EP |
97870110.0 |
Claims
1. Antigen derived from the gp160 env precursor protein of a new
HIV-1 group O strain comprising at least one amino acid sequence
chosen from the following group of sequences:
9 VQQMKI, (SEQ ID NO 53) KIGPMSWYSMG, (SEQ ID NO 54) MGLEKN, (SEQ
ID NO 55) IQQMKI, (SEQ ID NO 56) KIGPLAWYSMG, (SEQ ID NO 57)
MGLERN, (SEQ ID NO 58) QSVQEIKI, (SEQ ID NO 59) KIGPMAWYSIG, (SEQ
ID NO 60) IGIGTT, (SEQ ID NO 61) VQEIQT, (SEQ ID NO 62)
QTGPMAWYSIH, (SEQ ID NO 63) IHLRTP, (SEQ ID NO 64) IQEIKI, (SEQ ID
NO 65) KIGPMSWYSMG, (SEQ ID NO 66) MGIGQE, (SEQ ID NO 67) SVQELRI,
(SEQ ID NO 68) RIGPMAWYSMT, (SEQ ID NO 69) MTLERD, (SEQ ID NO 70)
SVQEIPI, (SEQ ID NO 136)
and/or at least one amino acid sequence chosen from the following
group of sequences:
10 RNQQLLNLWGCKGRLIC, (SEQ ID NO 71) CKGRLICYTSVQWNM, (SEQ ID NO
72) LWGCKGRIVC, (SEQ ID NO 73) SLWGCKGKLIC, (SEQ ID NO 74) CKGKSIC,
(SEQ ID NO 75) CKGKIVC, (SEQ ID NO 76) CRGRQVC, (SEQ ID NO 77)
CKGRLICYTSVH, (SEQ ID NO 79) CKGNLIC, (SEQ ID NO 80) CKGKMIC, (SEQ
ID NO 81) CKGRVVC, (SEQ ID NO 82)
or a fragment of said antigen, said fragment consisting of at least
8, preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 50 up to the maximum number of contiguous amino
acids of the amino acid sequence of said antigen, with said
fragment being characterized by the fact that it specifically
reacts with antibodies raised against said antigen.
2. Antigen according to claim 1, characterized by an amino acid
sequence comprising at least one of the following amino acid
sequences:
11 (SEQ ID NO 83) CERPGNNSIQQMKIGPLAWYSMGLERNKSSISRLAYC, (SEQ ID NO
84) CERPGNNSIQQMKIGPMAWYSMGLERNKSSI- SRLAYC, (SEQ ID NO 85)
CERPGNQSVQEIKIGPMAWYSIG- IGTTPANWSRIAYC, (SEQ ID NO 86)
CERPGNQSVQEIKIGPMAWYSIGIGTTPTYNWSRIAYC, (SEQ ID NO 87)
CVRPWNQTVQEIQTGPMAWYSIHLRTPLANLSRIAYC, (SEQ ID NO 88)
CQRPGNLTIQEIKIGPMSWYSMGIGQEDHSKSRNAYC, (SEQ ID NO 89)
CERPYYQSVQELRIGPMAWYSMTLERDRAGSDIRAAYC, (SEQ ID NO 90)
CERPGNHTVQQMKIGPMSWYSMGLEKNNTSSRRAFC, (SEQ ID NO 135)
CERTWNQSVQEIPIGPMAWYSMSVELDLNT- TGSRSADC,
and/or at least one amino acid sequence chosen from the following
group of sequences:
12 DQQLLNLWGCKGRIVCYTSVKWN, (SEQ ID NO 91)
NQQLLNLWGCKGRLVCYTSVKWNK, (SEQ ID NO 92) NQQLLNLWGCKGRLVCYTSVKWNN,
(SEQ ID NO 138) NQQRLNLWGCKGKMICYTSVPWN, (SEQ ID NO 93)
NQQLLNLWGCKGKSICYTSVKWN, (SEQ ID NO 94) NQQLLNLWGCKGRLICYTSVQWN,
(SEQ ID NO 95) NQQRLNLWGCKGKMICYTSVKWN, (SEQ ID NO 96)
NQQLLNLWGCKGNLICYTSVKWN, (SEQ ID NO 97) NQQLLNLWGCRGRQVCYTSVIWN,
(SEQ ID NO 98) SQQLLNLWGCKGRLICYTSVHWN, (SEQ ID NO 99)
NQQLLNLWGCKGRIVCYTSVKWN, (SEQ ID NO 100) NQQLLNSWGCKGKIVCYTAVKWN,
(SEQ ID NO 101) NQQLLSLWGCKGKLICYTSVKWN, (SEQ ID NO 102)
NQQLLNLWGCKGRLVCYTSVQWN, (SEQ ID NO 137)
or a fragment of said antigen, said fragment consisting of at least
8, preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 50 up to the maximum number of contiguous amino
acids of the amino acid sequence of said antigen, with said
fragment being characterized by the fact that it specifically
reacts with antibodies raised against said antigen.
3. Antigen according to any of claims 1 to 2, characterized by an
amino acid sequence comprising at least one of the amino acid
sequences represented by SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ
ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16,
SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO
26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO 34, SEQ ID
NO 36, SEQ ID NO 38, SEQ ID NO 40 as shown in the alignment on FIG.
1, and/or at least one of the amino acid sequences represented by
SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO
50, or SEQ ID NO 52 as shown in the alignment on FIG. 2, and/or the
amino acid sequence represented by SEQ ID NO 134, or a fragment of
said antigen, said fragment consisting of at least 8, preferably 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50
up to the maximum number of contiguous amino acids of any of the
sequences represented by SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ
ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16,
SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO
26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO 34, SEQ ID
NO 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ
ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, or SEQ ID NO
134 with said antigen fragment being characterized by the fact that
it specifically reacts with antibodies raised against the antigen
from which it is derived.
4. A polynucleic acid encoding an antigen according to any of
claims 1 to 3, and more particularly a polynucleic acid comprising
a nucleotide sequence chosen from the group of (I) a nucleotide
sequence represented by SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ
ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ
ID NO 17, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 23, SEQ ID NO 25,
SEQ ID NO 27, SEQ ID NO 29, SEQ ID NO 31, SEQ ID NO 33, SEQ ID NO
35, SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO 41, SEQ ID NO 43, SEQ ID
NO 45, SEQ ID NO 47, SEQ ID NO 49, SEQ ID NO 51, SEQ ID NO 106 or
(ii) a nucleotide sequence complementary to a sequence according to
(I), or (iii) a nucleotide sequence showing at least 95%,
preferably 96%, 97%, 98% and most preferably 99% homology to the
fill length of a sequence according to (I), or (iv) a nucleotide
sequence according to (I) whereby T is replaced by U, or (v) a
nucleotide sequence according to (I) whereby at least one
nucleotide is substituted by a nucleotide analogue.
5. A nucleic acid fragment consisting of a sequence of at least 15,
preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50
contiguous nucleotides of the sequence of a polynucleic acid
according to claim 4, with said nucleic acid fragment being
characterized by the fact that it selectively hybridizes to said
polynucleic acid and/or selectively amplifies said polynucleic
acid.
6. A virus strain belonging to HIV-1 group O, comprising in its
genome a nucleic acid according to claim 4, and more particularly
comprising in its genome the RNA equivalent of one of the DNA
sequences represented by SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ
ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ
ID NO 17, SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 23, SEQ ID NO 25,
SEQ ID NO 27, SEQ ID NO 29, SEQ ID NO 31, SEQ ID NO 33, SEQ ID NO
35, SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO 106 and/or one of the DNA
sequences represented by SEQ ID NO 41, SEQ ID NO 43, SEQ ID NO 45,
SEQ ID NO 47, SEQ ID NO 49, SEQ ID NO 51, and/or a variant sequence
of the above-mentioned DNA sequences, said variant sequence showing
at least 95% homology with the entire length of one of the
above-mentioned sequences.
7. A virus strain according to claim 6, deposited at the ECACC on
Jun. 13, 1997 under accession number V97061301, V97061302 or
V97061303, or deposited at the ECACC on Jul. 13, 1998, under
provisional accession number V98071301 or V98071302.
8. A polynucleic acid isolated from an HIV-1 group O strain
according to any of claims 6 to 7.
9. An antigen isolated from an HIV-1 group O strain according to
any of claims 6 to 7.
10. An antibody, preferably a monoclonal antibody, raised against
an antigen or antigen fragment according to any of claims 1 to 3,
or claim 9, with said antibody recognizing specifically the antigen
or the antigen fragment to which it has been raised.
11. A method for detecting the presence of an HIV-1 infection, said
method comprising the detection of antibodies against HIV-1,
including HIV-1 group O, using an antigen or antigen fragment
according to any of claims 1 to 3, or claim 9, and/or the detection
of viral antigen originating from HIV-1, including HIV-1 group O,
using an antibody according to claim 10, and/or the detection of
viral nucleic acid originating from HIV-1, including HIV-1 group O,
using a nucleic acid or nucleic acid fragment according to claims 4
or 5, or claim 8, in a biological sample.
12. A kit for the detection of the presence of an HIV-1 infection,
comprising at least one of the antigens or antigen fragments
according to any of claims 1 to 3, or claim 9, and/or at least one
of the nucleic acids or nucleic acid fragments according to claim 4
or 5, or claim 8 and/or an antibody according to claim 10.
13. A vaccine composition which provides protective immunity
against an HIV-1 infection, including an HIV-1 type O infection,
comprising as an active principle at least one antigen or antigen
fragment according to claims 1 to 3, or 9, or at least one nucleic
acid according to claims 4 to 5, or 8 or a virus like particle
(VLP) comprising at least one antigen or antigen fragment according
to claims 1 to 3, or 9, or an attenuated form of at least one of
the HIV-1 type O strains according to claims 6 to 7, said active
principle being combined with a pharmaceutically acceptable
carrier.
Description
[0001] The current invention relates to new HIV-1 group O antigens,
nucleic acids encoding them, and the use of said antigens and/or
nucleic acids as reagents in the diagnosis and prophylaxes of AIDS.
It also relates to new HIV-1 group strains comprising these
antigens.
[0002] The human immunodeficiency virus (HIV) is the responsible
agent for the acquired immunodeficiency syndrome (AIDS) in humans.
AIDS is usually associated with two distinct types of HIV: HIV-1
and HIV-2, initially described by Gallo et al. (1984) and
Barr-Sinoussi et al. (1983) on the one hand, and Clavel et al.
(1986) on the other hand. Although both types, HIV-1 and HIV-2,
cause a dysfunction of the immune system and induce similar
clinical symptoms in infected persons, they are genetically
distinct (Clavel et al. 1986) Epidemiological studies have shown
that the prevalence of HIV-2 infection is confined mainly to West
Africa, whereas HIV-1 infection is a world wide problem. Numerous
HIV-1 isolates have been obtained and sequenced from diverse
geographical locations. At present, at least ten distinct subgroups
or clades (A to J) of HIV-1 have been described, equidistantly
related in phylogenetic analysis of the env- and/or gag-gene
(Kostrikis et al. 1995; Louwagie et al. 1993; Myers et al
1995).
[0003] More recently, HIV-1 group O (for "Outlier") strains have
been described as divergent viruses, belonging to an independent
cluster (Charneau et al. 1994; Gurtler et al. 1994; Myers et al
1995; Sharp et al. 1994; Vanden Haesevelde et al. 1996), when
compared to the vast majority of worldwide HIV-1 strains classified
as group M (for "Major"). Although these two groups of viruses
share the same genomic structure, the elevated level of divergence
between them supports the hypothesis of independent origins.
[0004] Most of the currently described group O strains have been
characterized from Cameroonian patients or from patients who have
travelled in Cameroon (De Leys et al. 1990; Gurtler et al. 1994;
Loussert-Ajaka et al. 1995; Vanden Haesevelde et al. 1996). Group O
infection is not restricted to Cameroon and its neighbouring
countries, but it has also been documented in West, East, and
Southern Africa (Peeters et al. 1996; Peeters et al. submitted). In
addition, cases of group O infection have been described in several
European countries (France, Spain, Germany, Norway) and in the USA
(Centres for Disease control and Prevention 1996; Charneau et al.
1994; Hampl et al 1995; Soriano et al. 1996).
[0005] Several hypotheses have been developed to explain the
paradoxical observation that HIV-1 has been present in African
countries for many decades (probably about a century) and that it
has only become apparent over the past 15 years. The answer should
probably take in account numerous parameters such as demographic,
sociologic, ethologic, ethnologic, and virologic parameters. In a
mathematical model, May and Anderson (1990) suggest that initial
chains of infection were found in isolated populations at low rates
with some `sparks` thrown in the neighbouring villages, and the
exponential epidemic has started when there was a sufficient number
of fire-boxes. To date, no differences were observed between HIV-1
group M and O pathogenic potential even though a limited number of
patients infected by these latter strains have been reported.
However some of them have already died or reached stage IV in the
CDC classification (Charneau et al. 1994; Gurtler et al. 1994;
Loussert-Ajaka et al. 1995). It is possible that group O epidemics,
compared to group M, could be rampant at this time. In the next
years, it will therefore be extremely important to monitor the
prevalence of these viruses, in Africa but also in the developed
countries, to detect them as early as possible and to prevent a new
HIV epidemic.
[0006] HIV-1 group O strains present a public health challenge
since they are documented to give incomplete and atypical HIV-1
Western blot profiles (Charneau et al. 1994; Gurtler et al. 1994).
Some commercially available ELISA or rapid tests were unable to
detect HIV-antibodies in HIV-1 group O infected patients
(Loussert-Ajaka et al 1994; Simon et al. 1994). The distribution of
group O infections may be much more wide spread than currently
thought, because of a lack of adequate detection techniques.
Moreover, whereas HIV-1 group M strains have been extensively
studied and characterized as to their genetic variability, there is
at present no clear view on the genetic diversity of strains
belonging to HIV-1 group O.
[0007] At present, sequence information on the complete genome is
only available for the prototype isolates of HIV-1 group O, namely
ANT70 (Vanden Haesevelde et al. 1994), MVP-5180 (Gurtler et al.
1994), and VAU (Charneau et al. 1994). Some additional HIV-1 group
O strains have been sequenced in the gag and env regions (for
example WO 96/27013, WO 96/12809, EP 0727483).
[0008] HIV-viruses show a high degree of genetic variability. In
the case of HIV-1 viruses it is more or less accepted that at least
one nucleotide change occurs during one replication cycle. Certain
regions of the genome, for example those encoding structurally or
enzymatically important proteins, may be rather conserved, but
other regions, especially the env-region, may be subject of very
high genetic variability.
[0009] The envelope proteins of HIV are the viral proteins most
accessible to immune attack, and much attention has been directed
towards elucidating their structure and function. The env gene
encoding the envelope proteins consists of hypervariable sequences
(V-regions) alternated by more constant regions (C-regions)
(Starcich et al, 1986; Willey et al, 1986). The envelope protein is
first synthesized as a heavily glycosylated precursor protein
(gp160), which is later cleaved by a non-viral protease to generate
a transmembrane protein, also referred to as gp41, and an outer
surface protein often referred to as gp120. One particular region
of the gp120 glycoprotein derived from the HIV-1 virus type has
been studied extensively, namely the third hypervariable domain
(V3) also known as the principal neutralizing determinant (PND)
(Javaherian et al., 1989). The V3 domain of HIV-1 contains a loop
structure of 35 amino acids (V3-loop) which is formed by a
cysteine-cysteine disulfide bridge (Leonard et al. 1990). The gp41
protein contains an immunodominant domain (ID) as found in all
retroviruses. For HIV-viruses, this domain has been divided in two
distinct regions, corresponding to an immunosuppressive peptide
(ISU) of about 17 aa, and a cysteine loop being the principal
immunodominant domain (PID). The delineation of these respective
regions in the gp41 protein is demonstrated in FIG. 1.
[0010] The genetic variability of HIV-viruses considerably
complicates both diagnosis and prevention of HIV-infection. Sera
from patients infected with unknown types of HIV-virus, may contain
antibodies which are not detected by the current assay methods,
which are based on (poly)peptide sequences of known viral strains.
The detection of virus or viral antigen in certain samples, like
organs for transplantation, or blood transfusion samples, may be
missed due to the presence of hitherto unknown variant types.
Variation may occur in those genomic regions which are considered
to be important in future vaccines. Finally, it is not known at
present if different genomic types may influence the course of the
AIDS disease, i.e. its virulence and/or susceptibility for
therapeutics.
[0011] Therefore, there is a constant need for characterization and
sequencing of new HIV-strains, and especially of new HIV-1 group O
strains, which until now have only scarcely been characterized.
Information on the genetic variability of this "Outlier" group may
enable a more rational approach for optimization of diagnostic
tests and for development of vaccines. Especially the variability
of certain regions in the genome, known to be important target
regions for the immune response, or for certain therapeutic drugs,
is of utmost importance. New sequencing data may require the
revision of existing diagnostic assays, and/or the development of
new assays. Depending on the situation, it may be important to
obtain a general detection of all HIV-infected samples, with a low
number of false positives and false negatives, or to be able to
differentiate different types of HIV-infection (such as HIV-1 group
M, HIV-1 group O, HIV-2).
[0012] It is the aim of the current invention to provide new
nucleic acid and peptide sequences originating from HIV-1 group O
strains.
[0013] It is more specifically the aim of the current invention to
provide nucleic acid and peptide sequences corresponding to the
env-region of new HIV-1 group O strains, more particularly
corresponding to the gp160 env-precursor protein region, and most
particularly to the C2V3 region and the gp41 region.
[0014] It is also an aim of the present invention to provide for
new viral strains belonging to HIV-1 group O.
[0015] It is moreover an aim of the present invention to provide
for antigens derived from said new HIV-1 group O strains.
[0016] It is also an aim of the current invention to provide for
nucleic acids derived from said new HIV-1 group O strains.
[0017] It is also an aim of the present invention to provide
antibodies reacting specifically with the antigens from the new
HIV-1 group O strains.
[0018] It is moreover an aim of the present invention to provide
for probes hybridizing specifically with the nucleic acids of the
new HIV-1 group O strains.
[0019] It is moreover an aim of the present invention to use said
antigens and/or antibodies and/or probes in a test for detecting
the presence of HIV-infection and/or to differentiate different
types of HIV-infection.
[0020] It is thus also an aim of the present invention to provide
for assays enabling the detection and/or differentiation of
HIV-infections.
[0021] It is finally also an aim of the present invention to
provide for vaccine compositions providing protection against
AIDS.
[0022] The following definitions serve to illustrate the terms and
expressions used in the different embodiments of the present
invention as set out below:
[0023] The term "polynucleic acid" corresponds to either
double-stranded or single-stranded cDNA or genomic DNA or RNA,
containing at least 10, 20, 30, 40 or 50 contiguous nucleotides.
Single stranded polynucleic acid sequences are always represented
in the current invention from the 5' end to the 3' end.
[0024] Polynucleic acids according to the invention may be prepared
by any method known in the art for preparing polynucleic acids
(e.g. the phosphodiester method for synthesizing oligonucleotides
as described by Agarwal et al. (1972), the phosphotriester method
of Hsiung et al. (1979), or the automated diethylphosphoroamidite
method of Baeucage et al. (1981)). Alternatively, the polynucleic
acids of the invention may be isolated fragments of naturally
occurring or cloned DNA, cDNA or RNA.
[0025] The term "oligonucleotide" refers to a single stranded
nucleic acid comprising two or more nucleotides, and less than 100
nucleotides. The exact size of an oligonucleotide depends on the
ultimate function or use of said oligonucleotide. For use as a
probe or primer the oligonucleotides are preferably about 5-50
nucleotides long, more preferably 10-30 nucleotides long.
[0026] The oligonucleotides according to the present invention can
be formed by cloning of recombinant plasmids containing inserts
including the corresponding nucleotide sequences, if need be by
cleaving the latter out from the cloned plasmids upon using the
adequate nucleases and recovering them, e.g. by fractionation
according to molecular weight. The probes according to the present
invention can also be synthesized chemically, e.g. by automatic
synthesis on commercial instruments sold by a variety of
manufacturers.
[0027] The nucleotides as used in the present invention may be
ribonucleotides, deoxyribonucleotides and modified nucleotides such
as inosine or nucleotides containing modified groups which do not
essentially alter their hybridisation characteristics. Moreover, it
is obvious to the man skilled in the art that any of the
below-specified probes can be used as such, or in their
complementary form, or in their RNA form (wherein T is replaced by
U).
[0028] The oligonucleotides used as primers or probes may also
comprise or consist of nucleotide analogues such as
phosphorothioates (Matsukura et al., 1987), alkylphosphorothioiates
(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).
[0029] As most other variations or modifications introduced into
the original DNA sequences of the invention, these variations will
necessitate adaptions with respect to the conditions under which
the oligonucleotide should be used to obtain the required
specificity and sensitivity. However the eventual results of the
hybridisation or amplification will be essentially the same as
those obtained with the unmodified oligonucleotides.
[0030] 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,
immobilization to solid phase etc.
[0031] The term "probe" refers to single stranded sequence-specific
oligonucleotides which have a sequence which is sufficiently
complementary to hybridize to the target sequence to be
detected.
[0032] Preferably said probes are 90%, 95% or more homologous to
the exact complement of the target sequence to be detected. These
target sequences may be genomic DNA, genomic RNA or messenger RNA,
or amplified versions thereof.
[0033] The term "hybridizes to" refers to preferably stringent
hybridization conditions, allowing hybridisation between sequences
showing at least 90%, 95% or more homology with each other.
[0034] The term "primer" refers to a single stranded DNA
oligonucleotide sequence capable of acting as a point of initiation
for synthesis of a primer extension product which 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 primer is about
5-50 nucleotides long. Specific length and sequence will depend on
the complexity of the required DNA or RNA targets, as well as on
the conditions of primer use such as temperature and ionic
strength. The fact that amplification primers do not have to match
exactly with the corresponding template sequence to warrant proper
amplification is amply documented in the literature (Kwok et al.,
1990).
[0035] The amplification method used can be either polymerase chain
reaction (PCR; Saiki et al., 1988), ligase chain reaction (LCR;
Landgren et al., 1988; Wu & 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; Walker et al., 1992) or amplification by means of
Q.beta. replicase (Lizardi et al., 1988; Lomeli et al., 1989) or
any other suitable method to amplify nucleic acid molecules.
[0036] The term "complementary" nucleic acids as used in the
current invention means that the nucleic acid sequences can form a
perfect base paired double helix with each other.
[0037] The terms "polypeptide" and "peptide" are used
interchangeably throughout the specification and designate a linear
series of amino acids connected one to the other by peptide bonds
between the alpha-amino and carboxy groups of adjacent amino acids.
Polypeptides can be of a variety of lengths, either in their
natural (uncharged) forms or in a charged form (=salt form), and
either free of modifications such as glycosylation, side chain
oxidation, or phosphorylation or containing these modifications.
Preferably the peptides of the invention are less than 100 amino
acids in length, more preferably less than 50, and even less than
30 amino acids long. It is well understood in the art that amino
acid sequences contain acidic and basic groups, and that the
particular ionization state exhibited by the peptide is dependent
on the pH of the surrounding medium when the protein is in
solution, or that of the medium from which it was obtained if the
protein is in solid form. Also included in the definition are
proteins modified by additional substituents attached to the amino
acids side chains, such as glycosyl units, lipids, or inorganic
ions such as phosphates, as well as modifications relating to
chemical conversions of the chains, such as oxidation of sulfhydryl
groups. Thus, "polypeptide" or its equivalent terms is intended to
include the appropriate amino acid sequence referenced, subject to
those of the foregoing modifications which do not destroy its
functionality.
[0038] The polypeptides of the invention, and particularly the
fragments, can be prepared by classical chemical synthesis.
[0039] The synthesis can be carried out in homogeneous solution or
in solid phase.
[0040] For instance, the synthesis technique in homogeneous
solution which can be used is the one described by Houbenweyl in
the book entitled "Methode der organischen chemie" (Method of
organic chemistry) edited by E. Wunsh, vol. 15-I et II. THIEME,
Stuttgart 1974.
[0041] The polypeptides of the invention can also be prepared in
solid phase according to the methods described by Atherton and
Shepard in their book entitled "Solid phase peptide synthesis" (IRL
Press, Oxford, 1989).
[0042] The polypeptides according to this invention can also be
prepared by means of recombinant DNA techniques as described by
Maniatis et al., Molecular Cloning: A Laboratory Manual, New York,
Cold Spring Harbor Laboratory, 1982). In that case the polypeptides
are obtained as expression products of the nucleic acids encoding
said polypeptides. The expression occurs in a suitable host cell
(eukaryotic or prokaryotic) which has been transformed with a
vector in which the nucleic acid encoding the polypeptide has been
inserted (called "insert"). The nucleic acid insert may have been
obtained through classical genomic cloning techniques (screening of
genomic libraries, shotgun cloning etc . . . ), or by amplification
of the relevant part in the viral genome, using suitable primer
pairs and, for example, the polymerase chain reaction, or by DNA
synthesis.
[0043] The word "antigen" refers to a molecule which provokes an
immune response (also called "immunogen"), or which can be
recognized by the immune system (also called "antigen sensu
strictu"). The immune response or the immune recognition reaction
can be of the cellular or humoral type. The antigens of the current
invention are all polypeptides or peptides, and therefore, the
words "antigen" and "(poly)peptide" may be used interchangeably
throughout the current invention.
[0044] The term "antigenic determinant" or "epitope" refers to that
portion of an antigenic molecule 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 from the art.
[0045] The terms "homologous" and "homology" are used in the
current invention as synonyms for "identical" and "identity"; this
means that amino acid sequences which are e.g. said to be 55%
homologous, show 55% identical amino acids in the same position
upon alignment of the sequences. The same definition holds for
homologous nucleic acid sequences, i.e. nucleic acid sequences
which are e.g. said to be 55% homologous, show 55% identical base
pairs in the same position upon alignment of the sequences.
[0046] The aims of the present invention have been met by the
following embodiments.
[0047] The present invention provides for an antigen, derived from
the gp160-env precursor protein of a new HIV-1 group O strain, and
characterized by an amino acid sequence comprising at least one of
the following sequences:
1 VQQMIKI, (SEQ ID NO 53) KIGPMSWYSMG, (SEQ ID NO 54) MGLEKN, (SEQ
ID NO 55) IQQMKI, (SEQ ID NO 56) KIGPLAWYSMG, (SEQ ID NO 57)
MGLERN, (SEQ ID NO 58) QSVQEIKI, (SEQ ID NO 59) KIGPMAWYSIG, (SEQ
ID NO 60) IGIGTT, (SEQ ID NO 61) VQEIQT, (SEQ ID NO 62)
QTGPMAWYSIH, (SEQ ID NO 63) IHLRTP, (SEQ ID NO 64) IQEIKI, (SEQ ID
NO 65) KIGPMSWYSMG, (SEQ ID NO 66) MGIGQE, (SEQ ID NO 67) SVQELRI,
(SEQ ID NO 68) RIGPMAWYSMT, (SEQ ID NO 69) MTLERD, (SEQ ID NO 70)
SVQEIPI, (SEQ ID NO 136)
[0048] and/or at least one amino acid sequence chosen from the
following group of sequences RNQQLLNLWGCKGRLIC (SEQ ID NO 71),
2 RNQQLLNLWGCKGRLIC, (SEQ ID NO 71) CKGRLICYTSVQWNM, (SEQ ID NO 72)
LWGCKGRIVC, (SEQ ID NO 73) SLWGCKGKLIC, (SEQ ID NO 74) CKGKSIC,
(SEQ ID NO 75) CKGKIVC, (SEQ ID NO 76) CRGRQVC, (SEQ ID NO 77)
CKGRLICYTSVH, (SEQ ID NO 79) CKGNLIC, (SEQ ID NO 80) CKGKMIC, (SEQ
ID NO 81) CKGRVVC, (SEQ ID NO 82)
[0049] or a fragment of said antigen, said fragment consisting of
at least 8, preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous
amino acids of the amino acid sequence of said antigen, and being
characterized by the fact that it specifically reacts with
antibodies raised against said antigen.
[0050] The term "derived from" signifies that the antigen contains
a fragment of the gp160 env precursor protein.
[0051] The expression "specifically reacts with" means that the
antigen fragment is specifically recognized by antibodies raised
against the antigen from which it is derived. Specificity of
reaction may be preferably demonstrated using monoclonal antibodies
raised against the antigen of the invention. Specificity of
polyclonal antibodies may be obtained after absorption of said
antibodies with the corresponding antigens of other HIV-1 group O
strains, in order to eliminate non-specific antibodies (=cross
reactive antibodies) present in the polyclonal mixture. The
expression "specifically react with" also means that sera taken
from patients infected with the HIV-1 group O strain from which the
antigen of the invention originates, show a preferential reaction
with the antigen or antigen fragment of the invention, as compared
to the reactivity with a corresponding antigen or antigen fragment
of other HIV-1 group O strains (=control), under comparable
reaction conditions. This preferential reaction may be measured
quantitatively (e.g. ELISA absorption values) and should result in
reactivity values which are at least 20%, 30%, 40% and preferably
50% higher than the reactivity with the control antigen. In
practice, this means that the selected fragments of the
above-mentioned antigens will always show at least one amino acid
difference when compared in an alignment with the sequence of
corresponding antigens of other HIV-1 group O isolates, such as
ANT70, MVP5180, VAU or others.
[0052] The above-mentioned amino acid sequences SEQ ID NO 53 to 70
and 136 originate from the central region in the V3 loop of the
gp160-env precursor protein of new HIV-1 group O strains, while the
amino acid sequences represented by SEQ ID NO 71-77 and 79-82
originate from the gp41-principal immunodominant domain (PID) of
the gp160-env precursor protein of the same HIV-1 group O
strains.
[0053] The current invention also provides for antigens consisting
of any of the amino acid sequences represented by SEQ ID NO 53-70,
136, 71-77, 79-82, or consisting of an amino acid sequence
according to any of SEQ ID NO 53-70, 136, 71-77, 79-82, whereby
said sequence is extended at its N-terminal and/or C-terminal end
with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino
acids.
[0054] The invention further provides for an antigen as described
above, characterized by an amino acid sequence comprising at least
one of the following amino acid sequences:
3 (SEQ ID NO 83) CERPGNNSIQQMKIGPLAWYSMGLERNKSSISRLAYC, (SEQ ID NO
84) CERPGNNSIQQMKIGPMAWYSMGLERNKSSI- SRLAYC, (SEQ ID NO 85)
CERPGNQSVQEIKIGPMAWYSIG- IGTTPANWSRIAYC, (SEQ ID NO 86)
CERPGNQSVQEIKIGPMAWYSIGIGTTPTYNWSRIAYC, (SEQ ID NO 87)
CVRPWNQTVQEIQTGPMAWYSIHLRTPLANLSRIAYC, (SEQ ID NO 88)
CQRPGNLTIQEIKIGPMSWYSMGIGQEDHSKSRNAYC, (SEQ ID NO 89)
CERPYYQSVQELRIGPMAWYSMTLERDRAGSDIRAAYC, (SEQ ID NO 90)
CERPGNHTVQQMKIGPMSWYSMGLEKNNTSSRRAFC, (SEQ ID NO 135)
CERTWNQSVQEIPIGPMAWYSMSVELDLNT- TGSRSADC,
[0055] and/or at least one amino acid sequence chosen from the
following group of sequences:
4 DQQLLNLWGCKGRIVCYTSVKWN, (SEQ ID NO 91) NQQLLNLWGCKGRLVCYTSVKWNK,
(SEQ ID NO 92) NQQLLNLWGCKGRLVCYTSVKWNN, (SEQ ID NO 138)
NQQRLNLWGCKGKMICYTSVPWN, (SEQ ID NO 93) NQQLLNLWGCKGKSICYTSVKWN,
(SEQ ID NO 94) NQQLLNLWGCKGRLICYTSVQWN, (SEQ ID NO 95)
NQQRLNLWGCKGKMICYTSVKWN, (SEQ ID NO 96) NQQLLNLWGCKGNLICYTSVKWN,
(SEQ ID NO 97) NQQLLNLWGCRGRQVCYTSVIWN, (SEQ ID NO 98)
SQQLLNLWGCKGRLICYTSVHWN, (SEQ ID NO 99) NQQLLNLWGCKGRIVCYTSVKWN,
(SEQ ID NO 100) NQQLLNSWGCKGKIVCYTAVKWN, (SEQ ID NO 101)
NQQLLSLWGCKGKLICYTSVKWN, (SEQ ID NO 102) NQQLLNLWGCKGRLVCYTSVQWN,
(SEQ ID NO 137)
[0056] or a fragment of said antigen, said fragment consisting of
at least 8, preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 50 up to the maximum number of contiguous
amino acids of the amino acid sequence of said antigen, and being
characterized by the fact that it specifically reacts with
antibodies raised against said antigen.
[0057] The above-mentioned amino acid sequences SEQ ID NO 83 to 90
and 135 represent the V3 loop region of the gp160-env precursor
protein of new HIV-1 group O strains, while the amino acid
sequences SEQ ID NO 91 to 102, 137 and 138 originate from the
gp41-immunodominant domain (ID) of the gp160-env precursor protein
of the same HIV-1 group O strains.
[0058] The current invention also provides for antigens consisting
of any of the amino acid sequences represented by SEQ ID NO 83-102,
135, 137 and 138 or consisting of an amino acid sequence according
to any of SEQ ID NO 83-102, 135, 137 and 138, whereby said sequence
is extended at its N-terminal and/or C-terminal end with at least
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino acids.
[0059] The invention further provides for antigens as
above-defined, characterized by an amino acid sequence comprising
at least one of the amino acid sequences represented by SEQ ID NO
2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO
12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID
NO 22, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ
ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40 as
shown in the alignment on FIG. 1, and/or at least one of the amino
acid sequences represented by SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO
46, SEQ ID NO 48, SEQ ID NO 50, or SEQ ID NO 52 as shown in the
alignment on FIG. 2, and/or the amino acid sequence represented by
SEQ ID NO 134, or a fragment of said antigen, said fragment
consisting of at least 8, preferably 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up to the maximum number of
contiguous amino acids of any of the sequences represented by SEQ
ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ
ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20,
SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO
30, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID
NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ
ID NO 50, SEQ ID NO 52, or SEQ ID NO 134, with said antigen
fragment characterized by the fact that it specifically reacts with
antibodies raised against the antigen from which it is derived.
[0060] Furthermore, the invention provides for an antigen as
above-defined, characterized by an amino acid sequence consisting
of at least one of the following sequences: SEQ ID NO 2, SEQ ID NO
4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO
14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID
NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 32, SEQ
ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42,
SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO
52, or the amino acid sequence represented by SEQ ID NO 134 or a
fragment of said antigen, said fragment consisting of at least 8,
preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 50 up to the maximum number of contiguous amino acids
of any of the sequences represented by SEQ ID NO 2, SEQ ID NO 4,
SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14,
SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO
24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ ID NO 32, SEQ ID
NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ
ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52,
or SEQ ID NO 134, with said antigen fragment characterized by the
fact that it specifically reacts with antibodies raised against the
antigen from which it is derived.
[0061] It is to be noted that all the above-mentioned amino acid
sequences originate from HIV-1 group O strains, which have until
now never been described. More particularly, as is shown further in
the examples section, the new amino acid sequences originate from
the following strains:
[0062] The amino acid sequences represented by SEQ ID NO 2, 4, 42,
73, 59, 60, 61, 73, 85, 86, 100 originate from the gp160 env
precursor antigen isolated from a HIV-1 group O strain termed
MP340, or a quasi-species thereof.
[0063] The amino acid sequences represented by SEQ ID NO 6, 8, 44,
56, 57, 58, 82, 83, 84, 138 originate from the gp160 env precursor
antigen isolated from a HIV-1 group O strain termed FABA, or
alternatively termed MP331, or a quasi-species thereof.
[0064] The amino acid sequences represented by SEQ ID NO 10, 12,
46, 62, 63, 64, 73, 87, 100 originate from the gp106 env precursor
antigen isolated from a HIV-1 group O strain termed MP450, or a
quasi-species thereof.
[0065] The amino acid sequences represented by SEQ ID NO 14, 16,
48, 65, 66, 67, 76, 88, 101 originate from the gp160 env precursor
antigen isolated from a HIV-1 group O strain termed MP448, or a
quasi species thereof.
[0066] The amino acid sequences represented by SEQ ID NO 18, 50,
53, 54, 55, 73, 90, 91 originate from the gp160 env precursor
antigen isolated from a HIV-1 group O strain termed 189, or a
quasi-species thereof.
[0067] The amino acid sequences represented by SEQ ID NO 40, 52,
68, 69, 70, 71, 89, 95 originate from the gp160 env precursor
antigen isolated from a HIV-1 group O strain termed MP539, or a
quasi-species thereof.
[0068] The amino acid sequences represented by SEQ ID NO 20 and 92
originate from the gp160 env precursor antigen isolated from a
HIV-1 group O strain termed 320, or a quasi-species thereof.
[0069] The amino acid sequences represented by SEQ ID NO 22, 80 and
97 originate from the gp160 env precursor antigen isolated from a
HIV-1 group O strain termed BSD422, or a quasi-species thereof.
[0070] The amino acid sequences represented by SEQ ID NO 24, 79 and
99 originate from the gp160 env precursor antigen isolated from a
HIV-1 group O strain termed KGT008, or a quasi-species thereof.
[0071] The amino acid sequence represented by SEQ ID NO 26
originates from the gp160 env precursor antigen isolated from a
HIV-1 group O strain termed MP575, or a quasi-species thereof.
[0072] The amino acid sequences represented by SEQ ID NO 28, 72 and
95 originate from the gp160 env precursor antigen isolated from a
HIV-1 group O strain termed BSD189, or a quasi-species thereof.
[0073] The amino acid sequences represented by SEQ ID NO 30, 77 and
98 originate from the gp160 env precursor antigen isolated from a
HIV-1 group O strain termed BSD649, or a quasi-species thereof.
[0074] The amino acid sequences represented by SEQ ID NO 32, 81 and
96 originate from the gp160 env precursor antigen isolated from a
HIV-1 group O strain termed BSD242, or a quasi-species thereof.
[0075] The amino acid sequences represented by SEQ ID NO 34, 81 and
93 originate from the gp160 env precursor antigen isolated from a
HIV-1 group O strain termed 533, or a quasi-species thereof.
[0076] The amino acid sequences represented by SEQ ID NO 36, 75 and
94 originate from the gp160 env precursor antigen isolated from a
HIV-1 group O strain termed 772P94, or a quasi-species thereof.
[0077] The amino acid sequences represented by SEQ ID NO 38, 74 and
102 originate from the gp160 env precursor antigen isolated from a
HIV-1 group O strain termed MP95B, or a quasi-species thereof.
[0078] The amino acid sequences represented by SEQ ID NO 134, 135,
136, and 137 originate from the gp160 env precursor antigen
isolated from a HIV-1 group O strain termed MP645, or a
quasi-species thereof.
[0079] It is noted that the amino acid sequence represented by SEQ
ID NO 73 is characteristic for the gp41 immunodominant region of at
least the following new HIV-1 group O strains: MP340, MP450, and
189.
[0080] The current invention therefore specifically relates to
env-derived antigens comprising the characteristic sequence
represented by SEQ ID NO 73, as well as virus strains containing
these antigens.
[0081] It is also noted that the amino acid sequence represented by
SEQ ID NO 81 is characteristic for the gp41 immunodominant region
of at least the following new HIV-1 group O strains: BSD242 and
533.
[0082] The current invention therefore specifically relates to
env-derived antigens comprising the characteristic sequence
represented by SEQ ID NO 81, as well as virus strains containing
these antigens.
[0083] It is also noted that the amino acid sequence represented by
SEQ ID NO 95 is characteristic for the gp41 immunodominant region
of at least the following new HIV-1 group O strains: MP539 and
BSD189.
[0084] The current invention therefore specifically relates to
env-derived antigens comprising the characteristic sequence
represented by SEQ ID NO 95, as well as virus strains containing
these antigens.
[0085] The term "quasi-species" refers in general to the group of
related but genetically and possibly biologically different viruses
(also called "variants") that an infected individual harbors. The
term "related" means that the "variants" all arise from a single
infectious agent, in this case from a single HIV-1 group O strain.
It has been calculated that an HIV-infected patient carries about
10.sup.6 to 10.sup.8 genetically distinct HIV-variants, which are
generated by the high error rate of reverse transcriptase and the
high turnover rate in vivo. In the context of the current
description the term "quasi-species" refers also to a strain
isolated from the quasi-species "group" as above-defined.
[0086] The term "genetically different" means that the nucleic acid
sequence of the genome of one strain shows at least one nucleotide
difference with the corresponding sequence of another strain
belonging to the same quasi-species.
[0087] The term "biologically different" means that some strains of
a quasi-species may have different biological characteristics
compared to the biological characteristics of other strains from
the same quasi-species. These biological characteristics may
encompass for example the HIV-1 cell tropism, viral virulence, the
capacity to induce syncytia, etc.
[0088] Nucleic acid sequences originating from quasi-species differ
from each other but always show a high percentage of homology, most
often a homology of 90%, 95% or higher. The same holds for the
sequence of polypeptides originating from quasi-species. Homology
percentages on the protein level usually exceed 95%, 96%, 97%, 98%,
or even 99%. These percentages of homology count for the comparison
of sequence stretches which are at least 100 nucleotides (about 33
amino acids), and preferably 200, 300 or more nucleotides long (66,
100 or more amino acids). It has to be understood that, when very
short sequence stretches are compared (e.g. stretches of about 30
nucleotides, or 10 amino acids) the homology ranges may be much
lower, if these short sequence stretches contain the mutual
differences.
[0089] Examples of sequences originating from "quasi-species" are
provided further in the examples section, where gp41- and
C2V3-nucleotide and amino acid sequences of certain strains
belonging to the same "quasi-species" are compared to each other.
For example, for strains MP340, FABA, MP450 and MP448 gp41-nucleic
acid sequences have been determined on different samples,
originating from the same patient, i.e. on serum samples and on
peripheral blood monocyte (PBMC) samples. Table 2 shows that, in
these specific examples, homology percentages vary from 95% to 100%
between gp41-nucleic acid sequences determined on serum samples as
compared to PBMC-samples.
[0090] It is to be understood that the amino acid and nucleic acid
sequences of the current invention also encompass those sequences
which are not explicitly recited, but which have been determined on
"quasi-species" of the respective viral strains. As indicated
above, these "variant" sequences show a homology range of at least
90%, preferably 95% with the sequences which are specifically
recited in the current application.
[0091] The above-mentioned antigens are polypeptide or peptide
molecules, which are characterized by the above-mentioned amino
acid sequences. It has to be understood however, that these
(poly)peptides may be modified by for example glycosylation, side
chain oxidation or phosphorylation as explained above. A very
particular type of side chain oxidation is cyclisation by bridge
formation between the --SH groups of two cysteine residues in the
same (poly)peptide chain. The cyclic (poly)peptides formed in this
way by S--S bridging may be particularly suitable to expose
epitopes located in the loop structure. Epitopes presented in this
manner may be in a better shape to be recognized by the immune
system, and more particularly by antibodies possibly present in the
serum of HIV-infected persons.
[0092] A preferential embodiment of the current invention provides
for any of the above-mentioned (poly)peptides in a cyclic form.
[0093] Cyclisation may occur between two cysteine residues which
are present in the above-cited amino acid sequences. For example,
cyclic peptides with a loop structure of about 6 amino acids long
may be formed with the amino acid sequences represented by e.g. SEQ
ID NO 71 to 82, and 91 to 102, and 137. Another example are the
V3-loop peptides of about 35 amino acids long, which may be formed
by cyclisation of the cysteine residues of the amino acid sequences
represented by e.g. SEQ ID NO 83 to 90, and 135.
[0094] On the other hand, cyclisation may also be induced in amino
acid sequences which do not contain two cysteine residues
naturally, but which have been extended with one or two cysteine
residues at their extremities, or at in internal position inside
the amino acid chain. The current invention therefore also refers
to (poly)peptides characterized by any of the above-mentioned amino
acid sequences, modified by addition of one or several cysteine
residues, at the C-terminal and/or N-terminal extremity and/or
inside the (poly)peptide chain.
[0095] Another particular type of modification includes the
extension of the N-terminal and/or C-terminal end of the
(poly)peptide antigen by linker sequences, said linker sequences
comprising for example additional amino acids or other molecules
(such as for example biotin). The addition of linker sequences to
the polypeptide antigen may have several advantages such as:
[0096] a more efficient immobilisation on a solid substrate,
[0097] a more efficient presentation of the immunoreactive
epitope(s) in the (poly)peptide,
[0098] linkage to other antigenic determinants . . .
[0099] A preferential embodiment therefore includes antigens or
antigen fragments comprising any of the above-mentioned amino acid
sequences, extended with linker sequences.
[0100] It has to be understood that the above-mentioned
(poly)peptide antigens of the invention may be prepared by
different methods known in the art. They may be prepared by
synthetic means as described above, or they may be produced by
recombinant DNA technology. In the latter case, they are the result
of the expression of the nucleic acids encoding said antigens or
antigen fragments in an appropriate host cell.
[0101] The invention also relates to a recombinant vector for the
expression of any of the above-mentioned polypeptides, recombinant
host cells expresssing these polypeptides, and processes for the
recombinant expression of these polypeptides; said tools for
recombinant expression are well known by anyone skilled in the art,
and have been described in more detail for example in
WO96/13590.
[0102] The invention further provides for a (poly)nucleic acid
encoding any of the above-mentioned (poly)peptide antigens.
[0103] More particularly, the current invention provides for a
polynucleic acid comprising a nucleotide sequence chosen from the
group of
[0104] (I) a nucleotide sequence represented by SEQ ID NO 1, SEQ ID
NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID
NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 21, SEQ
ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 29, SEQ ID NO 31,
SEQ ID NO 33, SEQ ID NO 35, SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO
41, SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49, or SEQ
ID NO 51, SEQ ID NO 106 or
[0105] (ii) a nucleotide sequence complementary to a sequence
according to (I), or
[0106] (iii) a nucleotide sequence showing at least 95%, preferably
96%, 97%, 98% and most preferably 99% homology to a sequence
according to (I), or
[0107] (iv) a nucleotide sequence according to (I) whereby T is
replaced by U, or
[0108] (v) a nucleotide sequence according to (I) whereby at least
one nucleotide is substituted by a nucleotide analogue.
[0109] It is to be noted that, as will be shown further on in the
examples section, the above-mentioned polynucleic acids all
originate from the env-gene of new HIV-1 group O strains. The
nucleic acid sequences represented by SEQ ID NO 1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 39
correspond to the region encoding the gp41-immunodominant domain in
the env-gene, while the nucleic acid sequences represented by SEQ
ID NO 41, 43, 45, 47, 49, and 51 correspond to the region encoding
the C2V3 region in this same env-gene. The nucleotide sequence
represented by SEQ ID NO 106 is illustrated in FIG. 8A, and
comprises the full env-gene of a new HIV-1 group O strain, termed
MP645, together with additional accompanying genes.
[0110] The nucleotide sequences mentioned above under item (iii)
represent variant nucleic acid sequences which may be isolated e.g.
from strains belonging to the same quasi-species.
[0111] More particularly, the invention provides for a polynucleic
acid consisting of a nucleotide sequence chosen from the group
of
[0112] (I) a nucleotide sequence represented by SEQ ID NO 1, SEQ ID
NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID
NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 21, SEQ
ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 29, SEQ ID NO 31,
SEQ ID NO 33, SEQ ID NO 35, SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO
41, SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49, or SEQ
ID NO 51, SEQ ID NO 106 or
[0113] (ii) a nucleotide sequence complementary to a sequence
according to (I), or
[0114] (iii) a nucleotide sequence showing at least 95%, preferably
96%, 97%, 98% and most preferably 99% homology to a sequence
according to (I), or
[0115] (iv) a nucleotide sequence according to (I) whereby T is
replaced by U, or
[0116] (v) a nucleotide sequence according to (I) whereby at least
one nucleotide is substituted by a nucleotide analogue.
[0117] The invention further provides for a nucleic acid fragment
consisting of a sequence of at least 15, preferably 16, 17, 18, 19,
20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of the
sequence of a polynucleic acid as specified above, and
characterized by the fact that it selectively hybridizes to the
polynucleic acid from which it is derived.
[0118] The above-described nucleic acid fragment may be used as a
specific hybridization probe for the detection of the nucleic acids
of the current invention.
[0119] The term "selectively hybridizing" means that the
hybridization signal obtained after hybridization of the fragment
with the nucleic acid from which it is derived, is more intense
than the hybridization signal obtained when the fragment is
hybridized to the corresponding nucleic acid from another HIV-1
group O strain, under the same stringent hybridization and wash
conditions. In practice this means that the nucleic acid fragment
will show at least one mismatched nucleotide with the sequence of
the corresponding nucleic acid fragment of another HIV-1 group O
strain.
[0120] The term "stringent hybridization conditions" implies that
the hybridization takes place at a temperature which is situated
approximately between Tm and (Tm-10.degree. C.), whereby Tm
represents the calculated melting temperature of the target
nucleic. It is generally known that the stringency depends on the
percentage mismatches (=non-matching nucleotides upon alignment)
present in the hybridizing duplex. According to a simplified
formula, the hybridization temperature may be calculated as
follows: Tm-1.2 (% mismatch). A temperature decrease of 10.degree.
C. implies a maximum percentage of allowed mismatches of 8.3%.
[0121] The invention further provides for a nucleic acid fragment
consisting of a sequence of at least 15, preferably 16, 17, 18, 19,
20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides of the
sequence of a polynucleic acid as specified above, and
characterized by the fact that it selectively amplifies the
polynucleic acid from which it is derived.
[0122] The nucleic acid fragment as described above may be used as
a specific amplification primer of the nucleic acids of the current
invention.
[0123] The term "selective amplification" refers to the fact that
said nucleic acid fragment may initiate a specific amplification
reaction of the nucleic acids of the invention (e.g. a polymerase
chain reaction) in the presence of other nucleic acids, under
appropriate amplification conditions. It means that, under the
appropriate amplification conditions, only the nucleic acids of the
invention will be amplified, and not the other nucleic acids
possibly present.
[0124] Preferred embodiments of the invention comprise polynucleic
acids or fragments thereof as specified below.
[0125] A polynucleic acid comprising SEQ ID NO 1, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 1.
[0126] A polynucleic acid consisting of SEQ ID NO 1, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 1, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 1.
[0127] A polynucleic acid comprising SEQ ID NO 3, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 3.
[0128] A polynucleic acid consisting of SEQ ID NO 3, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 3, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 3.
[0129] A polynucleic acid comprising SEQ ID NO 5, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 5.
[0130] A polynucleic acid consisting of SEQ ID NO 5, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 5, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 5.
[0131] A polynucleic acid comprising SEQ ID NO 7, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 7.
[0132] A polynucleic acid consisting of SEQ ID NO 7, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 7, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 7.
[0133] A polynucleic acid comprising SEQ ID NO 9, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 9.
[0134] A polynucleic acid consisting of SEQ ID NO 9, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 9, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 9.
[0135] A polynucleic acid comprising SEQ ID NO 11, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 11.
[0136] A polynucleic acid consisting of SEQ ID NO 11, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, up to 50 contiguous nucleotides of SEQ ID NO 11, said
fragment characterized by the fact that it selectively hybridizes
to the polynucleic acid consisting of SEQ ID NO 11.
[0137] A polynucleic acid comprising SEQ ID NO 13, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 13.
[0138] A polynucleic acid consisting of SEQ ID NO 13, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50, up to the maximum number of contiguous nucleotides of
SEQ ID NO 13, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 13.
[0139] A polynucleic acid comprising SEQ ID NO 15, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 15.
[0140] A polynucleic acid consisting of SEQ ID NO 15, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 15, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 15.
[0141] A polynucleic acid comprising SEQ ID NO 17, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 17.
[0142] A polynucleic acid consisting of SEQ ID NO 17, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 17, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 17.
[0143] A polynucleic acid comprising SEQ ID NO 19, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 19.
[0144] A polynucleic acid consisting of SEQ ID NO 19, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25,50 up to the maximum number of contiguous nucleotides of SEQ
ID NO 19, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 19.
[0145] A polynucleic acid comprising SEQ ID NO 21, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 21.
[0146] A polynucleic acid consisting of SEQ ID NO 21, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 21, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 21.
[0147] A polynucleic acid comprising SEQ ID NO 23, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 23.
[0148] A polynucleic acid consisting of SEQ ID NO 23, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 23, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 23.
[0149] A polynucleic acid comprising SEQ ID NO 25, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 25.
[0150] A polynucleic acid consisting of SEQ ID NO 25, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, up to 50 contiguous nucleotides of SEQ ID NO 25, said
fragment characterized by the fact that it selectively hybridizes
to the polynucleic acid consisting of SEQ ID NO 25.
[0151] A polynucleic acid comprising SEQ ID NO 27, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 27.
[0152] A polynucleic acid consisting of SEQ ID NO 27, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 27, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 27.
[0153] A polynucleic acid comprising SEQ ID NO 29, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 29.
[0154] A polynucleic acid consisting of SEQ ID NO 29, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 29, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 29.
[0155] A polynucleic acid comprising SEQ ID NO 31, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 31.
[0156] A polynucleic acid consisting of SEQ ID NO 31, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 31, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 31.
[0157] A polynucleic acid comprising SEQ ID NO 33, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 33.
[0158] A polynucleic acid consisting of SEQ ID NO 33, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 33, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 33.
[0159] A polynucleic acid comprising SEQ ID NO 35, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 35.
[0160] A polynucleic acid consisting of SEQ ID NO 35, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 35, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 35.
[0161] A polynucleic acid comprising SEQ ID NO 37, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 37.
[0162] A polynucleic acid consisting of SEQ ID NO 37, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 37, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 37.
[0163] A polynucleic acid comprising SEQ ID NO 39, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 39.
[0164] A polynucleic acid consisting of SEQ ID NO 39, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 39, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 39.
[0165] A polynucleic acid comprising SEQ ID NO 41, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 41.
[0166] A polynucleic acid consisting of SEQ ID NO 41, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 41, said fragment characterized by the fact that if
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 41.
[0167] A polynucleic acid comprising SEQ ID NO 43, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 43.
[0168] A polynucleic acid consisting of SEQ ID NO 43, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 43, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 43.
[0169] A polynucleic acid comprising SEQ ID NO 45, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 45.
[0170] A polynucleic acid consisting of SEQ ID NO 45, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 45, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 45.
[0171] A polynucleic acid comprising SEQ ID NO 47, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 47.
[0172] A polynucleic acid consisting of SEQ ID NO 47, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 47, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 47.
[0173] A polynucleic acid comprising SEQ ID NO 49, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 49.
[0174] A polynucleic acid consisting of SEQ ID NO 49, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 49, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 49.
[0175] A polynucleic acid comprising SEQ ID NO 51, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 51.
[0176] A polynucleic acid consisting of SEQ ID NO 51, or a fragment
comprising at least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 50 up to the maximum number of contiguous nucleotides of
SEQ ID NO 51, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid consisting of SEQ ID
NO 51.
[0177] A polynucleic acid comprising SEQ ID NO 106, or a fragment
consisting of at least 15, preferably 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 up to 50 contiguous nucleotides of said polynucleic
acid, said fragment characterized by the fact that it selectively
hybridizes to the polynucleic acid comprising SEQ ID NO 106.
[0178] A polynucleic acid consisting of SEQ ID NO 106, or a
fragment comprising at least 15, preferably 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 50 up to the maximum number of contiguous
nucleotides of SEQ ID NO 106, said fragment characterized by the
fact that it selectively hybridizes to the polynucleic acid
consisting of SEQ ID NO 106.
[0179] The invention further provides for a virus strain belonging
to HIV-1 group O, comprising in its genome any of the
above-mentioned nucleic acids.
[0180] More particularly, the invention provides for a virus strain
belonging to HIV-1 group O, comprising in its genome the RNA
equivalent of
[0181] one of the DNA sequences represented by SEQ ID NO 1, SEQ ID
NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID
NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 21, SEQ
ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 29, SEQ ID NO 31,
SEQ ID NO 33, SEQ ID NO 35, SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO
106 and/or
[0182] one of the DNA sequences represented by SEQ ID NO 41, SEQ ID
NO 43, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49, SEQ ID NO 51,
and/or
[0183] a variant sequence of the above-mentioned DNA sequences,
said variant sequence showing at least 95% homology with the entire
length of one of the above-mentioned sequences.
[0184] More particularely, the invention relates to a strain of
HIV-1 group O as defined above, comprising in its genome the RNA
equivalent of the DNA sequence represented by SEQ ID NO 5 and/or
SEQ ID NO 43 or a variant sequence thereof, said variant sequence
showing at least 95% homology with SEQ ID NO 5 and/or SEQ ID NO 43.
An HIV-1 group O strain of this type, termed FABA (or synonymously
MP331) has been deposited at the ECACC on Jun. 13 1997, under
accession No V97061301.
[0185] An example of a variant sequence of SEQ ID NO 5 is SEQ ID NO
7. The latter sequence was determined on a serum sample of a
patient infected by the strain FABA, while the former sequence was
determined on peripheral blood mononuclear cells (PBMC's) taken
from the same patient. The nucleic acids represented by SEQ ID NO 5
and SEQ ID NO 7 show 95% homology, and can be said to belong to
strains from the same quasi-species.
[0186] The invention also relates to a strain of HIV-1 group O as
defined above, comprising in its genome the RNA equivalent of the
DNA sequence represented by SEQ ID NO 9 and/or SEQ ID NO 45 or a
variant sequence, said variant sequence showing at least 95%
homology with SEQ ID NO 9 and/or SEQ ID NO 45. An HIV-1 group O
strain of this type, termed MP450, has been deposited at the ECACC
on Jun. 13, 1997 under accession No. V97061302.
[0187] An example of a variant sequence of SEQ ID NO 9 is SEQ ID NO
11. The latter sequence was determined on a serum sample of a
patient infected by the strain MP450, while the former sequence was
determined on peripheral blood mononuclear cells (PBMC's) taken
from the same patient.
[0188] Furthermore, the invention also relates to a strain of HIV-1
group O as defined above, comprising in its genome the RNA
equivalent of the DNA sequence represented by SEQ ID NO 39 and/or
SEQ ID NO 51 or a variant sequence, said variant sequence showing
at least 95% homology with SEQ ID NO 39 and/or SEQ ID NO 51. An
HIV-1 group O strain of this type, termed MP539, has been deposited
at the ECACC on Jun. 13, 1997 under accession No.V97061303.
[0189] Furthermore, the invention also relates to a strain of HIV-1
group O as defined above comprising in its genome the RNA
equivalent of the DNA sequence represented by SEQ ID NO 1 and/or
SEQ ID NO 41 or a variant sequence, said variant sequence showing
at least 95% homology with SEQ ID NO 1 and/or SEQ ID NO 41. A
strain of this type is termed MP340 throughout this invention.
[0190] An example of a variant sequence of SEQ ID NO 1 is SEQ ID NO
3. The latter sequence was determined on a serum sample of a
patient infected by the strain MP340, while the former sequence was
determined on peripheral blood mononuclear cells (PBMC's) taken
from the same patient. The nucleic acids represented by SEQ ID NO 1
and SEQ ID NO 3 show 99% homology, and can be said to belong to
strains from the same quasi-species.
[0191] Furthermore, the invention also relates to a strain of HIV-1
group O as defined above comprising in its genome the RNA
equivalent of the DNA sequence represented by SEQ ID NO 13 and/or
SEQ ID NO 47 or a variant sequence, said variant sequence showing
at least 95% homology with SEQ ID NO 13 and/or SEQ ID NO 47. A
strain of this type is termed MP448 throughout this invention.
[0192] Furthermore, the invention also relates to a strain of HIV-1
group O as defined above comprising in its genome the RNA
equivalent of the DNA sequence represented by SEQ ID NO 17 and/or
SEQ ID NO 49 or a variant sequence, said variant sequence showing
at least 95% homology with SEQ ID NO 17 and/or SEQ ID NO 49. A
strain of this type is termed 189 throughout this invention.
[0193] Furthermore, the invention also relates to a strain of HIV-1
group O as defined above comprising in its genome the RNA
equivalent of the DNA sequence represented by SEQ ID NO 19 or a
variant sequence showing at least 95% homology with SEQ ID NO 19. A
strain of this type is termed 320 throughout this invention.
[0194] Furthermore, the invention also relates to a strain of HIV-1
group O as defined above comprising in its genome the RNA
equivalent of the DNA sequence represented by SEQ ID NO 21 or a
variant sequence showing at least 95% homology with SEQ ID NO 21. A
strain of this type is termed BSD422 throughout this invention.
[0195] Furthermore, the invention also relates to a strain of HIV-1
group O as defined above comprising in its genome the RNA
equivalent of the DNA sequence represented by SEQ ID NO 23 or a
variant sequence showing at least 95% homology with SEQ ID NO 23. A
strain of this type is termed KGT008 throughout this invention.
[0196] Furthermore, the invention also relates to a strain of HIV-1
group O as defined above comprising in its genome the RNA
equivalent of the DNA sequence represented by SEQ ID NO 25 or a
variant sequence showing at least 95% homology with SEQ ID NO 25. A
strain of this type, termed MP575, has been deposited at the ECACC
on Jul. 13, 1998, under provisional accession No. V98071301.
[0197] Furthermore, the invention also relates to a strain of HIV-1
group O as defined above comprising in its genome the RNA
equivalent of the DNA sequence represented by SEQ ID NO 27 or a
variant sequence showing at least 95% homology with SEQ ID NO 27. A
strain of this type is termed BSD189 throughout this invention.
[0198] Furthermore, the invention also relates to a strain of HIV-1
group O as defined above comprising in its genome the RNA
equivalent of the DNA sequence represented by SEQ ID NO 29 or a
variant sequence showing at least 95% homology with SEQ ID NO 29. A
strain of this type is termed BSD649 throughout this invention.
[0199] Furthermore, the invention also relates to a strain of HIV-1
group O as defined above comprising in its genome the RNA
equivalent of the DNA sequence represented by SEQ ID NO 31 or a
variant sequence showing at least 95% homology with SEQ ID NO 31. A
strain of this type is termed BSD242 throughout this invention.
[0200] Furthermore, the invention also relates to a strain of HIV-1
group O as defined above comprising in its genome the RNA
equivalent of the DNA sequence represented by SEQ ID NO 33 or a
variant sequence showing at least 95% homology with SEQ ID NO 33. A
strain of this type is termed 533 throughout this invention.
[0201] Furthermore, the invention also relates to a strain of HIV-1
group O as defined above comprising in its genome the RNA
equivalent of the DNA sequence represented by SEQ ID NO 35 or a
variant sequence showing at least 95% homology with SEQ ID NO 35. A
strain of this type is termed 772.P94 throughout this
invention.
[0202] Furthermore, the invention also relates to a strain of HIV-1
group O as defined above comprising in its genome the RNA
equivalent of the DNA sequence represented by SEQ ID NO 37 or a
variant sequence showing at least 95% homology with SEQ ID NO 37. A
strain of this type is termed MP95B throughout this invention.
[0203] Furthermore, the invention also relates to a strain of HIV-1
group O as defined above comprising in its genome the RNA
equivalent of the DNA sequence represented by SEQ ID NO 106 or a
variant sequence showing at least 95% homology with SEQ ID NO 106.
A strain of this type, termed MP645, has been deposited at the
ECACC on Jul. 13, 1998, under provisional accession No.
V98071302
[0204] Another embodiment of the current invention provides for a
nucleic acid molecule isolated from any of the HIV-1 group O
strains as defined above.
[0205] In addition, the current invention provides for an antigen
or antigen fragment isolated from any of the HIV-1 group O strains
as defined above.
[0206] It is to be understood that the current invention also
provides for nucleic acid sequences and antigen sequences which are
contained in the above-mentioned new HIV-1 group O viral strains,
and which extend beyond the explicitly cited sequences represented
by SEQ ID NO 1 to 102, 106, 135 to 138. The person skilled in the
art will realize that, starting from the partial sequences
disclosed above, it is perfectly possible to obtain the complete
genomic information of the respective viruses, by standard cloning
methods such as the construction of a cDNA library or the
construction of a genomic library or by the technique of the
polymerase chain reaction. Sometimes a combination of these methods
may be necessary to obtain the sequence of the full genome.
[0207] The following describes the strategies which may be followed
to obtain additional genomic sequence information on HIV-1 group O
strains, of which partial sequences have been disclosed above.
[0208] 1. Construction of a cDNA Library.
[0209] HIV-1 group O viruses are propagated and isolated using
standard methods e.g. by cultivation of peripheral blood
lymphocytes (PBMC) from the HIV-infected individual together with
stimulated lymphocytes from healthy donors, or alternatively by
infecting cell lines with the virus in a permanent way. Once virus
is detected in the culture supernatant using standard techniques
(e.g. measuring reverse transcriptase activity; measuring p24
antigen . . . ), virus is harvested from the culture supernantant
by centrifugation under conditions where the virus is pelleted. RNA
is obtained by disrupting the virus in a buffer containing 6M
guanidinium chloride and the RNA is pelleted through a 5.5M CsCl
cushion. The RNA which is resuspended in a suitable buffer is then
phenolized and precipitated with e.g. ethanol and lithium
chloride.
[0210] cDNA synthesis is performed on the complete RNA or part of
the RNA using commercially available kits. OligodT primers, random
primers, or HIV-1 specific primers may be used to prime the cDNA
synthesis which is done by a reverse transcriptase (RT) enzyme.
This leads to a first DNA strand which is complementary to the
initial RNA strand and which forms RNA::DNA hybrids. The RNA strand
is removed with Rnase H and the second DNA strand is then
synthesised with DNA polymerase I. The overhanging single stranded
cDNA ends are removed with T4 DNA polymerase. The resulting cDNA is
ligated to linkers which contain an appropriate restriction site.
After hydrolysis of the cDNA with the appropriate restriction
enzyme, the cDNA of suitable size is isolated (e.g. from agarose
gel after electrophoresis) and ligated in a suitable vector. The
vector containing the cDNA fragments can be propagated in competent
E. coli cells using standard methods.
[0211] Various techniques to screen for colonies containing HIV-1
specific sequences are known in the art. They involve screening of
e.g. a cDNA expression library (e.g. .lambda.gt11) with serum
(polyclonal or monoclonal serum) or the screening of a cDNA library
with .sup.32P labelled HIV-1 DNA fragments under non-stringent or
stringent hybridization conditions. Background signals are lowered
by washing the filters subsequently under more stringent
conditions. After identification of the E. coli containing the
suitable fragment, the fragment is isolated from the plasmid and is
introduced (as a complete entity or a fragment thereof) in
expression vectors. Using standard techniques, these vectors
produce the protein(s) encoded by the inserted DNA fragment. The
resulting proteins is further purified and used for the development
of diagnostic assays. Sequence information of the virus is obtained
from the plasmid containing viral DNA sequences.
[0212] 2. Construction of a Genomic Library
[0213] Chromosomal DNA is prepared from cells infected with the
HIV-1 group O virus (e.g. cells permanently producing the virus)
using standard techniques (Maniatis et al. 1982). This DNA may be
used to construct a genomic library (Zabarousky and Allikmets
1986). The chromosomal DNA which contains the proviral HIV-1 group
O DNA is partially digested with a selected restriction enzyme.
Fragments between 9 Kb and 23 Kb, isolated on a 40%-10% sucrose
gradient, are manipulated according to standard techniques in order
to introduce them in a vector system suitable for the cloning of
long DNA fragments e.g. lambda derived vectors or cosmids.
[0214] The vector with the DNA fragment is introduced in a suitable
E. coli strain and is further propagated onto plates. Plaques or
colonies from the genomic library are transferred to nylon or
nitrocellulose membranes and screened with enzyme or .sup.32P
labelled DNA fragments of the viral genome (plaque or colony
screening) under non-stringent or stringent hybridization
conditions. Colonies or plaques displaying positive signals are
purified from other colonies or plaques. The viral DNA is further
subcloned and sequenced. Genes or fragments of genes are further
manipulated using standard techniques in order to express important
viral proteins or epitopes which may be used for the development of
diagnostic assays.
[0215] 3. Polymerase Chain Reaction (PCR).
[0216] HIV-1 group O viral DNA fragments may also be obtained using
the polymerase chain reaction (PCR) (Kwok et al. 1987) which is a
standard technology used for the cloning of DNA fragments. PCR may
be performed on cellular DNA of cells infected with the virus or on
cDNA obtained from viral RNA derived from virus culture,
lymphocytes, serum, plasma, . . . . The PCR may use primers which
contain specific sequences of the virus based on sequences of the
virus which are already known, or alternatively, primers which
contain sequences derived from related viruses in regions known to
be conserved or not conserved among HIV variants. Annealing
conditions of the primers should preferentially not be too
stringent (e.g. Tm-20.degree. C.), however the best conditions
should be experimentally established. The resulting amplification
product is subsequently sequenced and new primers are designed
based on the newly generated sequence in order to further amplify
the viral DNA, again eventually in combination with primers derived
from the partially determined sequence of the isolate or from the
sequence of related viruses.
[0217] Example 4 provides the cloning and sequencing strategy
followed in order to obtain the polynucleic acid sequences encoding
the antigens Vif, Pol, Vpr, Tat (1st exon), Vpu, Rev (1st exon) and
gp160, or fragments thereof, from the HIV-1 group O viruses FABA
(MP331), MP448, MP539 and MP 645.
[0218] Therefore, in addition to the gp41 and V3 sequences
described above, the present invention further provides for a
polynucleic acid containing a polynucleic acid sequence encoding at
least part of the Vif, Pol, Vpr, Tat (1st exon), Vpu, Rev (1st
exon) or gp160 antigens from any of the following HIV-1 group O
strains of the invention: MP340, FABA(MP331), MP450, MP448, 189,
MP539, 320, BSD422, KGT008, MP575, BSD189, BSD649, BSD242, 533,
772P94, MP95B and MP645.
[0219] The terms "Vif, Pol, Vpr, Tat (1st exon), Vpu, Rev (1st
exon) and gp160" are terms for HIV-antigens familiar to the person
skilled in the art. Vif and Vpu have important roles during virion
morphogenesis. Vif is required for the production of fully
infectious viruses, while Vpu is necessary for the efficient
release of virus particles budding from the cell membrane in
cultured cells (Gottlinger et al, 1993). Vpu also mediates the
rapid degradation of the CD4 receptor molecule in the endoplasmatic
reticulum (Willey et al, 1992; Bour et al, 1995). The Vpr protein
is involved in the nuclear migration of the prointegration complex
(Heinzinger et al, 1994) and is also found in mature virions and
hence a structural component of the virus (Paxton et al, 1993). Tat
(transactivator) and Rev (regulator of virion expression) are
encoded in overlapping reading frames which generate small
regulatory proteins translated from multiple spliced mRNAs (Salfeld
et al, 1990; Solomin et al, 1990; Furtado et al, 1991). Both
proteins are essential for virus replication and are positive
regulators of gene expression (Arya et al, 1985; Feinberg et al,
1986). Pol is encoded by the coding gene pol, which overlaps with
the gag information but in a different reading frame. Pol is a
precursor protein which is autocleaved to form the following viral
enzymes: a protease, a reverse transcriptase with polymerase
activity and Rnase H activity, and an integrase Ross et al, 1991).
FIG. 8 illustrates the sequence of a large genomic fragment from a
number of HIV-1 group O strains of the current invention (MP645
(SEQ ID NO 106), MP331 (SEQ ID NO 103), MP448 (SEQ ID NO 104) and
MP539(SEQ ID NO 105)), and the location of the Vif, Pol, Vpr, Tat
(1st exon), Vpu, Rev (1st exon) and gp160 (partially) genes in
these sequences.
[0220] The present invention thus provides for a polynucleic acid
comprising a nucleotide sequence chosen from the group of
[0221] (I) a nucleotide sequence represented by any of SEQ ID NO
103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 106, or
[0222] (ii) a nucleotide sequence complementary to a sequence
according to (I), or
[0223] (iii) a nucleotide sequence showing at least 95%, preferably
96%, 97%, 98% and most preferably 99% homology to a sequence
according to (I), or
[0224] (iv) a nucleotide sequence according to (I) whereby T is
replaced by U, or
[0225] (v) a nucleotide sequence according to (I) whereby at least
one nucleotide is substituted by a nucleotide analogue, or
[0226] (vi) a fragment a sequence of at least 15, preferably 16,
17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides
of any of the nucleotide sequences according to (I) to (v), and
characterized by the fact that it selectively hybridizes to the
polynucleic acid from which it is derived, and/or selectively
amplifies the polynucleic acid from which it is derived.
[0227] More particularly, the present invention provides for a
polynucleic acid consisting of a nucleotide sequence chosen from
the group of
[0228] (I) a nucleotide sequence represented by any of SEQ ID NO
103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 106, or
[0229] (ii) a nucleotide sequence complementary to a sequence
according to (I), or
[0230] (iii) a nucleotide sequence showing at least 95%, preferably
96%, 97%, 98% and most preferably 99% homology to a sequence
according to (I), or
[0231] (iv) a nucleotide sequence according to (I) whereby T is
replaced by U, or
[0232] (v) a nucleotide sequence according to (I) whereby at least
one nucleotide is substituted by a nucleotide analogue
[0233] (vi) a fragment a sequence of at least 15, preferably 16,
17, 18, 19, 20, 21, 22, 23, 24, 25 up to 50 contiguous nucleotides
of any of the nucleotide sequences according to (I) to (v), and
characterized by the fact that it selectively hybridizes to the
polynucleic acid from which it is derived, and/or selectively
amplifies the polynucleic acid from which it is derived.
[0234] The above-described nucleic acid fragment may be used as a
specific hybridization probe for the detection of the nucleic acids
of the current invention.
[0235] The term "selectively hybridizing" means that the
hybridization signal obtained after hybridization of the fragment
with the nucleic acid from which it is derived, is more intense
than the hybridization signal obtained when the fragment is
hybridized to the corresponding nucleic acid from another HIV-1
group O strain, under the same stringent hybridization and wash
conditions. In practice this means that the nucleic acid fragment
will show at least one mismatched nucleotide with the sequence of
the corresponding nucleic acid fragment of another HIV-1 group O
strain.
[0236] The term "stringent hybridization conditions" implies that
the hybridization takes place at a temperature which is situated
approximately between Tm and (Tm-10.degree. C.), whereby Tm
represents the calculated melting temperature of the target
nucleic. It is generally known that the stringency depends on the
percentage mismatches (=non-matching nucleotides upon alignment)
present in the hybridizing duplex. According to a simplified
formula, the hybridization temperature may be calculated as
follows: Tm-1.2 (% mismatch). A temperature decrease of 10.degree.
C. implies a maximum percentage of allowed mismatches of 8.3%.
[0237] The nucleic acid fragment as described above may also be
used as a specific amplification primer of the nucleic acids of the
current invention.
[0238] The term "selective amplification" refers to the fact that
said nucleic acid fragment may initiate a specific amplification
reaction of the nucleic acids of the invention (e.g. a polymerase
chain reaction) in the presence of other nucleic acids, under
appropriate amplification conditions. It means that, under the
appropriate amplification conditions, only the nucleic acids of the
invention will be amplified, and not the other nucleic acids
possibly present.
[0239] Preferred embodiments of the invention comprise polynucleic
acids or fragments thereof as specified below.
[0240] A polynucleic acid comprising SEQ ID NO 103, or comprising a
fragment consisting of at least 15, preferably 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, up to 50 contiguous nucleotides of said
polynucleic acid, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid comprising SEQ ID NO
103.
[0241] A polynucleic acid comprising SEQ ID NO 104, or comprising a
fragment consisting of at least 15, preferably 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, up to 50 contiguous nucleotides of said
polynucleic acid, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid comprising SEQ ID NO
104.
[0242] A polynucleic acid comprising SEQ ID NO 105, or comprising a
fragment consisting of at least 15, preferably 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, up to 50 contiguous nucleotides of said
polynucleic acid, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid comprising SEQ ID NO
105.
[0243] A polynucleic acid comprising SEQ ID NO 106, or comprising a
fragment consisting of at least 15, preferably 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, up to 50 contiguous nucleotides of said
polynucleic acid, said fragment characterized by the fact that it
selectively hybridizes to the polynucleic acid comprising SEQ ID NO
106.
[0244] A polynucleic acid fragment consisting of a sequence of at
least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, up to
50 contiguous nucleotides of SEQ ID NO 103, said fragment
characterized by the fact that it selectively amplifies the
polynucleic acid from which it is derived.
[0245] A polynucleic acid fragment consisting of a sequence of at
least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, up to
50 contiguous nucleotides of SEQ ID NO 104, said fragment
characterized by the fact that it selectively amplifies the
polynucleic acid from which it is derived.
[0246] A polynucleic acid fragment consisting of a sequence of at
least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, up to
50 contiguous nucleotides of SEQ ID NO 105, said fragment
characterized by the fact that it selectively amplifies the
polynucleic acid from which it is derived.
[0247] A polynucleic acid fragment consisting of a sequence of at
least 15, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, up to
50 contiguous nucleotides of SEQ ID NO 106, said fragment
characterized by the fact that it selectively amplifies the
polynucleic acid from which it is derived.
[0248] The invention further-provides for an antigen comprising at
least part of Vif, Pol, Vpr, Tat (1st exon), Vpu, Rev (1st exon)
and/or gp160 encoded by the nucleic acid sequences as described
above from any of the following HIV-1 group O strains: MP340,
FABA(MP331), MP450, MP448, 189, MP539, 320, BSD422, KGT008, MP575,
BSD189, BSD649, BSD242, 533, 772P94, MP95B and MP645.
[0249] The current invention more particularly provides for an
antigen comprising at least one amino acid sequence chosen from the
following groups of sequences
[0250] Ii) an amino acid sequence represented by any of SEQ ID NO
107, SEQ ID NO 108, SEQ ID NO 109 and SEQ ID NO 110 representing
the Vif antigen,
[0251] (ii) an amino acid sequence represented by any of SEQ ID NO
111, SEQ ID NO 112, SEQ ID NO 113 and SEQ ID NO 114 representing
the Vpu antigen,
[0252] (iii) an amino acid sequence represented by any of SEQ ID NO
115, SEQ ID NO 116, SEQ ID NO 117 and SEQ ID NO 118 representing
the Vpr antigen,
[0253] (iv) an amino acid sequence represented by any of SEQ ID NO
119, SEQ ID NO 120, SEQ ID NO 121 and SEQ ID NO 122 representing
the Tat antigen,
[0254] (v) an amino acid sequence represented by any of SEQ ID NO
123, SEQ ID NO 124, SEQ ID NO 125 and SEQ ID NO 126 representing
the Rev antigen,
[0255] (vi) an amino acid sequence represented by any of SEQ ID NO
127, SEQ ID NO 128, SEQ ID NO 129 and SEQ ID NO 130 representing
the Pol antigen,
[0256] (vii) an amino acid sequence represented by any of SEQ ID NO
2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO
12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID
NO 22, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ
ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40,
SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO
50, SEQ ID NO 52, SEQ ID NO 132, and SEQ ID NO 134 representing at
least part of the Env antigen, or
[0257] (viii) a fragment of any of the above-mentioned antigens Ii)
to (vii), said fragment consisting of at least 8, preferably 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up
to the maximum number of contiguous amino acids of the amino acid
sequence of said antigen, and being characterized by the fact that
it specifically reacts with antibodies raised against said
antigen.
[0258] The current invention further provides for an antigen
consisting of an amino acid sequence chosen from the following
groups of sequences:
[0259] Ii) an amino acid sequence SEQ ID NO 107, SEQ ID NO 108, SEQ
ID NO 109 and SEQ ID NO 110 representing the Vif antigen,
[0260] (ii) an amino acid sequence SEQ ID NO 111, SEQ ID NO 112,
SEQ ID NO 113 and SEQ ID NO 114 representing the Vpu antigen,
[0261] (iii) an amino acid sequence SEQ ID NO 115, SEQ ID NO 116,
SEQ ID NO 117 and SEQ ID NO 118 representing the Vpr antigen,
[0262] (iv) an amino acid sequence SEQ ID NO 119, SEQ ID NO 120,
SEQ ID NO 121 and SEQ ID NO 122 representing the Tat antigen,
[0263] (v) an amino acid sequence SEQ ID NO 123, SEQ ID NO 124, SEQ
ID NO 125 and SEQ ID NO 126 representing the Rev antigen,
[0264] (vi) an amino acid sequence SEQ ID NO 127, SEQ ID NO 128,
SEQ ID NO 129 and SEQ ID NO 130 representing the Pol antigen,
[0265] (vii) an amino acid sequence represented by any of SEQ ID NO
2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO
12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID
NO 22, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ
ID NO 32, SEQ ID NO 34, SEQ ID NO 36,SEQ ID NO 38, SEQ ID NO 40,
SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO
50, SEQ ID NO 52, SEQ ID NO 132, and SEQ ID NO 134 representing at
least part of the Env antigen, or
[0266] (viii) a fragment of any of the above-mentioned antigens (I)
to (vii), said fragment consisting of at least 8, preferably 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50 up
to the maximum number of contiguous amino acids of the amino acid
sequence of said antigen, and being characterized by the fact that
it specifically reacts with antibodies raised against said
antigen.
[0267] The current invention thus also relates to:
[0268] (I) an antigen derived from the gp160 env precursor protein
of the new HIV-1 group 0 strain MP340 comprising at least one of
the sequences according to SEQ ID NO 2, 4, 42, 59, 60, 61, 73, 85,
86, 100, or fragments thereof with said fragments specifically
reacting with antibodies raised against the antigen they are
derived from;
[0269] (ii) a polynucleic acid encoding an antigen according to (I)
and comprising at least one of the nucleotide sequences according
to SEQ ID NO 1, 3, 41, including homologous sequences,
complementary sequences, and fragments hybridizing thereto;
[0270] (iii) a virus strain comprising in its genome a polynucleic
acid according to (ii), more particularly a virus strain termed
MP340, as well as polynucleic acids and antigens isolated
therefrom.
[0271] The current invention thus also relates to:
[0272] (I) an antigen derived from the gp160 env precursor protein
of the new HIV-1 group 0 strain MP331 (or FABA) comprising at least
one of the sequences according to SEQ ID NO 6, 8, 44, 56, 57, 58,
82, 83, 84, 138 or fragments thereof with said fragments
specifically reacting with antibodies raised against the antigen
they are derived from;
[0273] (ii) an antigen derived from the Vif, Vpu, Vpr, Tat, Rev,
Pol and Env protein of the new HIV-1 group 0 strain MP331 (or FABA)
comprising at least one of the sequences according to SEQ ID NO
107, 111, 115, 119, 123, 127, 131, or fragments thereof with said
fragments specifically reacting with antibodies raised against the
antigen they are derived from;
[0274] (iii) a polynucleic acid encoding an antigen according to
(I) or (ii) and comprising at least one of the nucleic acid
sequences according to SEQ ID NO 5, 7, 43, 103, including
homologous sequences, complementary sequences, and fragments
hybridizing thereto;
[0275] (iv) a virus strain comprising in its genome said a
polynucleic acid according to (iii), more particularly a virus
strain termed MP331 (FABA) deposited at the ECACC under accession
number V97061301, as well as polynucleic acids and antigens
isolated therefrom.
[0276] The current invention thus also relates to:
[0277] (I) an antigen derived from the gp160 env precursor protein
of the new HIV-1 group 0 strain MP450 comprising at least one of
the sequences according to SEQ ID NO 10, 12, 46, 62, 63, 64, 73,
87, 100, or fragments thereof with said fragments specifically
reacting with antibodies raised against the antigen they are
derived from;
[0278] (ii) a polynucleic acid encoding an antigen according to (I)
and comprising at least one of the nucleotide sequences according
to SEQ ID NO 9, 11, 45, including homologous sequences,
complementary sequences, and fragments hybridizing thereto;
[0279] (iii) a virus strain comprising in its genome a polynucleic
acid according to (ii), more particularly a virus strain termed
MP450 deposited at the ECACC under accession number V97061302, as
well as polynucleic acids and antigens isolated therefrom.
[0280] The current invention thus also relates to:
[0281] (I) an antigen derived from the gp160 env precursor protein
of the new HIV-1 group 0 strain MP448 comprising at least one of
the sequences according to SEQ ID NO 14, 16, 48, 65, 66, 67, 76,
88, 101, or fragments thereof with said fragments specifically
reacting with antibodies raised against the antigen they are
derived from;
[0282] (ii) an antigen derived from the Vif, Vpu, Vpr, Tat, Rev,
Pol and Env protein of the new HIV-1 group 0 strain MP448
comprising at least one of the sequences according to SEQ ID NO
108, 112, 116, 120, 124, 128, 132 or fragments thereof with said
fragments specifically reacting with antibodies raised against the
antigen they are derived from;
[0283] (iii) a polynucleic acid encoding an antigen according to
(I) or (ii) and comprising at least one of the nucleic acid
sequences according to SEQ ID NO 13, 15, 47, 104, including
homologous sequences, complementary sequences, and fragments
hybridizing thereto;
[0284] (iv) A virus strain comprising in its genome said
polynucleic acid according to (iii), more particularly a virus
strain termed MP448, as well as polynucleic acids and antigens
isolated therefrom.
[0285] The current invention thus also relates to:
[0286] (I) an antigen derived from the gp160 env precursor protein
of the new HIV-1 group 0 strain 189 comprising at least one of the
sequences according to SEQ ID NO 18, 50, 53, 54, 55, 73, 90, 91, or
fragments thereof with said fragments specifically reacting with
antibodies raised against the antigen they are derived from;
[0287] (ii) a polynucleic acid encoding an antigen according to (I)
and comprising at least one of the nucleotide sequences according
to SEQ ID NO 17, 49, including homologous sequences, complementary
sequences, and fragments hybridizing thereto;
[0288] (iii) a virus strain comprising in its genome a polynucleic
acid according to (ii), more particularly a virus strain termed
189, as well as polynucleic acids and antigens isolated
therefrom.
[0289] The current invention thus also relates to:
[0290] (I) an antigen derived from the gp160 env precursor protein
of the new HIV-1 group 0 strain MP539 comprising at least one of
the sequences according to SEQ ID NO 40, 52, 68, 69, 70, 71, 89, 95
or fragments thereof with said fragments specifically reacting with
antibodies raised against the antigen they are derived from;
[0291] (ii) an antigen derived from the Vif, Vpu, Vpr, Tat, Rev,
Pol and Env protein of the new HIV-1 group 0 strain MP539
comprising at least one of the sequences according to SEQ ID NO
109, 113, 117, 121, 125, 129, 133, or fragments thereof with said
fragments specifically reacting with antibodies raised against the
antigen they are derived from.
[0292] (iii) a polynucleic acid encoding an antigen according to
(I) or (ii) and comprising at least one of the nucleic acid
sequences according to SEQ ID NO 39, 51, 105, including homologous
sequences, complementary sequences, and fragments hybridizing
thereto;
[0293] (iv) A virus strain comprising in its genome said
polynucleic acid according to (iii), more particularly a virus
strain deposited at the ECACC under accession number V97061303, as
well as polynucleic acids and antigens isolated therefrom.
[0294] The current invention thus also relates to:
[0295] (I) an antigen derived from the gp160 env precursor protein
of the new HIV-1 group 0 strain 320 comprising at least one of the
sequences according to SEQ ID NO 20, 92, or fragments thereof with
said fragments specifically reacting with antibodies raised against
the antigen they are derived from;
[0296] (ii) a polynucleic acid encoding an antigen according to (I)
and comprising the nucleotide sequence according to SEQ ID NO 19
including homologous sequences, complementary sequences, and
fragments hybridizing thereto;
[0297] (iii) a virus strain comprising in its genome a polynucleic
acid according to (ii), more particularly a virus strain termed
320, as well as polynucleic acids and antigens isolated
therefrom.
[0298] The current invention thus also relates to:
[0299] (I) an antigen derived from the gp160 env precursor protein
of the new HIV-1 group 0 strain BSD422 comprising at least one of
the sequences according to SEQ ID NO 22, 80, 79, or fragments
thereof with said fragments specifically reacting with antibodies
raised against the antigen they are derived from;
[0300] (ii) a polynucleic acid encoding an antigen according to (I)
and comprising the nucleotide sequence according to SEQ ID NO 21
including homologous sequences, complementary sequences, and
fragments hybridizing thereto;
[0301] (iii) a virus strain comprising in its genome a polynucleic
acid according to (ii), more particularly a virus strain termed
BSD422, as well as polynucleic acids and antigens isolated
therefrom.
[0302] The current invention thus also relates to:
[0303] (I) an antigen derived from the gp160 env precursor protein
of the new HIV-1 group 0 strain KGT008 comprising at least one of
the sequences according to SEQ ID NO 24, 79, 99? or fragments
thereof with said fragments specifically reacting with antibodies
raised against the antigen they are derived from;
[0304] (ii) a polynucleic acid encoding an antigen according to (I)
and comprising the nucleotide sequence according to SEQ ID NO 23
including homologous sequences, complementary sequences, and
fragments hybridizing thereto;
[0305] (iii) a virus strain comprising in its genome a polynucleic
acid according to (ii), more particularly a virus strain termed
KGT008, as well as polynucleic acids and antigens isolated
therefrom.
[0306] The current invention thus also relates to:
[0307] (I) an antigen derived from the gp160 env precursor protein
of the new HIV-1 group 0 strain MP575 comprising the sequence
according to SEQ ID NO 26, or fragments thereof with said fragments
specifically reacting with antibodies raised against the antigen
they are derived from;
[0308] (ii) a polynucleic acid encoding an antigen according to Ii)
and comprising the nucleotide sequence according to SEQ ID NO 25
including homologous sequences, complementary sequences, and
fragments hybridizing thereto;
[0309] (iii) A virus strain comprising in its genome said
polynucleic acid according to (ii), more particularly a virus
strain deposited at the ECACC under provisional accession number
V98071301, as well as polynucleic acids and antigens isolated
therefrom.
[0310] The current invention thus also relates to:
[0311] (I) an antigen derived from the gp160 env precursor protein
of the new HIV-1 group 0 strain BSD189 comprising at least one of
the sequences according to SEQ ID NO 28, 72, 95, or fragments
thereof with said fragments specifically reacting with antibodies
raised against the antigen they are derived from;
[0312] (ii) a polynucleic acid encoding an antigen according to (I)
and comprising the nucleotide sequence according to SEQ ID NO 27
including homologous sequences, complementary sequences, and
fragments hybridizing thereto;
[0313] (iii) a virus strain comprising in its genome a polynucleic
acid according to (ii), more particularly a virus strain termed
BSD189, as well as polynucleic acids and antigens isolated
therefrom.
[0314] The current invention thus also relates to:
[0315] (I) an antigen derived from the gp160 env precursor protein
of the new HIV-1 group 0 strain BSD649 comprising at least one of
the sequences according to SEQ ID NO 30, 77, 98, or fragments
thereof with said fragments specifically reacting with antibodies
raised against the antigen they are derived from;
[0316] (ii) a polynucleic acid encoding an antigen according to (I)
and comprising the nucleotide sequence according to SEQ ID NO 29
including homologous sequences, complementary sequences, and
fragments hybridizing thereto;
[0317] (iii) a virus strain comprising in its genome a polynucleic
acid according to (ii), more particularly a virus strain termed
BSD649, as well as polynucleic acids and antigens isolated
therefrom.
[0318] The current invention thus also relates to:
[0319] (I) an antigen derived from the gp160 env precursor protein
of the new HIV-1 group 0 strain BSD242 comprising at least one of
the sequences according to SEQ ID NO 32, 81, 96, or fragments
thereof with said fragments specifically reacting with antibodies
raised against the antigen they are derived from;
[0320] (ii) a polynucleic acid encoding an antigen according to (I)
and comprising the nucleotide sequence according to SEQ ID NO 31
including homologous sequences, complementary sequences, and
fragments hybridizing thereto;
[0321] (iii) a virus strain comprising in its genome a polynucleic
acid according to (ii), more particularly a virus strain termed
BSD242, as well as polynucleic acids and antigens isolated
therefrom.
[0322] The current invention thus also relates to:
[0323] (I) an antigen derived from the gp160 env precursor protein
of the new HIV-1 group 0 strain 533 comprising at least one of the
sequences according to SEQ ID NO 34, 81, 93, or fragments thereof
with said fragments specifically reacting with antibodies raised
against the antigen they are derived from;
[0324] (ii) a polynucleic acid encoding an antigen according to (I)
and comprising the nucleotide sequence according to SEQ ID NO 33
including homologous sequences, complementary sequences, and
fragments hybridizing thereto;
[0325] (iii) a virus strain comprising in its genome a polynucleic
acid according to (ii), more particularly a virus strain termed
533, as well as polynucleic acids and antigens isolated
therefrom.
[0326] The current invention thus also relates to:
[0327] (I) an antigen derived from the gp160 env precursor protein
of the new HIV-1 group 0 strain 772P94 comprising at least one of
the sequences according to SEQ ID NO 36, 75, 94, or fragments
thereof with said fragments specifically reacting with antibodies
raised against the antigen they are derived from;
[0328] (ii) a polynucleic acid encoding an antigen according to (I)
and comprising the nucleotide sequence according to SEQ ID NO 35
including homologous sequences, complementary sequences, and
fragments hybridizing thereto;
[0329] (iii) a virus strain comprising in its genome a polynucleic
acid according to (ii), more particularly a virus strain termed
772P94, as well as polynucleic acids and antigens isolated
therefrom.
[0330] The current invention thus also relates to:
[0331] (I) an antigen derived from the gp160env precursor protein
of the new HIV-1 group 0 strain MP95B comprising at least one of
the sequences according to SEQ ID NO 38, 74, 102, or fragments
thereof with said fragments specifically reacting with antibodies
raised against the antigen they are derived from;
[0332] (ii) a polynucleic acid encoding an antigen according to (I)
and comprising the nucleotide sequence according to SEQ ID NO 37
including homologous sequences, complementary sequences, and
fragments hybridizing thereto;
[0333] (iii) a virus strain comprising in its genome a polynucleic
acid according to (ii), more particularly a virus strain termed
MP95B, as well as polynucleic acids and antigens isolated
therefrom.
[0334] The current invention thus also relates to:
[0335] (I) an antigen derived from the gp160 env precursor protein
of the new HIV-1 group 0 strain MP645 comprising at least one of
the sequences according to SEQ ID NO 135, 136, 137, or fragments
thereof with said fragments specifically reacting with antibodies
raised against the antigen they are derived from;
[0336] (ii) an antigen derived from the Vif, Vpu, Vpr, Tat, Rev,
Pol and Env protein of the new HIV-1 group 0 strain MP645
comprising one of the sequences according to SEQ ID NO 110, 114,
118, 122, 126, 120, 134, or fragments thereof with said fragments
specifically reacting with antibodies raised against the antigen
they are derived from.
[0337] (iii) a polynucleic acid encoding an antigen according to
(I) or (ii) and comprising the nucleic acid sequence according to
SEQ ID NO 106, including homologous sequences, complementary
sequences, and fragments hybridizing thereto;
[0338] (iv) A virus strain comprising in its genome said
polynucleic acid according to (iii), more particularly a virus
strain deposited at the ECACC under provisional accession number
V98071302, as well as polynucleic acids and antigens isolated
therefrom.
[0339] In another embodiment, the invention provides for an
antibody, preferably a monoclonal antibody, raised against an
antigen or antigen fragment as described above. Such an antibody
recognizes specifically the antigen or the antigen fragment to
which it has been raised.
[0340] According to an alternative embodiment, the present
invention also relates to an antigen-binding fragment of the
antibody, said fragment being of the F(ab').sub.2, Fab or single
chain Fv type, or any type of recombinant antibody derived from
said specific antibodies or monoclonal antibodies, provided that
said antibody fragment or recombinant antibody still recognizes
specifically the antigen or antigen fragment to which it has been
raised.
[0341] The expression "antibody recognizing specifically" means
that the binding between the antigen as a ligand and a molecule
containing an antibody combining site, such as a Fab portion of a
whole antibody, is specific, signifying that no cross-reaction
occurs.
[0342] The expression "antibody specifically raised against a
compound" means that the sole immunogen used to produce said
antibody was said compound.
[0343] The possible cross-reactivity of polyclonal antisera may be
eliminated by preabsorption of the polyclonal antiserum against the
cross-reacting antigenic determinants.
[0344] In a preferential embodiment, the above-mentioned antibodies
are neutralizing antibodies, i.e. antibodies capable of in vitro
inhibition of viral growth, determined according to methods known
in the art.
[0345] Neutralizing antibodies may be used as a reagent in a
so-called "passive vaccine" composition, i.e. a composition
conferring temporary protection against an infection, upon
injection in an individual. The invention also relates to passive
vaccine compositions, comprising any of the above-mentioned
neutralizing antibodies.
[0346] The monoclonal antibodies of the invention can be produced
by any hybridoma liable to be formed according to classical methods
from splenic cells of an animal, particularly of a mouse or rat,
immunized with the antigen of the invention, defined above on the
one hand, and of cells of a myeloma cell line on the other hand,
and to be selected by the ability of the hybridoma to produce the
monoclonal antibodies recognizing the antigen which has been
initially used for the immunization of the animals.
[0347] The monoclonal antibodies according to a preferred
embodiment 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.
[0348] Also fragments derived from these monoclonal antibodies such
as Fab, F(ab)'.sub.2 and ssFv ("single chain variable fragment"),
providing they have retained the original binding properties, form
part 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.
[0349] The antibodies involved in the invention can be labelled by
an appropriate label of the enzymatic, fluorescent, or radioactive
type.
[0350] The invention also relates to the use of the antigens of the
invention, or fragments thereof, for the selection of recombinant
antibodies by the process of repertoire cloning (Perrson et al.,
1991).
[0351] The present invention further relates to an anti-idiotype
antibody raised against any of the antibodies as defined above.
[0352] The term "anti-idiotype antibodies" refers to monoclonal
antibodies raised against the antigenic determinants of the
variable region of monoclonal antibodies themselves raised against
the antigens of the invention. These antigenic determinants of
immunoglobulins are known as idiotypes (sets of idiotopes) and can
therefore be considered to be the "fingerprint" of an antibody (for
review see de Prval, 1978; Fleishmann and Davie, 1984). The methods
for production of monoclonal anti-idiotypic antibodies have been
described by Gheuens and McFarlin (1982). Monoclonal anti-idiotypic
antibodies have the property of forming an immunological complex
with the idiotype of the monoclonal antibody against which they
were raised. In this respect the monoclonal antibody is often
referred to as Ab1, and the anti-idiotypic antibody is referred to
as Ab2. These anti-idiotype Ab2s may be used as substitutes for the
polypeptides of the invention or as competitors for binding of the
polypeptides of the invention to their target.
[0353] The present invention further relates to antisense peptides
derived from the antigens of the invention as described above.
[0354] More particularly, the term "antisense peptide" is reviewed
by Blalock (1990) and by Roubos (1990). In this respect, the
molecular recognition theory (Blalock, 1990) states that not only
the complementary nucleic acid sequences interact but that, in
addition, interacting sites in proteins are composed of
complementary amino acid sequences (sense ligand with receptor or
sense ligand with antisense peptides). Thus, two peptides derived
from complementary nucleic acid sequences in the same reading frame
will show a total interchange of their hydrophobic and hydrophilic
amino acids when the amino terminus of one is aligned with the
carboxy terminus of the other. This inverted hydropathic pattern
might allow two such peptides to assume complementary conformations
responsible for specific interaction.
[0355] The antisense peptides can be prepared as described in Ghiso
et al. (1990). By means of this technology it is possible to
logically construct a peptide having a physiologically relevant
interaction with a known peptide by simple nucleotide sequence
analysis for complementarity, and synthesize the peptide
complementary to the binding site.
[0356] The present invention further relates to a diagnostic method
for detecting the presence of an HIV-1 infection, said method
comprising
[0357] the detection of antibodies against HIV-1, including HIV-1
group O, using any of the antigens or antigen fragments of the
invention as described above, and/or
[0358] the detection of viral antigen originating from HIV-1,
including HIV-1 group O, using any of the antibodies of the
invention as described above and/or
[0359] the detection of viral nucleic acids originating from HIV-1,
including HIV-1 group O, using any of the nucleic acids or nucleic
acid fragments of the invention as described above, in a biological
sample.
[0360] Preferably the above-mentioned diagnostic method for
detecting the presence of an HIV-1 infection also includes the
detection of an HIV-1 group O infection, and more particularly also
includes the detection of an infection caused by any of the HIV-1
group O strains of the current invention.
[0361] The term "biological sample" refers to any biological sample
(tissue or fluid) possibly containing HIV nucleic acids, and/or HIV
antigens and/or antibodies against HIV, and refers more
particularly to blood, serum, plasma, organs or tissue samples.
[0362] In most instances, the [HIV-1 group O]-reagents (=antigens
and/or antibodies and/or nucleic acids) of the invention will be
used in methods which combine them with other HIV-reagents
(=antigens and/or antibodies and/or nucleic acids). The addition of
the HIV-1 type O reagents of the current invention to methods and
kits for detection of HIV-infection in general, may result in
methods and kits showing
[0363] a higher sensitivity, and/or
[0364] a higher discriminating power between different types of
HIV-infection, for example HIV-1 group M, HIV-1 group O and HIV-2
infection.
[0365] The term "sensitivity" refers to the ratio of positively
reacting samples/the number of truly infected samples.
[0366] More specifically, the present invention relates to a method
for in vitro diagnosis of a HIV-1 infection, including a HIV-1
group O infection, comprising at least the step of contacting a
biological sample with:
[0367] a HIV-1 group O antigen, or antigen fragment, as defined
above, under conditions allowing the formation of an immunological
complex, and/or,
[0368] a HIV-1 group O nucleic acid, or nucleic acid fragment, as
defined above, under conditions allowing the formation of a
hybridization complex, with the nucleic acids of said sample being
possibly amplified prior to hybridization, and/or,
[0369] an antibody specifically directed against an HIV-1 group O
antigen as defined above, under conditions allowing the formation
of an immunological complex, and/or,
[0370] an anti-idiotype antibody as defined above, under conditions
allowing the formation of an antibody-anti-idiotypic complex,
and/or,
[0371] an antisense peptide as defined above, under conditions
allowing the formation of an antigen-antisense peptide complex,
[0372] and subsequently detecting the complexes formed.
[0373] In a more specific embodiment, the invention relates to a
method for detecting the presence of antibodies against HIV-1 in a
biological sample, in particular antibodies against an HIV-1 group
O strain, preferably a serum sample, comprising the following
steps:
[0374] contacting the biological sample taken from a patient with
at least one antigen or antigen fragment as described above, under
conditions enabling the formation of an immunological complex,
and
[0375] detecting the immunological complex formed between said
antigen or antigen fragment and the antibodies possibly present in
the sample.
[0376] Conditions allowing the formation of an immunological
complex are known to the person skilled in the art.
[0377] In a special embodiment, the antigens being used in the
above-described method for detection of anti-HIV-1 group O
antibodies, can be replaced by anti-idiotype antibodies as
described above, acting as their equivalents.
[0378] Conditions allowing the formation of an
antibody-anti-idiotypic complex are known in the art.
[0379] The invention further relates to a method for detecting the
presence of an antigen or an antigen fragment of HIV-1, in
particular an antigen or antigen fragment of an HIV-1 group O
strain, in a biological sample comprising the following steps:
[0380] contacting the biological sample taken from a patient with
at least one antibody as described above under conditions enabling
the formation of an immunological complex, and
[0381] detecting the immunological complex formed between said
antibody and the antigen or antigen fragment possibly present in
the sample.
[0382] In a special embodiment, the antibodies being used in the
above-described method for detection of HIV-1 group O antigens, may
be replaced by anti-sense peptides as described above, acting as
their equivalents.
[0383] Conditions allowing the formation of an antigen-antisense
peptide complex are known in the art.
[0384] Design of immunoassays 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 labelled antibody or polypeptide; the
labels may be, for example, enzymatic, fluorescent,
chemoluminescent, radioactive, or dye molecules. Assays which
amplify the signals from the immune complex are also known,
examples of which are assays which utilize biotin and avidin or
streptavidin, and enzyme-labelled and mediated immunoassays, such
as ELISA assays.
[0385] An advantageous embodiment provides for a method for
detection of anti-HIV-1 group O antibodies in a sample, whereby the
antigens or antigen fragments of the invention are immobilized on a
solid support, for example on a membrane strip. Different antigens
or antigen fragments of the invention may be immobilized together
or next to each other (e.g. in the form of parallel lines). The
antigens of the invention may also be combined with other antigens,
e.g. antigens from other HIV-1 group O strains, or from HIV-1 group
M or from HIV-2 strains.
[0386] The combination of different antigens in one single
detection method as described above has certain advantages, such
as:
[0387] achieving a higher test sensitivity: e.g. by combining
several antigenic determinants from different HIV-strains, the
total number of positively reacting sera originating from
HIV-infected patients will be greater, and/or
[0388] enabling differentiation between individuals infected by
different strains of HIV, more particularly enabling
differentiation between HIV-1 group M, HIV-1 group O and HIV-2
infected patients.
[0389] The invention thus also relates to a solid support onto
which the antigens of the invention, possibly in combination with
other antigens, have been immobilized.
[0390] Another embodiment of the invention provides for a method
for detecting the presence of HIV-1 nucleic acids, including HIV-1
group O nucleic acids, in a biological sample, comprising:
[0391] (I) possibly extracting the polynucleic acids contained in
the sample,
[0392] (ii) possibly amplifying the HIV-1 polynucleic acids,
including the HIV-1 group O polynucleic acids, with a suitable
primer pair,
[0393] (iii) detecting the amplified nucleic acids, after
hybridization with a probe as described above.
[0394] The expression "a suitable primer pair" refers to a pair of
primers allowing the amplification of the target region to which
the probes of the current invention hybridize. Depending on the
application, the primer sequences may be chosen such that they
amplify specifically the nucleic acids of the current invention,
or, on the other hand, it may be preferred to obtain a more general
amplification, e.g. of all or nearly all HIV-1 group O sequences,
or of all or nearly all HIV-1 sequences, and even HIV-2
sequences.
[0395] In case a general amplification of HIV-1 group M and HIV-1
group O sequences is preferred, the following pair of primers may
be used to amplify part of the gp41 region:
5 (SEQ ID NO 139) 5'-GGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCG-3- ', and
(SEQ ID NO 140)) 5'-TCTGAAACGACAGAGGTGAGTATCCCTGCCTAA-3'.
[0396] In case a more specific amplification of the gp41 region of
HIV-1 group O strains is preferred, the following pair of primers
may be used:
6 (SEQ ID NO 141) 5'-TGGATCCCACAGTGTACTGAAGGGTATAGTGCA-3'- , and
(SEQ ID NO 142)) 5'-CATTTAGTTATGTCAAGCCA- ATTCCAAA-3'.
[0397] The invention also relates to a method for genotyping HIV-1
or HIV-1 type O strains, comprising the following steps:
[0398] possibly extracting the nucleic acids from the sample,
[0399] amplifying the HIV-1 or HIV-1 type O nucleic acids using a
suitable primer pair,
[0400] hybridizing the nucleic acids of the sample with at least
one probe as described above,
[0401] detecting the hybrids formed,
[0402] inferring the presence of one or more HIV-1 or HIV-1 type O
genotypes from the hybridization pattern obtained.
[0403] The term "genotyping" refers to the typing of HIV-strains
according to the sequence of their nucleic acids. Depending on the
application, it may be the intention of a genotyping assay to
differentiate between large groups of HIV-strains (e.g. HIV-1 group
M; HIV-1 group O or HIV-2) or to subtype smaller entities (such as
e.g. the clades withing HIV-1 group M (A to J)). Subtyping within
HIV-1 group O may also be accomplished using the nucleic acids of
the current invention.
[0404] Conditions allowing hybridization are known in the art and
e.g. exemplified in Maniatis et al. (1982). However, according to
the hybridization solution (SSC, SSPE, etc.), the probes used
should be hybridized at their appropriate temperature in order to
attain sufficient specificity (in some cases differences at the
level of one nucleotide mutation are to be discriminated).
[0405] Amplification of nucleic acids present in a sample prior to
detection in vitro may be accomplished by first extracting the
nucleic acids present in the sample according to any of the
techniques known in the art, and second, amplifying the target
nucleic acid by any amplification method as specified above. In
case of extraction of RNA, generation of cDNA is necessary;
otherwise cDNA or genomic DNA is extracted.
[0406] The term "labelled" refers to the use of labelled nucleic
acids. This may include the use of labelled nucleotides
incorporated during the polymerase step of the amplification such
as illustrated by Saiki et al. (1988) or Bej et al. (1990) or
labelled primers, or by any other method known to the person
skilled in the art. Labels may be isotopic (.sup.32P, .sup.35S,
etc.) or non-isotopic (biotin, digoxigenin, etc.).
[0407] Suitable assay methods for purposes of the present invention
to detect hybrids formed between oligonucleotide probes according
to the invention and the nucleic acid sequences in a sample may
comprise any of the assay formats known in the art. For example,
the detection can be accomplished using a dot blot format, the
unlabelled amplified sample being bound to a membrane, the membrane
being incubated with at least one labelled probe under suitable
hybridization and wash conditions, and the presence of bound probe
being monitored. Probes can be labelled with radioisotopes or with
labels allowing chromogenic or chemiluminescent detection such as
horse-radish peroxidase coupled probes.
[0408] An alternative is a "reverse" dot-blot format, in which the
amplified sequence contains a label. In this format, the unlabelled
oligonucleotide probes are bound to a solid support and exposed to
the labelled sample under appropriate stringent hybridization and
subsequent washing conditions. It is to be understood that also any
other assay method which relies on the formation of a hybrid
between the nucleic acids of the sample and the oligonucleotide
probes according to the present invention may be used.
[0409] According to an advantageous embodiment, the process of
detecting HIV-1 type O nucleic acids contained in a biological
sample comprises the steps of contacting amplified copies of the
nucleic acids present in the sample, with a solid support on which
probes as defined above, have been previously immobilized.
Preferably, the amplified nucleic acids are labelled in order to
subsequently detect hybridization.
[0410] In a very specific embodiment, the probes have been
immobilized on a membrane strip in the form of parallel lines. This
type of reverse hybridization method is specified further as a Line
Probe Assay (LiPA), and has been described more extensively in for
example WO 94/12670.
[0411] The invention thus also relates to a solid support onto
which the nucleic acids of the invention have been immobilized.
[0412] The invention also provides for a composition comprising at
least one of the antigens or antigen fragments as above described,
and/or at least one of the nucleic acids or nucleic acid fragments
as above described, and/or an antibody as above described.
[0413] Examples of such compositions may be e.g. a diagnostic kit,
an immunogenic composition, e.a.
[0414] In particular, the invention provides for a kit for the
detection of the presence of an HIV-1 infection, comprising at
least one of the antigens or antigen fragments as described above
and/or at least one of the nucleic acids or nucleic acid fragments
as described above and/or an antibody as described above.
[0415] More specifically, the current invention provides for a
diagnostic kit for determining the presence of HIV-1 nucleic acids,
including HIV-1 type O nucleic acids, in a biological sample, said
kit comprising at least one nucleic acid fragment as described
above. This nucleic acid fragment may be used as a primer or a
probe in said kit.
[0416] In addition, the current invention provides for a kit for
genotyping HIV-1 strains, including HIV-1 type O strains, in a
biological sample, said kit comprising at least one nucleic acid
fragment as described above. This nucleic acid fragment may be used
as a primer or a probe in said kit.
[0417] Moreover, the present invention also provides for a kit for
determining the presence of anti-(HIV-1 type O) antibodies present
in a biological sample, comprising at least one antigen or antigen
fragment as described above.
[0418] In addition, the present invention provides for a kit for
determining the presence of HIV-1 type O antigens present in a
biological sample, comprising at least one antibody as described
above.
[0419] The current invention also provides for a vaccine
composition which provides protective immunity against HIV-1
infection, in particular against HIV-1 group O infection,
comprising as an active principle at least one antigen or antigen
fragment as described above, or at least one nucleic acid as
described above, or a virus like particle (VLP) comprising at least
one antigen or antigen fragment as described above, or an
attenuated form of at least one of the HIV-1 type O strains as
described above, said active principle being combined with a
pharmaceutically acceptable carrier.
[0420] In a specific embodiment, polynucleic acid sequences coding
for any of the antigens or antigen fragments as defined above, are
used as a vaccine, either as naked DNA or as part of recombinant
vectors. In this case, it is the aim that said nucleic acids are
expressed into immunogenic protein/peptide and thus confer in vivo
protection to the vaccinated host (e.g. Ulmer et al., 1993).
[0421] The active ingredients of such a vaccine composition may be
administered orally, subcutaneously, conjunctivally,
intramuscularly, intra nasally, or via any other route known in the
art including for instance via the binding to carriers, via
incorporation into liposomes, by adding adjuvants known in the art,
etc.
FIGURE LEGEND
[0422] FIG. 1. Amino acid alignment of gp41 sequences from the
HIV-1 group O strains of the current invention, compared to gp41
sequences from some known prototype HIV-1 group O strains (Ant70,
MVP5180, VAU: boxed). If the name of a strain is followed by -P or
-PBMC, it means that the sequence was performed on strains present
in peripheral blood monocytes samples, in stead of serum samples.
Asteriks show perfectly conserved amino acids. Dots show well
conserved amino acids. Dashes refer to gaps introduced to maximize
the alignment. The immunodominant domain is underlined and within
this domain the dashed line indicates the immunosuppressive peptide
(ISU) and the dotted line indicates the principal immunodominant
domain (PID) by analogy to HIV-1 group M viruses. The number of
potential N-linked glycosylation sites which are shown by symbol
{circumflex over ( )} above the amino acid alignment, are indicated
at the right of the sequences on FIG. 1(contd. 1).
[0423] FIG. 2. Amino acid alignment of C2V3 sequences originating
from some of the HIV-1 group O strains ofthe current invention
(189, FABA, MP340, MP450, MP448, MP539), compared to C2V3 sequences
from some known prototype HIV-1 group O strains (Ant70, MVP5180 and
VAU: boxed). Asteriks show perfectly conserved amino acids. Dots
show well conserved amino acids. Dashes refer to gaps introduced to
maximize the alignment. The symbol + indicates the two conserved
cysteine residues flanking the V3 loop region.
[0424] FIG. 3. Nucleic acid alignment of gp41 sequences originating
from the HIV-1 group O strains of the current invention, compared
to gp41 sequences from some known prototype HIV-1 group O strains
(Ant70, MVP5180, VAU and VI686: in bold). Asteriks show positions
of conserved nucleic acids. Dashes refer to gaps introduced to
maximize alignment.
[0425] FIG. 4. Nucleic acid alignment of C2V3 sequences originating
from some of the HIV-1 group O strains of the current invention
(189, FABA, MP340, MP448, MP450 and MP539), compared to C2V3
sequences from some known prototype HIV-1 group O strains (MVP5180,
Ant70 and VI686: in bold). Asteriks show positions of conserved
nucleic acids. Dashes refer to gaps introduced to maximize
alignment.
[0426] FIG. 5. Phylogenetic tree analysis for the gp41-sequenced
region of the new HIV-1 group O strains of the current invention,
compared to the prototype HIV-1 group O strains (Ant70, MVP5180,
VAU and VI686), HIV-1 group M strains (U455, Z2Z6 and MN), and
SIVcpz-strains. SIVcpz-ANT has been used as an "outgroup" for the
analysis, and is therefore put between brackets []. The viruses of
the current invention are indicated in bold. Country of origin is
mentioned between parentheses. Phylogenetic relationships were
determined using the neighbor joining method, as described in
Materials and Methods. The numbers given at the branch points
represent bootstrap values out of 100 obtained for the neigbor
joining method.
[0427] FIG. 6. Nucleotide and amino acid sequences obtained from
the new HIV-1 group O strains of the current invention.
[0428] FIG. 7.
[0429] 7A. Comparison of the consensus amino acid sequences of the
potential gp41-immunosuppressive peptide (ISU) for HIV-1 group M
and O strains and ISU-peptide for SIVcpzGAB and SIVcpzANT.
[0430] 7B. Antigenicity/hydrophilicity plots of the consensus
ISU-peptide (17 amino acids flanked by Leucine (L) residues) for
HIV-1 group O and group M viruses. A value of 100% or nearly
predicts the considered peptide to be highly immunogenic.
[0431] FIG. 8.
[0432] 8A. Nucleic acid sequence (SEQ ID NO 106) and the
corresponding amino acid sequence translation of part of the genome
of HIV-1 group O virus MP645. The corresponding polypeptides Pol
(partially)(SEQ ID NO 120, Vif (SEQ ID NO 110), Vpr (SEQ ID NO
118), Tat (SEQ ID NO 122), Rev (SEQ ID NO 126), Vpu (SEQ ID NO 114)
and Env (SEQ ID NO 134) (partially) are underlined and their
corresponding name is indicated at the right of each open reading
frame.
[0433] 8B. Nucleic acid sequence (SEQ ID NO 103) and the
corresponding amino acid sequence translation of part of the genome
of HIV-1 group O virus MP331 (FABA). The corresponding polypeptides
Pol (partially) (SEQ ID NO 127), Vif (SEQ ID NO 107), Vpr (SEQ ID
NO 115), Tat (SEQ ID NO 119), Rev (SEQ ID NO 123), Vpu (SEQ ID NO
111) and Env (partially) (SEQ ID NO 131) are underlined and their
corresponding name is indicated at the right of each open reading
frame.
[0434] 8C. Nucleic acid sequence (SEQ ID NO 104) and the
corresponding amino acid sequence translation of part of the genome
of HIV-1 group O virus MP448. The corresponding polypeptides Pol
(partially) (SEQ ID NO 128), Vif (SEQ ID NO 108), Vpr (SEQ ID NO
116), Tat (SEQ ID NO 120), Rev (SEQ ID NO 124), Vpu (SEQ ID NO 112)
and Env (partially) (SEQ ID NO 132) are underlined and their
corresponding name is indicated at the right of each open reading
frame.
[0435] 8D. Nucleic acid sequence (SEQ ID NO 105) and the
corresponding amino acid sequence translation of part of the genome
of HIV-1 group O virus MP539. The corresponding polypeptides Pol
(partially) (SEQ ID NO 129), Vif (SEQ ID NO 109), Vpr (SEQ ID NO
117), Tat (SEQ ID NO 121), Rev (SEQ ID NO 125), Vpu (SEQ ID NO 113)
and Env (partially) (SEQ ID NO 133) are underlined and their
corresponding name is indicated at the right of each open reading
frame.
TABLE LEGEND
[0436] Table 1:
[0437] Divergence between HIV-1 group M and O viruses and the
chimpanzee viruses SIVcpzGAB and SIVcpzANT based on gp41 nucleic
acid sequences.
[0438] .sup.a: Divergence from group M viruses was calculated for
at least three randomly selected strains for each of the subtypes
from A to G.
[0439] Table 2: Percent divergence (=100%-% homology) between the
gp41 nucleic acid sequences of the different HIV-1 group O strains
of the current invention as specified in FIG. 3.
7TABLE 1 % genetic divergence Group O Group M CPZANT CPZGAB 37.5
(35.2-38.8) 31.2 (29.3-32.5) 32.6 CPZANT 36.5 (32.4-39.1) 33.7
(32.9-34.3) Group M 37.3 (35.0-40.8) 12.3 (2.2-16.6) Group O 14.7
(1.2-21.8)
[0440] Table 2 (Next Page)
8 MP340 FABA MP450 MP448 ANT70 MVP1580 VAU VI686 (PBMC) MP340
(PBMC) FABA (PBMC) MP450 (PBMC) MP448 189 ANT70 -- 18 16 9 13 13 12
13 12 12 10 9 14 MVP5180 -- 17 22 24 23 24 25 23 23 16 15 26 VAU --
18 21 21 20 21 20 20 18 17 22 VI686 -- 14 14 15 16 14 14 10 10 16
MP340 (PBMC) -- 1 14 15 2 2 14 14 2 MP340 -- 13 12 2 2 15 15 2 FABA
(PBMC) -- 5 13 13 15 15 13 FABA -- 13 13 15 15 15 MP450 (PBMC) -- 0
13 13 3 MP450 -- 13 13 3 MP448 (PBMC) -- 0 16 MP448 -- 16 189 --
320 BSD422 KGT008 MP575 BSD189 BSD649 BSD242 533 772P94 MP95B MP539
320 BSD422 KGT008 MP575 BSD189 BSD649 BSD242 533 772P94 MP95B MP539
ANT70 9 9 11 7 9 6 22 23 19 15 18 MVP5180 21 21 23 21 21 20 7 8 15
33 22 VAU 18 18 18 16 17 15 18 18 15 16 15 VI686 11 12 19 10 10 10
26 25 22 15 18 MP340 (PBMC) 14 15 17 13 11 12 26 24 20 20 20 MP340
14 14 17 13 11 12 27 24 20 20 19 FABA (PBMC) 14 18 17 13 14 13 28
26 20 20 22 FABA 15 18 17 14 14 14 29 27 20 20 22 MP450 (PBMC) 12
15 17 12 10 12 26 24 19 19 20 MP450 12 15 17 12 10 12 26 24 19 19
20 MP448 (PBMC) 10 12 12 10 11 11 19 19 19 15 17 MP448 11 12 12 9
11 11 18 19 18 14 17 189 15 17 19 14 13 14 28 26 22 22 22 320 -- 12
11 7 11 8 22 22 22 16 18 BSD422 -- 15 11 13 10 24 26 19 15 18
KGT008 -- 10 12 12 26 26 22 19 20 MP575 -- 8 7 25 25 20 17 17
BSD189 -- 9 26 26 19 17 18 BSD649 -- 24 23 19 16 18 BSD242 -- 5 16
16 25 533 -- 18 16 23 772P94 -- 17 19 MP95B -- 18 MP539 --
EXAMPLES
Example 1
Materials and Methods
[0441] Patients and Viruses
[0442] A total of 16 viruses have been characterized. Patients were
identified as being infected with an HIV-1 group O virus using a
specific serological testing algorithm, based on V3 peptides from
different M and O strains (consensusM, M-Mal, O-ANT-70, O-VI686,
O-MVP5180 (INNOLIA HIV-1 type O Research product, Innogenetics,
Belgium), as described elsewhere (Peeters et al., in press). Ten
patients were from Cameroon (BSD189, BSD242, BSD422, BSD649, MP340,
MP95B, MP448, MP575, MP539 and MP450), 2 from Gabon (189, 533) and
the others from Tchaad (320), Nigeria (KGT008), Senegal (FABA=331)
and Niger (772P94). For 3 patients from Cameroon (MP340, MP448 and
MP450) and for the patients from Nigeria and Senegal primary
uncultured peripheral blood mononuclear cells (PBMCs) were
available, while for the other patients from Cameroon, Gabon, Niger
and Tchaad only serum was available. Strain VI686 from Gabon has
been described previously (Janssens et al. 1994).
[0443] Nucleic Acid Extraction
[0444] DNA was extracted from PBMCs using the IsoQuick isolation
kit (Microprobe Corp., Garden Cove, Calif., USA), resuspended in
the appropriate volume of nuclease free water and {fraction (1/10)}
was used for amplifications. Viral RNA was extracted from 50 .mu.l
of plasma by the guanidinium thiocyanate-phenol-chloroform method
as described previously by Chomczynsky and Sacchi (1987),
resuspended in 5 .mu.l of nuclease free water and further used for
reverse transcription reaction.
[0445] RT, PCR and Sequencing
[0446] The reverse transcription reaction (RT) was performed in a
final volume of 20 .mu.l, containing 50 mM TrisHCl pH 8.3, 50 mM
KCl, 10 mM MgCl.sub.2, 10 mM DTT, 0.5 mM spermidine, 1 mM each
deoxynucleoside triphophate, 0.5 .mu.M outer reverse primer (41-4,
see further) and 5 U of Avian Myeloblastosis Virus Reverse
Transcriptase (Promega), for 30 min at 42.degree. C. Five
microliters from the RT reaction were used for PCR
amplification.
[0447] Nested PCR was used to amplify a fragment of approximately
420 bp from the gp41-region. Outer primers allow amplification of
HIV-1 sequences from group O and M (sense 41-1:
5'-GGGTTCTTGGGAGCAGCAGGAAGCACTA- TGGGCG-3' (SEQ ID NO 139),
antisense 41-4: 5'-TCTGAAACGACAGAGGTGAGTATCCCTG- CCTAA-3' (SEQ ID
NO 140)). Inner primers were determined according to the
HIV-1-Ant70 sequence (Vanden Haesevelde et al. 1994) (sense 41-6:
5'-TGGATCCCACAGTGTACTGAAGGGTATAGTGCA-3' (SEQ ID NO 141), antisense
41-7: 5'-CATTTAGTTATGTCAAGCCAATTCCAAA-3' (SEQ ID NO 142)). PCRs
were performed in a final volume of 100 .mu.l containing 10 mM
Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each
deoxynucleoside triphosphate, 2.5 U of Taq DNA polymerase (Promega)
and 0.4 .mu.M of each primer. After an initial denaturation step of
3 min at 94.degree. C., 30 to 35 cycles were performed at
94.degree. C. for 1 min to 20 s, 50.degree. C. for 1 to 30 s,
72.degree. C. for 1 min, followed by a final extension of 10 to 7
min. For the second round, 1 to 5 .mu.l of the first amplification
were subjected to the same cycling conditions for 35 to 40 PCR
cycles.
[0448] Amplification of the C2V3-region was obtained by nested PCR
using a set of primers selected from the HIV-1-Ant70 sequence.
Outer primers were: anti-sense V70-5
(5'-GTTCTCCATATATCTTTCATATCTCCCCCTA-3', SEQ ID NO 143) and sense
V70-1 (5'-TTGTACACATGGCATTAGGCCAACAGTAAGT-3', SEQ ID NO 144) and
inner primers were: sense V70-2 (5'-TGAATTCCTAATATTGAATGGGACACTC-
TCT-3', SEQ ID NO 145) and antisense V70-4
(5'-TGGATCCTACAATAAAAGAATTCTCCA- TGACA-3', SEQ ID NO 146).
Amplification conditions were as described above.
[0449] Each fragment was sequenced on both strands, as previously
described (Bibollet-Ruche 1997), using an Applied Biosystems
sequencer (model 373A, Applied Biosystems, Inc) and a dye-deoxy
terminator procedure, as specified by the manufacturer.
[0450] Sequence Analysis
[0451] Overlapping sequences were joined by using SeqEd-1.0
(Applied Biosystems, Inc). Sequences were aligned using CLUSTAL V
(Higgins et al 1992; Higgins and Sharp 1988) program. Evolutionary
distances were calculated by using the Kimura's two-parameters
method with correction for the multiple substitutions and excluding
positions with gaps in aligned sequences (Kimura 1983).
Phylogenetic relationships were computed from the distance matrix
by the neighbor-joining method (Saitou and Nei 1987). Phylogenetic
analyses were also performed by a parsimony approach and
implemented using DNAPARS. In both cases, reliability of the
branching orders was confirmed by the bootstrap approach
(Felsenstein 1985). Phylogenetic analyses were also performed for
proteic sequences using PROTPARS. These methods were implemented
using the PHYLIP 3.56 package (Felsenstein 1993). The results were
similar with both methods, for nucleotidic and proteic sequences,
in all essential aspects.
[0452] Antigenicity Profiles
[0453] Antigenicity of the ISU peptides have been calculated
according to programs developed by Garnier et al (1978) and Gibrat
et al (1987).
Example 2
Nucleic Acid Sequences and Phylogenetic Analysis
[0454] Analysis of gp41 Sequences
[0455] For the 16 viruses, 330 to 351 bp of the region spanning the
immunodominant domain of the transmembrane gp41 glycoprotein (by
analogy to HIV-1 group M viruses) was characterized. The different
sequences obtained are represented in FIG. 6. These sequences have
been aligned to the corresponding gp41-sequences of known HIV-1
group O strains, as shown in FIG. 3. Moreover, sequence identity
was calculated for intra and intergroup similarity by pairwise
alignment and comparison (Table 1). For the group M viruses, these
values were calculated for randomly selected strains for which
sequences were available in the database and which were
representative of subtypes A to G (the same sequences were further
used for phylogenetic analyses). Divergence between SIVcpz-GAB
(Huet et al. 1990) and the group M (mean 31,2%) was lower than
divergence between SIVcpz-GAB and the group O (mean 37%). These
results were similar when calculated with the second chimpanzee
virus, SIVcpz-ANT (Vanden Haesevelde et al. 1996), with mean
divergence of 33.7% and 36.5% with group M and O respectively
(Table 1). Intragroup divergence for group O viruses ranged from
1.2 to 21.8% (mean 14.7%) while divergence within group M clades (A
to G) ranged from 2.2 to 16.6% (mean 12.3%).
[0456] Phylogenetic trees were constructed with the 16 new
sequences described here, the four group O strains of which the
gp41 sequences have already been characterized, nl. Ant70 (Vanden
Haesevelde et al. 1994), MVP5180 (Gurtler et al. 1994), VAU
(Charneau et al. 1994) and VI686 (Janssens et al. 1994) and group M
viruses representative for A (U455), B (MN) and D (Z2Z6) subtypes
(see FIG. 5). Trees were constructed from nucleotidic sequences
according to the alignment in FIG. 3, by the neighbor-joining and
by the maximum-parsimony methods. Consistent results were obtained
by the two methods: group M and group O viruses clustered
separately (bootstrap values 98 and 100 respectively).
[0457] Within the newly characterized HIV-1 type O strains new
subclusters may appear, such as for example the subcluster
consisting of strains MP340, 189 and MP450, which segregate with a
bootstrap value of 100, or the subcluster consisting of strains
MVP5180, 533, BSD242 (bootstrap value 98). These subclusters may
define different genotypes within group O in analogy to the
different group M genotypes (clades). Phylogenetic analyses were
also performed for the deduced amino acid sequences for group O and
M viruses (data not shown) and the results were consistent with the
nucleotidic sequences, although lower bootstrap values were
observed (approximately 65%), probably due to the short size of the
sequences (109a.a., gaps excluded, data not shown). The countries
from which these viruses were sampled are indicated alongside the
strains and no links were found between the manner of clustering
and the geographical origins of the strains.
[0458] Table 2 shows the percentage divergence between
gp41-nucleotide sequences originating from HIV-1 group O strains of
the invention and known HIV-1 group O strains (Ant70, MVP5180, VAU
and VI686).
[0459] Analysis of C2V3 Sequences
[0460] For 6 of the 16 new virus strains (189, FABA, MP340, MP448,
MP450 and MP539), C2V3 sequences were determined. FIG. 4 shows the
alignment of these new C2V3 sequences with the corresponding
sequences of known HIV-1 group O strains.
Example 3
Analysis of Deduced Amino Acid Sequences
[0461] gp41 Amino Acid Sequences
[0462] FIG. 1 depicts the alignment of the deduced gp41 amino acid
sequences. Although different from group M, this region of the
transmembrane glycoprotein is also highly conserved among group O
viruses, especially for the region corresponding to the
immunodominant region in group M isolates. This region has been
divided into two domains, corresponding to an immunosuppressive
peptide (ISU-peptide) of 17 amino acids (aa) and to an
immunodominant cysteine loop of 7aa (Oldstone et al. 1991), also
called the principal immunodominant domain (PID). The consensus
cysteine loop for group O viruses (CKGR/KLV/IC) is quite closely
related to the group M consensus (CSGKLIC), suggesting that they
could have similar functions in both virus groups. This PID
sequence, corresponding to a B-cell epitope recognized by nearly
all sera from patients or animals (Bertoni et al. 1994; Chong et
al. 1991; Gnann et al. 1987; Norrby et al. 1987; Pancino et al.
1993), seems to be a very conserved structure, in contrast to the
variability observed for the neutralizing epitope of the V3 loop of
HIV-1 gp120 (Zwart et al. 1991; Zwart et al. 1993). Deletion of
this PID peptide or disruption of the cysteine loop severely
impairs the gp160 processing, leading to a loss of infectivity in
HIV-1 (Dedera et al. 1992; Pancino et al. 1993; Schulz et al. 1992)
and also in the far related type D simian retrovirus (Brody and
Hunter 1992).
[0463] Comparison of ISU-peptides from the group M, SIVcpz and
group O viruses are presented in FIG. 7A. The consensus peptide
from group O is quite divergent from the SIVcpz/group M peptide by
the presence of an arginine (R, positively charged) in position 2
instead of a phenylalanine (Q, hydrophilic), a leucine (L) in
position 5 and 8 instead of a valine (V), and a very different
stretch TLIQN instead of RYLKD in position 10-14. Prediction of the
secondary structure revealed an alpha helix in each case, and the
predicted isoelectric point is 9,7 and 11,3 for group M/SIVcpz and
group O strains respectively. FIG. 7B represents the predicted
hydrophilicity/antigenicity plot and revealed the presence of a
second peak at position 3 to 7 (ARLLA) for group O in addition to
the peak at position 12 (L) conserved in group M/SIVcpz and group O
viruses.
[0464] The divergence of the ISU peptide between group O and M
viruses may suggest different functionalities of the ISU peptide in
both groups. For several retroviruses this ISU peptide has been
shown to suppress a broad range of immune reactions such as the
inhibition of IL-2-dependant and concanavaline A-induced
proliferation of T lymphocytes (Denner et al. 1994; Ruegg et al.
1989) but also B lymphocyte proliferation for HIV-1 group M (Denner
et al. 1996). The principal B-epitope of this ISU peptide has been
mapped at the C-terminal end, centered around the leucine residue
in position 12 for group M (Denner et al. 1994). The data of FIG. 7
show a similar antigenicity peak for group O viruses but, in
contrast to group M and SIVcpz, the presence of a second peak is
observed towards the N-terminal part of this peptide. Moreover,
antibody response against this region in HIV-1 group M infected
individuals has been shown to be vigorous and independent of the
stage of the disease (Cumming et al. 1990; Zwart et al. 1994). This
result raises the possibility fo the design of peptides for the
specific detection of antibodies against group O viruses, which
could improve the discrimination between the two HIV-1 groups, and
could also be useful for the detection of divergent group O viruses
given the high conservation of these peptides among the different
strains we have characterized.
[0465] A third interesting feature concerns the number and the
position of the N-linked glycosylation sites in the immunodominant
domain (FIG. 1). In group M viruses, 4 sites are conserved in the
gp41 extracellular domain and they have been shown to play
important roles in the intracellular processing of the gp160 and in
the fusogenic properties of gp41 (Dedera et al. 1992; Vanini et al.
1993). Most of the group O viruses described here contain only
three potential N-linked glycosylation sites whereas only a few
strains contain four. Only the position of the first site is highly
conserved (position 60 in the alignment) and the positions of the
two or three other sites vary among the strains, in contrast to
group M viruses.
[0466] In HIV-1 group M viruses, it is known that removal of the
thirth glycosylation site abolishes the cleavage of the gp160
precursor and transport to the plasma membrane of infected cells.
An immediate consequence of this result, obtained in vitro after
transfection of this mutant in HeLa/CD4 cells, is that it fails to
induce syncytia (Dash et al. 1994). Roles of the other N-linked
glycosylation sites of the gp41 have not been fully explored but
their high conservation suggests important functions. In the case
of the gp120, the highly conserved sites surrounding the V3 loop
have been shown to influence infectivity (Lee et al. 1992), protein
folding (Li et al. 1993) and immunogenicity (Benjouad et al 1992)
of this domain. For group O viruses, only the first site is
conserved in all the strains which were characterized. Even if the
importance of N-glycosylation has not been studied for HIV-1 group
O viruses, it can be speculated that differences will be observed
when compared to group M viruses.
[0467] In conclusion, comparison of the gp41 amino acid sequences
clearly discriminate the two HIV-1 groups, either by the pattern of
N-linked glycosylation sites or by the characteristics of the ISU
peptide of the ectodomain of the transmembrane glycoprotein. The
differences observed between the two HIV-1 groups in the gp41
region raise the possibility that these viruses use different
mechanisms, for example in the intracellular processing of the
envelope precursor or in the domains involved in the membrane
fusion. Several studies have suggested that the immunodominant
region of the gp41 in group M viruses, which is a target for
neutralizing but also enhancing antibodies, could be a promising
candidate for vaccine development (Cumming et al. 1990; Muster et
al. 1993; Zwart et al. 1994). The data disclosed in the current
invention suggest that a vaccine developed against HIV-1 group M
viruses could be ineffective against group O strains, especially
when these viruses could use different pathways to induce
immunodeficiency in infected patients.
[0468] Analysis of C2V3 Amino Acid Sequences
[0469] FIG. 2 depicts the alignment of the deduced C2V3 sequences
of 6 of the HIV-1 group O strains of the invention as compared to
known HIV-1 group O strains. The delineation of the C2 and V3
region is according to Starcich et al. (1986) and Willey et al.
(1986). The C2 region belongs to the rather conserved regions which
are important in protein folding and protein function. The V3
region is one of the hypervariable regions, for which amino acid
conservation between different HIV-1 isolates is below 50%.
Hypervariable regions also contain short deletions or insertions
(Myers et al. 1992).
[0470] The V3-region of gp120, also known as the principal
neutralizing determinant (PND), contains a loop of 35 amino acids,
formed by a cysteine-cysteine disulfide bridge. The PND is
implicated in several important biological functions, such as:
[0471] 1. it determines the HIV-1 cell tropism (Hwang et al. 1991,
Shioda et al. 1991)
[0472] 2. it affects fusion (Feed et al. 1991) and viral virulence
(Fouchier et al. 1992)
[0473] 3. it stimulates cytotoxicity (Takahashi et al. 1992)
[0474] 4. it is the primary target for antibody neutralization
(Palker et al. 1989).
[0475] The fact that the V3 domain of gp120 may induce a protective
immune response, has made this region of particular interest for
vaccine research. Although the V3 region as a whole is
hypervariable, there is a rather high sequence conservation in its
tetrapeptide sequence Gly-Pro-Gly-Arg (GPGR) at the crown of the
loop (LaRosa et al. 1990). This motif corresponds to the binding
site of the neutralizing antibodies, and single amino acid changes
within this epitope highly reduce antibody binding (Meloen et al.
1989).
[0476] The alignment in FIG. 2 shows that sequence variability
within the V3 loop is high between the different HIV-1 group O
strains, including those of the current invention. These new
sequences, and more particularly the sequences represented by SEQ
ID NO 53 to 70 and SEQ ID NO 83 to 90 are important in the design
of new and better (=more sensitive and/or more specific)
HIV-diagnostic assays. Moreover, the various sequences in the V3
region will be important with regard to the development of
HIV-vaccines providing an as broad as possible protection against
HIV-infection.
Example 4
Cloning Strategy to Obtain Vif, Vpu, Vpr, Tat, Rev, Env and
Pol.
[0477] Polymerase Chain Reaction Amplification and Sequencing
[0478] The sequences of the nested primer sets were designed on
HIV-1 nucleic acid sequences in conserved regions flanking the Vif
and Vpu genes. DNA from cultured and uncultured PBMCs was extracted
using IsoQuick (Microprobe, Garden Cove, Calif.) according to the
manufacturer instructions and quantified spectrophotometrically.
Approximately 1 .mu.g of DNA was used for a first round of
amplification with an outer primer pair (VIF1, 5'
GGGTTTATTACAGGGACAGCAGAG 3' (SEQ ID NO 147) and VPU1, 5'
GGTTGGGGTCTGTGGGTACACAGG 3') (SEQ ID NO 148) in a final volume of
100 .mu.l of containing 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM
MgCl.sub.2 a 0.2 mM concentration of each deoxynucleoside
triphosphate, 2.5 U of Taq DNA polymerase (Promega, Madison, Wis.),
and a 0.4 .mu.M concentration of each primer. Five microliters from
this first round was used for a second round with an inner primer
pair (VIF2, 5' GCAAAACTACTCTGGAAAGGTG 3' (SEQ ID NO 149) and VPU2,
5' GCWTCTTTCCACACAGGTACCCC 3' (SEQ ID NO 150) where W represents an
A or a T) under the same reaction conditions. The two rounds of PCR
were run for 35 cycles each under the following cycling conditions:
94.degree. C. for 30 sec, 50.degree. C. for 30 sec, and 72.degree.
C. for 2 min. The two rounds of PCR were preceded by a denaturation
step of 3 min at 94.degree. C. and followed by a final extension
step of 7 min at 72.degree. C.
[0479] Sequencing of the amplified products was done directly after
purification by TAE-low melting point agarose gel electrophoresis
(Bibollet-Ruche et al, 1997) using an Applied Biosystems (Foster
City, Calif.) 373 Stretch sequencer and a Dye-Deoxy terminator
procedure (dye terminator cycle sequencing ready reaction, with
AmpliTaq DNA polymerase; Perkin-Elmer. Norwalk Conn.) as specified
by the manufacturer. Inner polymerase chain reaction (PCR) primers
(VIF2 and VPU2) and inner sequencing primers (OVIF, 5'
CATATTGGGGATTGATGCCAG 3' (SEQ ID NO 151); OVPU,
5'GCATYAGCGTTACTTACTGC 3': Y=C or T (SEQ ID NO 152)) were used.
Overlapping sequences were joined using SeqEd (Applied Biosystems)
to obtain the full-length sequence. Direct sequencing was performed
on PCR-generated fragments. Ambiguities observed at a limited
number of positions in some sequences were resolved when joining
the overlapping fragments.
[0480] Analyses of Accessory Protein Sequences
[0481] Open reading frames for the different accessory proteins
(Pol, Vif, Vpr, the first exon of Tat, Vpu and Env) were determined
and the deduced protein sequences were obtained using the Translate
program option of the PCgene software package. The resulting
sequences are indicated in FIGS. 8(B to D). FIG. 8A shows the
sequences of HIV-1 group O strain MP645 which was obtained
following a similar approach as the one described above for MP448,
MP539 and MP331.
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Sequence CWU 1
1
152 1 340 DNA Human 1 aacctgctaa gagcaataca ggcccagcaa gaattgctga
ggctatctgt atggggtatc 60 agacaactcc gagctcgcct gctagcctta
gaaaccttaa tacagaatca gcagctccta 120 aacctatggg gttgtaaggg
aaggatagtc tgctacacat cagtaaaatg gaacgataca 180 tggagacatg
tcactaatat gagtgaagtt tgggacaaac taacctggca ggaatgggat 240
cggcagatag acaacataag ctatgttata tatgatgaaa tacaaagagc acaagtacag
300 caagaacaaa atgagaagaa gttgctggag ttagatgaat 340 2 113 PRT Human
2 Asn Leu Leu Arg Ala Ile Gln Ala Gln Gln Glu Leu Leu Arg Leu Ser 1
5 10 15 Val Trp Gly Ile Arg Gln Leu Arg Ala Arg Leu Leu Ala Leu Glu
Thr 20 25 30 Leu Ile Gln Asn Gln Gln Leu Leu Asn Leu Trp Gly Cys
Lys Gly Arg 35 40 45 Ile Val Cys Tyr Thr Ser Val Lys Trp Asn Asp
Thr Trp Arg His Val 50 55 60 Thr Asn Met Ser Glu Val Trp Asp Lys
Leu Thr Trp Gln Glu Trp Asp 65 70 75 80 Arg Gln Ile Asp Asn Ile Ser
Tyr Val Ile Tyr Asp Glu Ile Gln Arg 85 90 95 Ala Gln Val Gln Gln
Glu Gln Asn Glu Lys Lys Leu Leu Glu Leu Asp 100 105 110 Glu 3 340
DNA Human misc_feature (1)..(340) N = any nucleotide 3 aacctgctaa
sancaataca ggcccakcaa gaattgctga ggctatctgt atggggtatc 60
agacaamtcc gagstygcct gstagcctta gaaaccttaa tacasaatca gcasctccta
120 aacctatggg gttgtaaagg aaggatastn tgctacacat cagtaaaatg
gaacnataca 180 tggaaacatg tcactnatat gagtgaagtt tgggacaaac
taacctggca ggaatgggat 240 cggcngatag acaacataag ctatgttata
tatgatgnna tacaaagagc acaagtacag 300 caagaacaaa atgagaagaa
gttgctggag ttagatgaat 340 4 113 PRT Human Misc_feature (1)..(113)
Xaa = unknown 4 Asn Leu Leu Xaa Xaa Ile Gln Ala Xaa Gln Glu Leu Leu
Arg Leu Ser 1 5 10 15 Val Trp Gly Ile Arg Gln Xaa Arg Xaa Xaa Leu
Xaa Ala Leu Glu Thr 20 25 30 Leu Ile Xaa Asn Gln Xaa Leu Leu Asn
Leu Trp Gly Cys Lys Gly Arg 35 40 45 Ile Xaa Cys Tyr Thr Ser Val
Lys Trp Asn Xaa Thr Trp Lys His Val 50 55 60 Thr Xaa Met Ser Glu
Val Trp Asp Lys Leu Thr Trp Gln Glu Trp Asp 65 70 75 80 Arg Xaa Ile
Asp Asn Ile Ser Tyr Val Ile Tyr Asp Xaa Ile Gln Arg 85 90 95 Ala
Gln Val Gln Gln Glu Gln Asn Glu Lys Lys Leu Leu Glu Leu Asp 100 105
110 Glu 5 352 DNA Human 5 aacctgctaa aagcaataca ggcccagcag
caattgctga ggttatctgt atggggtatc 60 aaacaactcc gagctcgcct
gctagcctta gaaaccttaa tacagaatca gcaactccta 120 aacctatggg
gctgtaaagg aaggctagtc tgctacacat cagtaaaatg gaacaataca 180
tggacaaaaa acatcacaaa catcacagac ctagacgaga tttgggacaa atttacatgg
240 caccaatggg atcaacagat aaaccacata agtgatgtca tatatgaaga
aataccaaag 300 gcacaagtac agcagggacc aaatgagagg aagttgctgg
agttagatga at 352 6 117 PRT Human 6 Asn Leu Leu Lys Ala Ile Gln Ala
Gln Gln Gln Leu Leu Arg Leu Ser 1 5 10 15 Val Trp Gly Ile Lys Gln
Leu Arg Ala Arg Leu Leu Ala Leu Glu Thr 20 25 30 Leu Ile Gln Asn
Gln Gln Leu Leu Asn Leu Trp Gly Cys Lys Gly Arg 35 40 45 Leu Val
Cys Tyr Thr Ser Val Lys Trp Asn Asn Thr Trp Thr Lys Asn 50 55 60
Ile Thr Asn Ile Thr Asp Leu Asp Glu Ile Trp Asp Lys Phe Thr Trp 65
70 75 80 His Gln Trp Asp Gln Gln Ile Asn His Ile Ser Asp Val Ile
Tyr Glu 85 90 95 Glu Ile Pro Lys Ala Gln Val Gln Gln Gly Pro Asn
Glu Arg Lys Leu 100 105 110 Leu Glu Leu Asp Glu 115 7 352 DNA Human
misc_feature (1)..(352) N = any nucleotide 7 nacctgttaa gagcaataca
ggcccagcag caattggtga ggttatctgt atggggtatc 60 agacaaatcc
gaggtngcct ggtagcctta gaaaccttaa tacagaatca gcaantcctn 120
aacctatggg gctgtaaagg aagggtagtt tgntacacat cagtaaaatg gaacaataca
180 tggacaaaaa acatcacaaa catcacagac ctagacgaga tttgggacaa
atttacatgg 240 cagcaatggg atcaacagat aaacaacata agtgatgtcc
tatatgaaga aatacaaaag 300 gcacaagtac agcaggaaca aaatgagagg
aagttgctgg agttagatga at 352 8 117 PRT Human Misc_feature
(1)..(117) Xaa = Unknown 8 Xaa Leu Leu Arg Ala Ile Gln Ala Gln Gln
Gln Leu Val Arg Leu Ser 1 5 10 15 Val Trp Gly Ile Arg Gln Ile Arg
Gly Xaa Leu Val Ala Leu Glu Thr 20 25 30 Leu Ile Gln Asn Gln Gln
Xaa Xaa Asn Leu Trp Gly Cys Lys Gly Arg 35 40 45 Val Val Xaa Tyr
Thr Ser Val Lys Trp Asn Asn Thr Trp Thr Lys Asn 50 55 60 Ile Thr
Asn Ile Thr Asp Leu Asp Glu Ile Trp Asp Lys Phe Thr Trp 65 70 75 80
Gln Gln Trp Asp Gln Gln Ile Asn Asn Ile Ser Asp Val Leu Tyr Glu 85
90 95 Glu Ile Gln Lys Ala Gln Val Gln Gln Glu Gln Asn Glu Arg Lys
Leu 100 105 110 Leu Glu Leu Asp Glu 115 9 340 DNA Human 9
aacctgctaa gagcaataca ggcccagcag caattgctga ggctatctgt atggggtatc
60 agacaactcc gagctcgcct gctagcctta gaaaccttaa tacagaatca
gcagctccta 120 aacctatggg gttgtaaggg aaggatagtc tgctacacat
cagtaaaatg gaacaataca 180 tggagaaatg tcactaatat gagtgaagtt
tgggacacac taacctggca ggaatgggat 240 cggcagatag acaacataag
ctatgttata tatgatgaaa tacaaagagc acaagtacag 300 caggaacaaa
atgagaagaa gttgctggag ttagatgaat 340 10 113 PRT Human 10 Asn Leu
Leu Arg Ala Ile Gln Ala Gln Gln Gln Leu Leu Arg Leu Ser 1 5 10 15
Val Trp Gly Ile Arg Gln Leu Arg Ala Arg Leu Leu Ala Leu Glu Thr 20
25 30 Leu Ile Gln Asn Gln Gln Leu Leu Asn Leu Trp Gly Cys Lys Gly
Arg 35 40 45 Ile Val Cys Tyr Thr Ser Val Lys Trp Asn Asn Thr Trp
Arg Asn Val 50 55 60 Thr Asn Met Ser Glu Val Trp Asp Thr Leu Thr
Trp Gln Glu Trp Asp 65 70 75 80 Arg Gln Ile Asp Asn Ile Ser Tyr Val
Ile Tyr Asp Glu Ile Gln Arg 85 90 95 Ala Gln Val Gln Gln Glu Gln
Asn Glu Lys Lys Leu Leu Glu Leu Asp 100 105 110 Glu 11 340 DNA
Human 11 aacctgctaa gagcaataca ggcccagcag caattgctga ggctatctgt
atggggtatc 60 agacaactcc gagctcgcct gctagcctta gaaaccttaa
tacagaatca gcagctccta 120 aacctatggg gttgtaaggg aaggatagtc
tgctacacat cagtaaaatg gaacaataca 180 tggagaaatg tcactaatat
gagtgaagtt tgggacacac taacctggca ggaatgggat 240 cggcagatag
acaacataag ctatgttata tatgatgaaa tacaaagagc acaagtacag 300
caggaacaaa atgagaagaa gttgctggag ttagatgaat 340 12 113 PRT Human 12
Asn Leu Leu Arg Ala Ile Gln Ala Gln Gln Gln Leu Leu Arg Leu Ser 1 5
10 15 Val Trp Gly Ile Arg Gln Leu Arg Ala Arg Leu Leu Ala Leu Glu
Thr 20 25 30 Leu Ile Gln Asn Gln Gln Leu Leu Asn Leu Trp Gly Cys
Lys Gly Arg 35 40 45 Ile Val Cys Tyr Thr Ser Val Lys Trp Asn Asn
Thr Trp Arg Asn Val 50 55 60 Thr Asn Met Ser Glu Val Trp Asp Thr
Leu Thr Trp Gln Glu Trp Asp 65 70 75 80 Arg Gln Ile Asp Asn Ile Ser
Tyr Val Ile Tyr Asp Glu Ile Gln Arg 85 90 95 Ala Gln Val Gln Gln
Glu Gln Asn Glu Lys Lys Leu Leu Glu Leu Asp 100 105 110 Glu 13 331
DNA Human 13 aacctgctaa gagcaataca ggcccagcag cacttgctga ggctatctgt
atggggtatc 60 agacaactcc gagctcgcct gctagcctta gaaaccttaa
tacagaatca gcaactccta 120 aactcatggg gctgtaaggg aaagatagtc
tgttacacag cagtaaaatg gaacaagaca 180 tggacaggaa atgaaagtat
ttgggaccac ctcacatggc agcaatggga tcagcagata 240 gacaatgtaa
gctccaccat atatgaggaa atactaaaag cacaagtaca gcaggaacag 300
aatgagcaaa agttgctgga gttagatgaa t 331 14 110 PRT Human 14 Asn Leu
Leu Arg Ala Ile Gln Ala Gln Gln His Leu Leu Arg Leu Ser 1 5 10 15
Val Trp Gly Ile Arg Gln Leu Arg Ala Arg Leu Leu Ala Leu Glu Thr 20
25 30 Leu Ile Gln Asn Gln Gln Leu Leu Asn Ser Trp Gly Cys Lys Gly
Lys 35 40 45 Ile Val Cys Tyr Thr Ala Val Lys Trp Asn Lys Thr Trp
Thr Gly Asn 50 55 60 Glu Ser Ile Trp Asp His Leu Thr Trp Gln Gln
Trp Asp Gln Gln Ile 65 70 75 80 Asp Asn Val Ser Ser Thr Ile Tyr Glu
Glu Ile Leu Lys Ala Gln Val 85 90 95 Gln Gln Glu Gln Asn Glu Gln
Lys Leu Leu Glu Leu Asp Glu 100 105 110 15 331 DNA Human 15
aacctgctaa gagcaataca ggcccagcag cacttgctga ggctatctgt atggggtatc
60 agacaactcc gagctcgcct gctagcctta gaaaccttaa tacagaatca
gcaactccta 120 aactcatggg gctgtaaggg aaagatagtc tgttacacag
cagtaaaatg gaacaggaca 180 tggacaggaa atgaaagtat ttgggaccac
ctcacatggc agcaatggga tcagcagata 240 gacaatgtaa gctccaccat
atatgaggaa atactaaaag cacaagtaca gcaggaacag 300 aatgagmaaa
arttgctgga gttagatgaa t 331 16 110 PRT Human misc_feature
(1)..(110) Xaa = Unknown 16 Asn Leu Leu Arg Ala Ile Gln Ala Gln Gln
His Leu Leu Arg Leu Ser 1 5 10 15 Val Trp Gly Ile Arg Gln Leu Arg
Ala Arg Leu Leu Ala Leu Glu Thr 20 25 30 Leu Ile Gln Asn Gln Gln
Leu Leu Asn Ser Trp Gly Cys Lys Gly Lys 35 40 45 Ile Val Cys Tyr
Thr Ala Val Lys Trp Asn Arg Thr Trp Thr Gly Asn 50 55 60 Glu Ser
Ile Trp Asp His Leu Thr Trp Gln Gln Trp Asp Gln Gln Ile 65 70 75 80
Asp Asn Val Ser Ser Thr Ile Tyr Glu Glu Ile Leu Lys Ala Gln Val 85
90 95 Gln Gln Glu Gln Asn Glu Xaa Xaa Leu Leu Glu Leu Asp Glu 100
105 110 17 340 DNA Human 17 aacctgctaa aagcaataca ggcccagcag
gaattgctga ggctatctgt atggggtatc 60 agacaactcc gagctcgcct
gctagcctta gaaaccttaa tacaggatca gcagctccta 120 aacctatggg
gttgtaaggg aaggatagtc tgctacacat cagtaaaatg gaacgataca 180
tggagacatg tcactaatat gagtgaagtt tgggacaaat taacctggca ggaatgggat
240 cggcagatag acaacataag ctatgttata tatgatgaaa tacaaagagc
acaagtacag 300 caaggaccaa atgagaagaa gttgctggag ttagatgaat 340 18
113 PRT Human 18 Asn Leu Leu Lys Ala Ile Gln Ala Gln Gln Glu Leu
Leu Arg Leu Ser 1 5 10 15 Val Trp Gly Ile Arg Gln Leu Arg Ala Arg
Leu Leu Ala Leu Glu Thr 20 25 30 Leu Ile Gln Asp Gln Gln Leu Leu
Asn Leu Trp Gly Cys Lys Gly Arg 35 40 45 Ile Val Cys Tyr Thr Ser
Val Lys Trp Asn Asp Thr Trp Arg His Val 50 55 60 Thr Asn Met Ser
Glu Val Trp Asp Lys Leu Thr Trp Gln Glu Trp Asp 65 70 75 80 Arg Gln
Ile Asp Asn Ile Ser Tyr Val Ile Tyr Asp Glu Ile Gln Arg 85 90 95
Ala Gln Val Gln Gln Gly Pro Asn Glu Lys Lys Leu Leu Glu Leu Asp 100
105 110 Glu 19 348 DNA Human 19 aacctgctaa gagcaataca ggcccagcag
caattgctga ggctatctgt atggggtatc 60 agacaactcc gagctcgcct
gctagcctta gaaaccttaa tacagaacca gcaactccta 120 aacctatggg
gctgtaaggg aaggctagtc tgctacacat cagtaaaatg gaacaagaca 180
tggataaata aaactgacac tgagatagag aatatttggg aaaatctgac atggcaggaa
240 tgggatcagc aaataagcaa cataagctcc accatatatg aggaaataca
aaaggcacaa 300 atacaacagg aacataatga gaaaaagttg ctggagctag atgaatgg
348 20 116 PRT Human 20 Asn Leu Leu Arg Ala Ile Gln Ala Gln Gln Gln
Leu Leu Arg Leu Ser 1 5 10 15 Val Trp Gly Ile Arg Gln Leu Arg Ala
Arg Leu Leu Ala Leu Glu Thr 20 25 30 Leu Ile Gln Asn Gln Gln Leu
Leu Asn Leu Trp Gly Cys Lys Gly Arg 35 40 45 Leu Val Cys Tyr Thr
Ser Val Lys Trp Asn Lys Thr Trp Ile Asn Lys 50 55 60 Thr Asp Thr
Glu Ile Glu Asn Ile Trp Glu Asn Leu Thr Trp Gln Glu 65 70 75 80 Trp
Asp Gln Gln Ile Ser Asn Ile Ser Ser Thr Ile Tyr Glu Glu Ile 85 90
95 Gln Lys Ala Gln Ile Gln Gln Glu His Asn Glu Lys Lys Leu Leu Glu
100 105 110 Leu Asp Glu Trp 115 21 349 DNA Human 21 aacctgctaa
gagcaataca ggctcagcat caactgctga agctatctgt atggggtatc 60
agacaactcc gagctcgcct gctagcctta gaaaccttta tacagaatca gcaactccta
120 aacctatggg gctgtaaagg aaacctaatc tgctacacat cagtaaaatg
gaacgaaaca 180 tggaaaggag ataggacttt tactgacatg gaaaatattt
ggaacaacct aacatggcag 240 gaatgggatc agcagataag caacataagc
tccaccatat atgacgaaat acaaaaggca 300 caagtacagc aggaacaaaa
tgagaaaaag ttactagagt taagtgaat 349 22 116 PRT Human 22 Asn Leu Leu
Arg Ala Ile Gln Ala Gln His Gln Leu Leu Lys Leu Ser 1 5 10 15 Val
Trp Gly Ile Arg Gln Leu Arg Ala Arg Leu Leu Ala Leu Glu Thr 20 25
30 Phe Ile Gln Asn Gln Gln Leu Leu Asn Leu Trp Gly Cys Lys Gly Asn
35 40 45 Leu Ile Cys Tyr Thr Ser Val Lys Trp Asn Glu Thr Trp Lys
Gly Asp 50 55 60 Arg Thr Phe Thr Asp Met Glu Asn Ile Trp Asn Asn
Leu Thr Trp Gln 65 70 75 80 Glu Trp Asp Gln Gln Ile Ser Asn Ile Ser
Ser Thr Ile Tyr Asp Glu 85 90 95 Ile Gln Lys Ala Gln Val Gln Gln
Glu Gln Asn Glu Lys Lys Leu Leu 100 105 110 Glu Leu Ser Glu 115 23
346 DNA Human 23 aacctgctaa gagcaataca ggcccagcag caattgctga
ggctatctgt atggggtatc 60 agacaactcc gagctcgcct gctagcctta
gaaaccttaa tacagagtca gcaactccta 120 aacctgtggg gctgtaaggg
aaggctaatc tgctacacct cagtgcattg gaataagaca 180 tggacaaata
agacagataa ggatttggag gatatgtggg acaacctaac atggcagcaa 240
tgggatcagc agataagtaa cataagcgcc accatatatg aggaaataca aaaggcacaa
300 gtacaacaag aatacaatga gagaaagttg ttggagttag atgaat 346 24 115
PRT Human 24 Asn Leu Leu Arg Ala Ile Gln Ala Gln Gln Gln Leu Leu
Arg Leu Ser 1 5 10 15 Val Trp Gly Ile Arg Gln Leu Arg Ala Arg Leu
Leu Ala Leu Glu Thr 20 25 30 Leu Ile Gln Ser Gln Gln Leu Leu Asn
Leu Trp Gly Cys Lys Gly Arg 35 40 45 Leu Ile Cys Tyr Thr Ser Val
His Trp Asn Lys Thr Trp Thr Asn Lys 50 55 60 Thr Asp Lys Asp Leu
Glu Asp Met Trp Asp Asn Leu Thr Trp Gln Gln 65 70 75 80 Trp Asp Gln
Gln Ile Ser Asn Ile Ser Ala Thr Ile Tyr Glu Glu Ile 85 90 95 Gln
Lys Ala Gln Val Gln Gln Glu Tyr Asn Glu Arg Lys Leu Leu Glu 100 105
110 Leu Asp Glu 115 25 340 DNA Human misc_feature (1)..(340) N =
any nucleotide 25 aacctgctaa gagcaataca ggcccagcag caattgctga
ggctatctgt atggggtatc 60 anacaactcc gagctcgcct gctagcatya
gaaaccttaa tacagaatcw gcaactcctg 120 aacctatggg gctgtaaggg
aakgctagtc tgctacacat cagtamaatg gaacaggaca 180 tggacaaaca
atactaattt agattcaatt tgggaaaatc taacatggca ggaatgggat 240
cagcagataa gcaacataag ctccaccata tatgaagaaa tacaaaaggc acaartacag
300 caggaatacr atgagaaaaa gttgctagag ttagatkaat 340 26 113 PRT
Human Misc_feature (1)..(113) Xaa = unknown 26 Asn Leu Leu Arg Ala
Ile Gln Ala Gln Gln Gln Leu Leu Arg Leu Ser 1 5 10 15 Val Trp Gly
Ile Xaa Gln Leu Arg Ala Arg Leu Leu Ala Xaa Glu Thr 20 25 30 Leu
Ile Gln Asn Xaa Gln Leu Leu Asn Leu Trp Gly Cys Lys Gly Xaa 35 40
45 Leu Val Cys Tyr Thr Ser Val Xaa Trp Asn Arg Thr Trp Thr Asn Asn
50 55 60 Thr Asn Leu Asp Ser Ile Trp Glu Asn Leu Thr Trp Gln Glu
Trp Asp 65 70 75 80 Gln Gln Ile Ser Asn Ile Ser Ser Thr Ile Tyr Glu
Glu Ile Gln Lys 85 90 95 Ala Gln Xaa Gln Gln Glu Tyr Xaa Glu Lys
Lys Leu Leu Glu Leu Asp 100 105 110 Xaa 27 340 DNA Human 27
aacctgctaa gagcaataca ggcccagcag caattgctga ggctatctgt atggggtatc
60 agacaactcc gagctcgcct gttggcctta gaaaccttaa tacagaatca
gcaactccta 120 aacctatggg gatgtaaggg aaggctaatc tgctacacat
cagtacaatg gaacatgaca 180 tggacaaaca attctaatct ggaaacaatt
tgggacaacc taacatggca ggaatgggat 240 cagcagataa acagcataag
ctctgtcata tatgaggaaa tacaaagggc acaagtacag 300 caggaacaaa
acgagaaaaa gttgctggag ttagaggaat 340 28 113 PRT Human 28 Asn Leu
Leu Arg Ala Ile Gln Ala Gln Gln Gln Leu Leu Arg Leu Ser 1 5 10 15
Val Trp Gly Ile Arg Gln Leu Arg Ala Arg Leu Leu Ala Leu Glu Thr
20
25 30 Leu Ile Gln Asn Gln Gln Leu Leu Asn Leu Trp Gly Cys Lys Gly
Arg 35 40 45 Leu Ile Cys Tyr Thr Ser Val Gln Trp Asn Met Thr Trp
Thr Asn Asn 50 55 60 Ser Asn Leu Glu Thr Ile Trp Asp Asn Leu Thr
Trp Gln Glu Trp Asp 65 70 75 80 Gln Gln Ile Asn Ser Ile Ser Ser Val
Ile Tyr Glu Glu Ile Gln Arg 85 90 95 Ala Gln Val Gln Gln Glu Gln
Asn Glu Lys Lys Leu Leu Glu Leu Glu 100 105 110 Glu 29 331 DNA
Human 29 aacctgctga gagcaataca ggcccagcag caattgctga ggctatctgt
atggggtatc 60 agacaactcc gagctcgcct gctagcctta gaaaccttaa
tacagaatca gcaactccta 120 aacctatggg gctgtagagg aaggcaagtc
tgctacacat cagtaatatg gaatgagaca 180 tggataggaa acgaaaccat
ttgggaagaa ctaacatggc aggaatggga tcggcagata 240 agcaacataa
gctccaccat atatgatgaa atacaaaagg cacaagtaca gcaggaacaa 300
aatgagaaaa aattgctgga gttagatgaa t 331 30 110 PRT Human 30 Asn Leu
Leu Arg Ala Ile Gln Ala Gln Gln Gln Leu Leu Arg Leu Ser 1 5 10 15
Val Trp Gly Ile Arg Gln Leu Arg Ala Arg Leu Leu Ala Leu Glu Thr 20
25 30 Leu Ile Gln Asn Gln Gln Leu Leu Asn Leu Trp Gly Cys Arg Gly
Arg 35 40 45 Gln Val Cys Tyr Thr Ser Val Ile Trp Asn Glu Thr Trp
Ile Gly Asn 50 55 60 Glu Thr Ile Trp Glu Glu Leu Thr Trp Gln Glu
Trp Asp Arg Gln Ile 65 70 75 80 Ser Asn Ile Ser Ser Thr Ile Tyr Asp
Glu Ile Gln Lys Ala Gln Val 85 90 95 Gln Gln Glu Gln Asn Glu Lys
Lys Leu Leu Glu Leu Asp Glu 100 105 110 31 334 DNA Human
misc_feature (1)..(334) N = any nucleotide 31 aacctgctga gagcgataca
ggcccagcaa cacttgctga ggttatctgt atggggtatt 60 agacaactcc
gagctcgcct gcaagcctta gaaaccctta tacagaatca gcaacgccta 120
aacctatggg gctgtaaggg aaagatgatc tgttacacat cagtaaaatg gaacacatca
180 tggggagact ataatgacag tatttggggc aactanacat ggcaacaatg
ggaccaagaa 240 ataagcaatg taagctccat tatatatgac aaaatacaag
aagcacagga ccaacaggag 300 aggaatgtaa aagcattgtt ggagctggat gaat 334
32 111 PRT Human Misc_feature (1)..(111) Xaa = unknown 32 Asn Leu
Leu Arg Ala Ile Gln Ala Gln Gln His Leu Leu Arg Leu Ser 1 5 10 15
Val Trp Gly Ile Arg Gln Leu Arg Ala Arg Leu Gln Ala Leu Glu Thr 20
25 30 Leu Ile Gln Asn Gln Gln Arg Leu Asn Leu Trp Gly Cys Lys Gly
Lys 35 40 45 Met Ile Cys Tyr Thr Ser Val Lys Trp Asn Thr Ser Trp
Gly Asp Tyr 50 55 60 Asn Asp Ser Ile Trp Gly Asn Xaa Thr Trp Gln
Gln Trp Asp Gln Glu 65 70 75 80 Ile Ser Asn Val Ser Ser Ile Ile Tyr
Asp Lys Ile Gln Glu Ala Gln 85 90 95 Asp Gln Gln Glu Arg Asn Val
Lys Ala Leu Leu Glu Leu Asp Glu 100 105 110 33 334 DNA Human 33
aacctgctga gagcgataca ggcccagcaa cacttgctga ggttatctgt atggggtatc
60 agacaactcc gagctcgcct gcaagcctta gaaaccctta tacagaatca
gcaacgccta 120 aacctatggg gctgtaaggg aaagatgatc tgttacacat
cagtaccatg gaacacatca 180 tggggaaact ataatgacag tatttgggat
aagtatacat ggcaacaatg ggaccgagaa 240 atagacaatg taagctacat
tatatatgaa aaaatacaag aagcacaaga ccaacaggag 300 aagaatgtaa
aagcattgtt ggagctagat gaat 334 34 111 PRT Human 34 Asn Leu Leu Arg
Ala Ile Gln Ala Gln Gln His Leu Leu Arg Leu Ser 1 5 10 15 Val Trp
Gly Ile Arg Gln Leu Arg Ala Arg Leu Gln Ala Leu Glu Thr 20 25 30
Leu Ile Gln Asn Gln Gln Arg Leu Asn Leu Trp Gly Cys Lys Gly Lys 35
40 45 Met Ile Cys Tyr Thr Ser Val Pro Trp Asn Thr Ser Trp Gly Asn
Tyr 50 55 60 Asn Asp Ser Ile Trp Asp Lys Tyr Thr Trp Gln Gln Trp
Asp Arg Glu 65 70 75 80 Ile Asp Asn Val Ser Tyr Ile Ile Tyr Glu Lys
Ile Gln Glu Ala Gln 85 90 95 Asp Gln Gln Glu Lys Asn Val Lys Ala
Leu Leu Glu Leu Asp Glu 100 105 110 35 331 DNA Human 35 aacctgctga
gagcaataca ggcccagcaa catctgctga ggttatctgt atggggtatt 60
agacaactcc gagctcgcct gcaagcctta gaaaccctta tgcaaaatca gcaactccta
120 aacctatggg gctgtaaagg aaaatcaatc tgctacacat cagtaaaatg
gaacaacaca 180 tggggaggaa atctctcaat ttgggacagc ttaacatggc
agcaatggga tcaacaggta 240 gccaatgtaa gctctttgat atatgacaaa
atacaagaag cacaagaaca acaggaggaa 300 aatgaaaggg ccttgctgga
gttagatgaa t 331 36 110 PRT Human 36 Asn Leu Leu Arg Ala Ile Gln
Ala Gln Gln His Leu Leu Arg Leu Ser 1 5 10 15 Val Trp Gly Ile Arg
Gln Leu Arg Ala Arg Leu Gln Ala Leu Glu Thr 20 25 30 Leu Met Gln
Asn Gln Gln Leu Leu Asn Leu Trp Gly Cys Lys Gly Lys 35 40 45 Ser
Ile Cys Tyr Thr Ser Val Lys Trp Asn Asn Thr Trp Gly Gly Asn 50 55
60 Leu Ser Ile Trp Asp Ser Leu Thr Trp Gln Gln Trp Asp Gln Gln Val
65 70 75 80 Ala Asn Val Ser Ser Leu Ile Tyr Asp Lys Ile Gln Glu Ala
Gln Glu 85 90 95 Gln Gln Glu Glu Asn Glu Arg Ala Leu Leu Glu Leu
Asp Glu 100 105 110 37 331 DNA Human 37 aacctgttga gagcgataca
ggcccagcaa cacctgctga ggttatctgt atggggtata 60 agacaactcc
gagctcgcct gcaagcctta gaaaccttta tacagaacca gcaactccta 120
agcctatggg gatgtaaggg aaagctaatc tgttacacat ctgtaaaatg gaacacatca
180 tggggaggaa atgagagtat ttggaacaat ctaacatggc agcagtggga
tcaacagata 240 gacaacataa gttccatcat atatgatgaa atacaaaagg
cacaagagca acaggaacaa 300 aatgagaaaa gcttgctgga gttagatgaa t 331 38
110 PRT Human 38 Asn Leu Leu Arg Ala Ile Gln Ala Gln Gln His Leu
Leu Arg Leu Ser 1 5 10 15 Val Trp Gly Ile Arg Gln Leu Arg Ala Arg
Leu Gln Ala Leu Glu Thr 20 25 30 Phe Ile Gln Asn Gln Gln Leu Leu
Ser Leu Trp Gly Cys Lys Gly Lys 35 40 45 Leu Ile Cys Tyr Thr Ser
Val Lys Trp Asn Thr Ser Trp Gly Gly Asn 50 55 60 Glu Ser Ile Trp
Asn Asn Leu Thr Trp Gln Gln Trp Asp Gln Gln Ile 65 70 75 80 Asp Asn
Ile Ser Ser Ile Ile Tyr Asp Glu Ile Gln Lys Ala Gln Glu 85 90 95
Gln Gln Glu Gln Asn Glu Lys Ser Leu Leu Glu Leu Asp Glu 100 105 110
39 340 DNA Human 39 aacctgctaa gagcaataca ggcccagcaa gagctgctga
ggctatctgt atggggtatc 60 agacaactcc gagctcgcct gctagcctta
gaaaccttta tacggaatca gcaactccta 120 aacctctggg gctgtaaggg
aaggctaatt tgctatacat cagtacaatg gaacaaaaca 180 tggggtaatt
tgamwgataa tgagtcaatt tgggatgaca tracatggca ggagtgggat 240
aagcgggtag akaatgtaag ygccaccata tttgaagaaa tacgaagggc acaagaacaa
300 caggaacaaa atgagaaggc tttgctagaa ttagatgaat 340 40 113 PRT
Human Misc_feature (1)..(113) Xaa = unknown 40 Asn Leu Leu Arg Ala
Ile Gln Ala Gln Gln Glu Leu Leu Arg Leu Ser 1 5 10 15 Val Trp Gly
Ile Arg Gln Leu Arg Ala Arg Leu Leu Ala Leu Glu Thr 20 25 30 Phe
Ile Arg Asn Gln Gln Leu Leu Asn Leu Trp Gly Cys Lys Gly Arg 35 40
45 Leu Ile Cys Tyr Thr Ser Val Gln Trp Asn Lys Thr Trp Gly Asn Leu
50 55 60 Xaa Asp Asn Glu Ser Ile Trp Asp Asp Xaa Thr Trp Gln Glu
Trp Asp 65 70 75 80 Lys Arg Val Xaa Asn Val Xaa Ala Thr Ile Phe Glu
Glu Ile Arg Arg 85 90 95 Ala Gln Glu Gln Gln Glu Gln Asn Glu Lys
Ala Leu Leu Glu Leu Asp 100 105 110 Glu 41 315 DNA Human 41
taagattatg ggaaaaaata tctcggacag tgcagaaaat atcatagtga ccctaaattc
60 tactgtaaac ataacctgtg agagaccagg gaatcagtca gtacaagaga
taaaaatagg 120 tccaatggcc tggtacagca ttggcatagg gacaacaccc
gcaaactggt caaggatagc 180 ttattgccag tataatatca ctgattggga
aaaagcctta aaacaaacag ctgaaaggta 240 cttagaactt gtaaaccata
caagaaatga tactgttagc ataacattca atagcagcac 300 tggtggagat ctaga
315 42 103 PRT Human 42 Ile Met Gly Lys Asn Ile Ser Asp Ser Ala Glu
Asn Ile Ile Val Thr 1 5 10 15 Leu Asn Ser Thr Val Asn Ile Thr Cys
Glu Arg Pro Gly Asn Gln Ser 20 25 30 Val Gln Glu Ile Lys Ile Gly
Pro Met Ala Trp Tyr Ser Ile Gly Ile 35 40 45 Gly Thr Thr Pro Ala
Asn Trp Ser Arg Ile Ala Tyr Cys Gln Tyr Asn 50 55 60 Ile Thr Asp
Trp Glu Lys Ala Leu Lys Gln Thr Ala Glu Arg Tyr Leu 65 70 75 80 Glu
Leu Val Asn His Thr Arg Asn Asp Thr Val Ser Ile Thr Phe Asn 85 90
95 Ser Ser Thr Gly Gly Asp Leu 100 43 327 DNA Human 43 attataggaa
aaaacatttc ggacagtggg aaaaatatca tagtgaccct aaatcctact 60
gtaaacctga cttgtgagag accaggaaat aattcaatac aacagatgaa aataggtcca
120 ctggcctggt acagcatggg cctagagaga aacaaaagct caatctctag
attagcttat 180 tgcaggtata ataccactac gtgggaacaa gccttacaac
aaacagctga aaggtatcta 240 gaacttgtga acaacacgga caatattaca
ataatgttca atcgcagcac tgatggagat 300 tcagaggtaa cccatatgca ttttaac
327 44 109 PRT Human 44 Ile Ile Gly Lys Asn Ile Ser Asp Ser Gly Lys
Asn Ile Ile Val Thr 1 5 10 15 Leu Asn Pro Thr Val Asn Leu Thr Cys
Glu Arg Pro Gly Asn Asn Ser 20 25 30 Ile Gln Gln Met Lys Ile Gly
Pro Leu Ala Trp Tyr Ser Met Gly Leu 35 40 45 Glu Arg Asn Lys Ser
Ser Ile Ser Arg Leu Ala Tyr Cys Arg Tyr Asn 50 55 60 Thr Thr Thr
Trp Glu Gln Ala Leu Gln Gln Thr Ala Glu Arg Tyr Leu 65 70 75 80 Glu
Leu Val Asn Asn Thr Asp Asn Ile Thr Ile Met Phe Asn Arg Ser 85 90
95 Thr Asp Gly Asp Ser Glu Val Thr His Met His Phe Asn 100 105 45
356 DNA Human 45 ataagaatta tgggaaaaaa tatctcgaac agtgcagtaa
atatcatagt gaccctgaat 60 tctactgtaa acataacctg tgtgagacca
tggaatcaga cagtacaaga gatacaaaca 120 ggtccaatgg cctggtatag
cattcacttg aggacaccac tcgcaaactt gtcaaggata 180 gcttattgca
agtataatgc cgctgattgg gaaaaagcct taaaacaaac agctgaaagg 240
tacttagaac ttgtaaataa tacaagtaat aataatgtta ccataatatt caataacagc
300 actggtggag atccagagac aacccagtta cattttaact gtcatggagt tcttta
356 46 116 PRT Human 46 Ile Met Gly Lys Asn Ile Ser Asn Ser Ala Val
Asn Ile Ile Val Thr 1 5 10 15 Leu Asn Ser Thr Val Asn Ile Thr Cys
Val Arg Pro Trp Asn Gln Thr 20 25 30 Val Gln Glu Ile Gln Thr Gly
Pro Met Ala Trp Tyr Ser Ile His Leu 35 40 45 Arg Thr Pro Leu Ala
Asn Leu Ser Arg Ile Ala Tyr Cys Lys Tyr Asn 50 55 60 Ala Ala Asp
Trp Glu Lys Ala Leu Lys Gln Thr Ala Glu Arg Tyr Leu 65 70 75 80 Glu
Leu Val Asn Asn Thr Ser Asn Asn Asn Val Thr Ile Ile Phe Asn 85 90
95 Asn Ser Thr Gly Gly Asp Pro Glu Thr Thr Gln Leu His Phe Asn Cys
100 105 110 His Gly Val Leu 115 47 317 DNA Human 47 ataagactga
tggcaaaaaa tatttcggct actggccaaa atatcatagt gaccctaaat 60
actactataa acatgacctg ccagagacca ggaaatctaa caatacagga aataaagata
120 ggtccaatgt cctggtacag catgggcata gggcaggaag accactctaa
gtcaagaaac 180 gcttattgtg agtataatat cactgattgg gtacaggcct
taaaacagac agctgaaagg 240 tatttagaat tagtaaacaa tacaaatact
aatataaaca tgacattcga gaacagtact 300 ggaggagatc cagaggt 317 48 103
PRT Human 48 Leu Met Ala Lys Asn Ile Ser Ala Thr Gly Gln Asn Ile
Ile Val Thr 1 5 10 15 Leu Asn Thr Thr Ile Asn Met Thr Cys Gln Arg
Pro Gly Asn Leu Thr 20 25 30 Ile Gln Glu Ile Lys Ile Gly Pro Met
Ser Trp Tyr Ser Met Gly Ile 35 40 45 Gly Gln Glu Asp His Ser Lys
Ser Arg Asn Ala Tyr Cys Glu Tyr Asn 50 55 60 Ile Thr Asp Trp Val
Gln Ala Leu Lys Gln Thr Ala Glu Arg Tyr Leu 65 70 75 80 Glu Leu Val
Asn Asn Thr Asn Thr Asn Ile Asn Met Thr Phe Glu Asn 85 90 95 Ser
Thr Gly Gly Asp Pro Glu 100 49 319 DNA Human 49 taagaataat
gggaaaaaat atttcagaca atgggaaaaa tatcatagta accctaaatt 60
ctactctaaa aatgacctgt gagagaccag ggaatcatac agtacaacag atgaagatag
120 gtccaatgtc ctggtatagc atgggcttag agaaaaacaa taccagctca
agaagagctt 180 tttgcaagta taatgccact aattgggaaa aaaccttaaa
acaaatggct gaaaggtatt 240 tagaactcgt aaacaataca agtaataaca
cagtgacaat gatattcaat acaagcagtg 300 atggagatcc agaggtacc 319 50
104 PRT Human 50 Ile Met Gly Lys Asn Ile Ser Asp Asn Gly Lys Asn
Ile Ile Val Thr 1 5 10 15 Leu Asn Ser Thr Leu Lys Met Thr Cys Glu
Arg Pro Gly Asn His Thr 20 25 30 Val Gln Gln Met Lys Ile Gly Pro
Met Ser Trp Tyr Ser Met Gly Leu 35 40 45 Glu Lys Asn Asn Thr Ser
Ser Arg Arg Ala Phe Cys Lys Tyr Asn Ala 50 55 60 Thr Asn Trp Glu
Lys Thr Leu Lys Gln Met Ala Glu Arg Tyr Leu Glu 65 70 75 80 Leu Val
Asn Asn Thr Ser Asn Asn Thr Val Thr Met Ile Phe Asn Thr 85 90 95
Ser Ser Asp Gly Asp Pro Glu Val 100 51 331 DNA Human 51 aagaaggatg
ggggaaaaca atccttcaga tcggaagaag atcctagtga ccctaaattc 60
ccctataaac ataacctgcg agagaccata ctatcagtca gtacaagagt taaggatagg
120 tccaatggct tggtacagca tgacattaga acgagacagg gcaggcagtg
acataagggc 180 agcttattgc aagtataatg cctctgactg gagaaataca
ttaaaaggag tagctgagag 240 atatttagaa cttagaaatg aggaaggccc
ggtgaacgtg accttcaatg gaagtgcggg 300 tggagatcca gagatacgct
ttctgcattt t 331 52 109 PRT Human 52 Arg Met Gly Glu Asn Asn Pro
Ser Asp Arg Lys Lys Ile Leu Val Thr 1 5 10 15 Leu Asn Ser Pro Ile
Asn Ile Thr Cys Glu Arg Pro Tyr Tyr Gln Ser 20 25 30 Val Gln Glu
Leu Arg Ile Gly Pro Met Ala Trp Tyr Ser Met Thr Leu 35 40 45 Glu
Arg Asp Arg Ala Gly Ser Asp Ile Arg Ala Ala Tyr Cys Lys Tyr 50 55
60 Asn Ala Ser Asp Trp Arg Asn Thr Leu Lys Gly Val Ala Glu Arg Tyr
65 70 75 80 Leu Glu Leu Arg Asn Glu Glu Gly Pro Val Asn Val Thr Phe
Asn Gly 85 90 95 Ser Ala Gly Gly Asp Pro Glu Ile Arg Phe Leu His
Phe 100 105 53 6 PRT Human PEPTIDE (1)..(6) 53 Val Gln Gln Met Lys
Ile 1 5 54 11 PRT Human PEPTIDE (1)..(11) 54 Lys Ile Gly Pro Met
Ser Trp Tyr Ser Met Gly 1 5 10 55 6 PRT Human PEPTIDE (1)..(6) 55
Met Gly Leu Glu Lys Asn 1 5 56 6 PRT Human PEPTIDE (1)..(6) 56 Ile
Gln Gln Met Lys Ile 1 5 57 11 PRT Human PEPTIDE (1)..(11) 57 Lys
Ile Gly Pro Leu Ala Trp Tyr Ser Met Gly 1 5 10 58 6 PRT Human
PEPTIDE (1)..(6) 58 Met Gly Leu Glu Arg Asn 1 5 59 8 PRT Human
PEPTIDE (1)..(8) 59 Gln Ser Val Gln Glu Ile Lys Ile 1 5 60 11 PRT
Human PEPTIDE (1)..(11) 60 Lys Ile Gly Pro Met Ala Trp Tyr Ser Ile
Gly 1 5 10 61 6 PRT Human PEPTIDE (1)..(6) 61 Ile Gly Ile Gly Thr
Thr 1 5 62 6 PRT Human PEPTIDE (1)..(6) 62 Val Gln Glu Ile Gln Thr
1 5 63 11 PRT Human PEPTIDE (1)..(11) 63 Gln Thr Gly Pro Met Ala
Trp Tyr Ser Ile His 1 5 10 64 6 PRT Human PEPTIDE (1)..(6) 64 Ile
His Leu Arg Thr Pro 1 5 65 6 PRT Human PEPTIDE (1)..(6) 65 Ile Gln
Glu Ile Lys Ile 1 5 66 11 PRT Human PEPTIDE (1)..(11) 66 Lys Ile
Gly Pro Met Ser Trp Tyr Ser Met Gly 1 5 10 67 6 PRT Human PEPTIDE
(1)..(6) 67 Met Gly Ile Gly Gln Glu 1 5 68 7 PRT Human PEPTIDE
(1)..(7) 68 Ser Val Gln Glu Leu Arg Ile 1 5 69 11 PRT Human PEPTIDE
(1)..(11) 69 Arg Ile Gly Pro Met Ala Trp Tyr Ser Met Thr 1 5 10 70
6 PRT Human PEPTIDE (1)..(6) 70 Met Thr Leu Glu Arg Asp 1 5 71 17
PRT Human PEPTIDE (1)..(17) 71 Arg Asn Gln Gln Leu Leu Asn Leu Trp
Gly Cys Lys Gly Arg Leu Ile 1 5 10 15 Cys 72 15 PRT Human PEPTIDE
(1)..(15) 72 Cys Lys Gly Arg Leu Ile Cys Tyr Thr Ser Val Gln Trp
Asn Met 1 5
10 15 73 10 PRT Human PEPTIDE (1)..(10) 73 Leu Trp Gly Cys Lys Gly
Arg Ile Val Cys 1 5 10 74 11 PRT Human PEPTIDE (1)..(11) 74 Ser Leu
Trp Gly Cys Lys Gly Lys Leu Ile Cys 1 5 10 75 7 PRT Human PEPTIDE
(1)..(7) 75 Cys Lys Gly Lys Ser Ile Cys 1 5 76 7 PRT Human PEPTIDE
(1)..(7) 76 Cys Lys Gly Lys Ile Val Cys 1 5 77 7 PRT Human PEPTIDE
(1)..(7) 77 Cys Arg Gly Arg Gln Val Cys 1 5 78 1 DNA Human 78 g 1
79 12 PRT Human PEPTIDE (1)..(12) 79 Cys Lys Gly Arg Leu Ile Cys
Tyr Thr Ser Val His 1 5 10 80 7 PRT Human PEPTIDE (1)..(7) 80 Cys
Lys Gly Asn Leu Ile Cys 1 5 81 7 PRT Human PEPTIDE (1)..(7) 81 Cys
Lys Gly Lys Met Ile Cys 1 5 82 7 PRT Human PEPTIDE (1)..(7) 82 Cys
Lys Gly Arg Val Val Cys 1 5 83 37 PRT Human PEPTIDE (1)..(37) 83
Cys Glu Arg Pro Gly Asn Asn Ser Ile Gln Gln Met Lys Ile Gly Pro 1 5
10 15 Leu Ala Trp Tyr Ser Met Gly Leu Glu Arg Asn Lys Ser Ser Ile
Ser 20 25 30 Arg Leu Ala Tyr Cys 35 84 37 PRT Human PEPTIDE
(1)..(37) 84 Cys Glu Arg Pro Gly Asn Asn Ser Ile Gln Gln Met Lys
Ile Gly Pro 1 5 10 15 Met Ala Trp Tyr Ser Met Gly Leu Glu Arg Asn
Lys Ser Ser Ile Ser 20 25 30 Arg Leu Ala Tyr Cys 35 85 37 PRT Human
PEPTIDE (1)..(37) 85 Cys Glu Arg Pro Gly Asn Gln Ser Val Gln Glu
Ile Lys Ile Gly Pro 1 5 10 15 Met Ala Trp Tyr Ser Ile Gly Ile Gly
Thr Thr Pro Ala Asn Trp Ser 20 25 30 Arg Ile Ala Tyr Cys 35 86 38
PRT Human PEPTIDE (1)..(38) 86 Cys Glu Arg Pro Gly Asn Gln Ser Val
Gln Glu Ile Lys Ile Gly Pro 1 5 10 15 Met Ala Trp Tyr Ser Ile Gly
Ile Gly Thr Thr Pro Thr Tyr Asn Trp 20 25 30 Ser Arg Ile Ala Tyr
Cys 35 87 37 PRT Human PEPTIDE (1)..(37) 87 Cys Val Arg Pro Trp Asn
Gln Thr Val Gln Glu Ile Gln Thr Gly Pro 1 5 10 15 Met Ala Trp Tyr
Ser Ile His Leu Arg Thr Pro Leu Ala Asn Leu Ser 20 25 30 Arg Ile
Ala Tyr Cys 35 88 37 PRT Human PEPTIDE (1)..(37) 88 Cys Gln Arg Pro
Gly Asn Leu Thr Ile Gln Glu Ile Lys Ile Gly Pro 1 5 10 15 Met Ser
Trp Tyr Ser Met Gly Ile Gly Gln Glu Asp His Ser Lys Ser 20 25 30
Arg Asn Ala Tyr Cys 35 89 38 PRT Human PEPTIDE (1)..(38) 89 Cys Glu
Arg Pro Tyr Tyr Gln Ser Val Gln Glu Leu Arg Ile Gly Pro 1 5 10 15
Met Ala Trp Tyr Ser Met Thr Leu Glu Arg Asp Arg Ala Gly Ser Asp 20
25 30 Ile Arg Ala Ala Tyr Cys 35 90 36 PRT Human PEPTIDE (1)..(36)
90 Cys Glu Arg Pro Gly Asn His Thr Val Gln Gln Met Lys Ile Gly Pro
1 5 10 15 Met Ser Trp Tyr Ser Met Gly Leu Glu Lys Asn Asn Thr Ser
Ser Arg 20 25 30 Arg Ala Phe Cys 35 91 23 PRT Human PEPTIDE
(1)..(23) 91 Asp Gln Gln Leu Leu Asn Leu Trp Gly Cys Lys Gly Arg
Ile Val Cys 1 5 10 15 Tyr Thr Ser Val Lys Trp Asn 20 92 24 PRT
Human PEPTIDE (1)..(24) 92 Asn Gln Gln Leu Leu Asn Leu Trp Gly Cys
Lys Gly Arg Leu Val Cys 1 5 10 15 Tyr Thr Ser Val Lys Trp Asn Lys
20 93 23 PRT Human PEPTIDE (1)..(23) 93 Asn Gln Gln Arg Leu Asn Leu
Trp Gly Cys Lys Gly Lys Met Ile Cys 1 5 10 15 Tyr Thr Ser Val Pro
Trp Asn 20 94 23 PRT Human PEPTIDE (1)..(23) 94 Asn Gln Gln Leu Leu
Asn Leu Trp Gly Cys Lys Gly Lys Ser Ile Cys 1 5 10 15 Tyr Thr Ser
Val Lys Trp Asn 20 95 23 PRT Human PEPTIDE (1)..(23) 95 Asn Gln Gln
Leu Leu Asn Leu Trp Gly Cys Lys Gly Arg Leu Ile Cys 1 5 10 15 Tyr
Thr Ser Val Gln Trp Asn 20 96 23 PRT Human PEPTIDE (1)..(23) 96 Asn
Gln Gln Arg Leu Asn Leu Trp Gly Cys Lys Gly Lys Met Ile Cys 1 5 10
15 Tyr Thr Ser Val Lys Trp Asn 20 97 23 PRT Human PEPTIDE (1)..(23)
97 Asn Gln Gln Leu Leu Asn Leu Trp Gly Cys Lys Gly Asn Leu Ile Cys
1 5 10 15 Tyr Thr Ser Val Lys Trp Asn 20 98 23 PRT Human PEPTIDE
(1)..(23) 98 Asn Gln Gln Leu Leu Asn Leu Trp Gly Cys Arg Gly Arg
Gln Val Cys 1 5 10 15 Tyr Thr Ser Val Ile Trp Asn 20 99 23 PRT
Human PEPTIDE (1)..(23) 99 Ser Gln Gln Leu Leu Asn Leu Trp Gly Cys
Lys Gly Arg Leu Ile Cys 1 5 10 15 Tyr Thr Ser Val His Trp Asn 20
100 23 PRT Human PEPTIDE (1)..(23) 100 Asn Gln Gln Leu Leu Asn Leu
Trp Gly Cys Lys Gly Arg Ile Val Cys 1 5 10 15 Tyr Thr Ser Val Lys
Trp Asn 20 101 23 PRT Human PEPTIDE (1)..(23) 101 Asn Gln Gln Leu
Leu Asn Ser Trp Gly Cys Lys Gly Lys Ile Val Cys 1 5 10 15 Tyr Thr
Ala Val Lys Trp Asn 20 102 23 PRT Human PEPTIDE (1)..(23) 102 Asn
Gln Gln Leu Leu Ser Leu Trp Gly Cys Lys Gly Lys Leu Ile Cys 1 5 10
15 Tyr Thr Ser Val Lys Trp Asn 20 103 1339 DNA Human 103 attaaagtag
taccaagaag aaaggcaaaa ataatcagac attatggaaa acagatggca 60
ggtgctgata gtatggcaag tggacagaca gaaagtgaaa gcgtggaaca gcctggtgaa
120 ataccataag tacaggtcta ggaaggccaa ggactggtgt tacagacacc
attttgaatc 180 tagaaatcca agagtcagtt caagtgtaca tattccagta
gggatggctt gggtaatagt 240 gaccacatat tggggattga tgccagggga
gagagaggaa cagttgggac atggggttag 300 tatagaatgg cagtacaaaa
agtatacaac acagattgac cctgaaacag cagacaggat 360 gatacatctg
tattatttta cctgttttac agattcagca gtcaggaaag ccatcttagg 420
gcagagaata ctgaccaagt gtgaataccc tgcaggacat agtcaggtag ggacattgca
480 actactagct ctaagagtag tagtaaaagc aaaaagaaat aagcctcccc
tacccagtgt 540 ccagaaatta acagaagata gatggagcga gcacctgagg
atcaggggcc agctagagag 600 cctttcaatg aatgggcact agagatccta
gaagagctaa aagcagaggc agtaagacat 660 ttccctaggc agtggctaca
ggccttggga cagtacattt atgagactta tggggacact 720 tgggtaggag
ttatggcaat tacaagaatc ttacaacaaa tactatttgc ccattttaga 780
attggatgtc aacatagtag aataggaatt aacccaacta atacaagagg aagaggaaga
840 agaaatggat ccagtagatc ctgagatgcc cccttggcat caccctggga
gtcagcccca 900 gatcccttgt aacaattgct attgcaaaag atgctgctat
cattgccttg tttgtttcac 960 aagaaagggt ttggggatct cctatggcag
gaagaagcgg cgacaacgaa gagctgctgc 1020 gagccatccg gataataaag
atcttgtacc agagcagtaa gtaacgctaa tgcatcatag 1080 ggacctgcta
gtattaataa ttattagtgc tttgctgctt ataaatgtaa ttatatggat 1140
gtttattctt agacaatatt tagaacagaa gaaacaggac agaagggaaa gagacatact
1200 tgaaaggtta agaagaatag cagaaattaa agatgatagt gactatgaaa
gcaatgaaga 1260 ggaggaacag gaagttagag atcttataca tagtcatggc
tttgataatc ccatgtttga 1320 gctctgatca gaagtatgc 1339 104 1282 DNA
Human 104 attaaagtag taccaagaag aaaggcaaaa ataatcagag attatggaaa
acaaatggca 60 ggtactgata gtatggcaag tagacagaca gaaagtgaaa
acgtggaaca gcttggtgaa 120 ataccataag tacaggtcta ggaaggccaa
ggactggtac tacagacatc attatgaatc 180 tagaaatcca agaatcagtt
caggtgtata tattccagta gggccggctt gtatagtagt 240 gaacacatat
tggggattga tgccaggaga aagagatgaa catctgggac atggggttag 300
tatagaatgg cagtacaaga agtatacaac acagattgac cctgaaacag cagacaggat
360 gatacatcta tactatttta cctgttttac agaatcagca atcaggaaag
ccatcctagg 420 gcagagagta ctgaccaagt gtgaataccc tgcaggacat
agccaggtag ggacactaca 480 actactagct ctaagagttg tagtaaaaga
gagaaaacat aggcctcccc tacccagtgt 540 ccagaaatta acagaagata
gatggaacaa gcacctgagg atcagggacc agctagagag 600 ccattcaatg
aatggacact agagctccta gaagagctaa aagcagaagc agtaagacat 660
tttcctaggc cttggctaca ggccttggga caatacattt atgatactta tggggacact
720 tgggtaggag ttatggcaat tataagactc ttacaattaa tgatatttgc
ccattttaga 780 agaaatggat ccagtagacc ctgagatgcc cccttggcat
caccctggaa gtcagcccca 840 gaatccttgt aataaatgct attgcaaaaa
atgctgctat cattgctatg tttgtttcac 900 aagcaagggt ttgggaatct
cctatggcag gaagaagcga cgacgaccag cagctgctgc 960 aagccgcccg
gataataaag atcttgtacc agagcagtaa gtaacgctaa tgcagcaaaa 1020
ggacctgcta ttattagtaa ttattagtgc tttgctgctt ataaatataa ttctatggat
1080 gtttaatctt agaaaatatt tagaacaaaa gaaacaagac agaagggaaa
gagaaatact 1140 tgaaaggata agaagaataa gagaaattag agatgatagt
gactatgaaa gcaatgaaga 1200 ggaagaacaa gaagttaggg gtcatcttgt
gcatatgttt ggctttgcta atcccgtgtt 1260 tgagatctaa tgacctatat gc 1282
105 1339 DNA Human 105 atcaaggtag taccaagaag aaaagcaaaa atactcaggg
attatggaaa acagatggca 60 ggtgctgata gtatggcaag tggacagaca
gaaagtgaaa gcatggaata gcctggtaaa 120 ataccataag tacaggtcta
gaaagaccca gaactgggat tatagacatc attatgaaat 180 cagaaatcca
agaatcagct caggtgtata tattccagta ggtgaagcta agatagtagt 240
gactacatat tggggattaa tgccagggga aagagatgag catttgggac atggagtcag
300 tatagaatgg caatacaaaa attatagtac acagattgac cctgaaacag
cagataaaat 360 aatacatctg cattatttca cctgttttac agagtcagca
atcaggagag ccattttagg 420 gcagagagtg ctgaccaggt gtgaataccc
tgcaggacat agtcaggtag ggacactgca 480 actcctagca ttaagagcag
tagtaaaaga caaaagaagt aaacctcccc tacccagtgt 540 ccagaagtta
acaggagaca gatggaacag gcacctgaga atcagggacc agcaagagag 600
ccattcaatg aatgggcatt agagaccctg gaagaaataa aagcagaagc agtaagacac
660 tttcctaggc cttggctaca aagcttagga caatacatct atgagactta
tggagacacc 720 tgggaaggag ttatggcaat cataagaatc ttacaacagt
tgatatttgc ccattttaga 780 attggatgcc aacatagtag aataggaatt
accccatcta acgcaagagg aagaggaaga 840 agaaatggat ccagtagatc
ctgaggtgcc cccctggcat caccctggaa gtcagccccc 900 aaccccttgc
aacgcttgct attgcaaaag atgctgttat cattgctatc tttgtttcac 960
aaagaagggt ttgggaatct cccatggcag gaagaagcga cgacgaccag cagctgctgc
1020 aagctcttcg aataataaag atcttgtacc agagcagtaa gtaaagctaa
tgcatcataa 1080 ggacttgcta atcttaatag ttgctagtat tttgcttttt
acaaatatag tgatatggac 1140 atttattctt aagaaatatt tagagcagaa
ggaacaagat agaagggaaa gagaactact 1200 gaaaagaata aaaagaataa
gagaagtcag ggatgatagt gattatgaaa gcaatggaga 1260 tggaggacaa
gaagttatac atcttgtgca tactcatggt tttgttaacc ccatgtttga 1320
gctctgacaa gctatatcg 1339 106 3600 DNA Human 106 ttcacaattt
taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaataatag 60
acataatagc atcagatata caaactaaag aactacaaaa acaaattaca aaaattcaaa
120 attttcgggt ttattacagg gacagcagag atccaatttg gaaaggacca
gcaaaactac 180 tctggaaagg tgaaggggca gtagtaatac aggacaatag
tgatataaag gtagtaccaa 240 gaagaaaagc aaaaatcatt aaggattatg
gaaaacagat ggcaggtgat gattgtgtgg 300 caagtagaca gaatgaggat
tagaacatgg aacagtctag taaagcatca tatgtatatt 360 tctaagaaag
ctacagattg ggtttataaa catcactatg atagtagaca tccaaaagta 420
agctcagaag tacacattcc actaggggat gctaaattgg taataagaac atattggggt
480 ctacatacag gagaaagaga ctggcatttg ggtcatgggg tctccataga
atggaaacag 540 agaagatata gcacacaaat agatcctgac ctagcagacc
aactgattca cctgcattat 600 tttaactgtt tttcagaatc tgccataaga
aaagccatac taggacaagt agttagacct 660 aggtgtgatt atccagcagg
acatagtaag gtaggatctc tacaatattt ggcactgaaa 720 gcattagtaa
caccaacaag gacaaagcca cctttgccta gtgttaagaa attaacagaa 780
gacagatgga acaagcccca gaagaccagg gggcacagag ggagcggtcc aatgtatgga
840 cattagatct attagaggag cttaaacatg aagctgttag acattttcct
aggccttggc 900 tccagggatt aggacaatat atctatgaaa catatgggga
tacctgggaa ggagttgaag 960 ctataataag aattttgcaa caactactgt
ttgcccattt tagaattgga tgccaacata 1020 gtaggatagg aattaaccca
tctaacccaa gaggaaaagg aagaagaaat ggatccagta 1080 gatcctgaga
tacccccttg gcatcaccct ggaagtcagc cccagacccc ttgtaataac 1140
tgctcttgca aaaaatgctg ctaccattgc tatgtgtgtt tcacaagaaa gggtttggaa
1200 atctcctatg gcaggaagaa gcgacgaaga tcagccgctg aaacgcgtca
tccagataat 1260 caagatattg taccagagca gtaagtaacg ctaatgcagc
ttagggacca gctaacatta 1320 ataattatta gtgctttgtt gcttgtaaat
gtagttctat ggacatttat tcttagacaa 1380 tatttaaagc aaaagaaaca
agatagaagg ggaagagaaa tacttgaaag gttaagaaga 1440 ataagacaaa
ttgaagatga cagtgactat gaaagcgatg gaaaagagga acaggaagtt 1500
agggatcttg tgcatagtta tggctttgat aaccccatgt ttgagccatg accaacgcta
1560 tgcaacagtg tatgctgggg tacctgtatg ggaagaggca aacccagtat
tattttgtgc 1620 ttcagatgct aacctaacaa gcactgagaa gcataatatc
tgggcatcac aagcctgtgt 1680 tcccacagac cccactccac atgaatatcc
tttacacaat gtgacagata actttaatat 1740 atggaaaaat tacatggtag
aacaaatgca ggatgacatt attagcttat gggaacagag 1800 tttaaaacct
tgtgttcaaa tgactttcct gtgtgtacaa atgaattgta caagtgtaag 1860
taatagtagt gtaagtaata gtagtgtaag taatagtagt gtaagtaata gtagtgtaag
1920 tgatagtact atacccaaga agaaaaataa cagcagctca gaggaccttc
tgaaacagtg 1980 tgattttaat gcaaccacag ttctcaaaga caaaaaggag
aaaaaacaga ctctatttta 2040 tgtatcagat ttgatgaaac tgacaaatgt
cacaaatgac acaatgtata cattaattaa 2100 ttgtaactcc acaaccatta
agcaagcctg tccaaaggta acttttgagc caattccaat 2160 acactattgt
gctccagcgg ggtatgccat ctttaagtgt aacaacacag agtttaatgg 2220
aacgggccca tgcaacaaca ttacagtagt tacttgtaca catggtatca ggccaacagt
2280 gagtacgcaa ctaatattaa acgggacact ctctgaagga aaaataagaa
ttatgggaag 2340 aaatatcacg gacagtggaa aaaatattat agttacccta
aattatacta taaacataac 2400 ttgtgagaga acatggaatc agtcagtaca
agagatacct ataggtccaa tggcctggta 2460 cagcatgagc gtagagaaag
acaaaaacac aactggctcg aggtcagcag attgccagta 2520 taacacctct
gaatggacaa gagccttaga acaaacagct gaaaggtatt tagaactgat 2580
gaacaataca ggtaatactg ataatactac agtgatattc aatcatagca ctggtggaga
2640 tccagaggta tccttcctac attttaattg tcatggagag ttcttctatt
gtaacacatc 2700 tgggatgttt aattatacct tttcatgtaa aggaactaac
tgtacccaag ttggttccca 2760 aaatgaatat aataatcata caaccaagat
accttgcagg ataaaacagg tggtaaggtc 2820 atggataagg ggagggtcgg
gactctatgc acctcccagg caaggtcccc taaaatgtag 2880 ctcaaacata
actggaatga ttctacaatt ggataagcca tggaacagaa gtgggcacaa 2940
caatgacacc acatttagac caataggagg agaaatgaaa gatatatgga gaactgaatt
3000 gttcaaatac aaagtagtaa aggtaaaacc ttttagtgtg gcacctacaa
aaattgcaag 3060 gccagtcata ggcacgggca ctcaaagaga aaagagagca
gtaggattgg gaatgctatt 3120 cttaggggtt ctaagtgcag caggtagcac
tatgggcgca gcggcaacaa cgctggcggt 3180 acagacccac actttgatga
agggtatagt gcaacagcag gacaacctgc taagagcaat 3240 acaggcccag
cagcaattgc tgaggctatc tgtatggggt atcagacaac tccgagctcg 3300
cctgctagca ttagaaacct taatacagaa tcagcaactc ctgaacctat ggggctgtaa
3360 gggaaggcta gtctgctaca catcagtaca atggaacagg acatggacaa
acaatactaa 3420 tttagattca atttgggaaa atctaacatg gcaggaatgg
gatcagcaga taagcaacat 3480 aagctccacc atatatgagg aaatacaaaa
ggcacaaata cagcaggaat acaatgagaa 3540 aaagttgcta gagttagatg
aatgggcttc tatttggaat tggcttgaca taactaaatg 3600 107 192 PRT Human
107 Met Glu Asn Arg Trp Gln Val Leu Ile Val Trp Gln Val Asp Arg Gln
1 5 10 15 Lys Val Lys Ala Trp Asn Ser Leu Val Lys Tyr His Lys Tyr
Arg Ser 20 25 30 Arg Lys Ala Lys Asp Trp Cys Tyr Arg His His Phe
Glu Ser Arg Asn 35 40 45 Pro Arg Val Ser Ser Ser Val His Ile Pro
Val Gly Met Ala Trp Val 50 55 60 Ile Val Thr Thr Tyr Trp Gly Leu
Met Pro Gly Glu Arg Glu Glu Gln 65 70 75 80 Leu Gly His Gly Val Ser
Ile Glu Trp Gln Tyr Lys Lys Tyr Thr Thr 85 90 95 Gln Ile Asp Pro
Glu Thr Ala Asp Arg Met Ile His Leu Tyr Tyr Phe 100 105 110 Thr Cys
Phe Thr Asp Ser Ala Val Arg Lys Ala Ile Leu Gly Gln Arg 115 120 125
Ile Leu Thr Lys Cys Glu Tyr Pro Ala Gly His Ser Gln Val Gly Thr 130
135 140 Leu Gln Leu Leu Ala Leu Arg Val Val Val Lys Ala Lys Arg Asn
Lys 145 150 155 160 Pro Pro Leu Pro Ser Val Gln Lys Leu Thr Glu Asp
Arg Trp Ser Glu 165 170 175 His Leu Arg Ile Arg Gly Gln Leu Glu Ser
Leu Ser Met Asn Gly His 180 185 190 108 192 PRT Human 108 Met Glu
Asn Lys Trp Gln Val Leu Ile Val Trp Gln Val Asp Arg Gln 1 5 10 15
Lys Val Lys Thr Trp Asn Ser Leu Val Lys Tyr His Lys Tyr Arg Ser 20
25 30 Arg Lys Ala Lys Asp Trp Tyr Tyr Arg His His Tyr Glu Ser Arg
Asn 35 40 45 Pro Arg Ile Ser Ser Gly Val Tyr Ile Pro Val Gly Pro
Ala Cys Ile 50 55 60 Val Val Asn Thr Tyr Trp Gly Leu Met Pro Gly
Glu Arg Asp Glu His 65 70 75 80 Leu Gly His Gly Val Ser Ile Glu Trp
Gln Tyr Lys Lys Tyr Thr Thr 85 90 95 Gln Ile Asp Pro Glu Thr Ala
Asp Arg Met Ile His Leu Tyr Tyr Phe 100 105 110 Thr Cys Phe Thr Glu
Ser Ala Ile Arg Lys Ala Ile Leu Gly Gln Arg 115 120 125 Val Leu Thr
Lys Cys Glu Tyr Pro Ala Gly His Ser Gln Val Gly Thr 130 135 140 Leu
Gln Leu Leu Ala Leu Arg Val Val Val Lys Glu Arg Lys His Arg 145 150
155 160 Pro Pro Leu Pro Ser Val Gln Lys Leu Thr Glu Asp Arg Trp Asn
Lys 165 170 175 His Leu Arg Ile Arg Asp Gln Leu Glu Ser His Ser Met
Asn Gly His 180 185 190
109 192 PRT Human 109 Met Glu Asn Arg Trp Gln Val Leu Ile Val Trp
Gln Val Asp Arg Gln 1 5 10 15 Lys Val Lys Ala Trp Asn Ser Leu Val
Lys Tyr His Lys Tyr Arg Ser 20 25 30 Arg Lys Thr Gln Asn Trp Asp
Tyr Arg His His Tyr Glu Ile Arg Asn 35 40 45 Pro Arg Ile Ser Ser
Gly Val Tyr Ile Pro Val Gly Glu Ala Lys Ile 50 55 60 Val Val Thr
Thr Tyr Trp Gly Leu Met Pro Gly Glu Arg Asp Glu His 65 70 75 80 Leu
Gly His Gly Val Ser Ile Glu Trp Gln Tyr Lys Asn Tyr Ser Thr 85 90
95 Gln Ile Asp Pro Glu Thr Ala Asp Lys Ile Ile His Leu His Tyr Phe
100 105 110 Thr Cys Phe Thr Glu Ser Ala Ile Arg Arg Ala Ile Leu Gly
Gln Arg 115 120 125 Val Leu Thr Arg Cys Glu Tyr Pro Ala Gly His Ser
Gln Val Gly Thr 130 135 140 Leu Gln Leu Leu Ala Leu Arg Ala Val Val
Lys Asp Lys Arg Ser Lys 145 150 155 160 Pro Pro Leu Pro Ser Val Gln
Lys Leu Thr Gly Asp Arg Trp Asn Arg 165 170 175 His Leu Arg Ile Arg
Asp Gln Gln Glu Ser His Ser Met Asn Gly His 180 185 190 110 192 PRT
Human 110 Met Glu Asn Arg Trp Gln Val Met Ile Val Trp Gln Val Asp
Arg Met 1 5 10 15 Arg Ile Arg Thr Trp Asn Ser Leu Val Lys His His
Met Tyr Ile Ser 20 25 30 Lys Lys Ala Thr Asp Trp Val Tyr Lys His
His Tyr Asp Ser Arg His 35 40 45 Pro Lys Val Ser Ser Glu Val His
Ile Pro Leu Gly Asp Ala Lys Leu 50 55 60 Val Ile Arg Thr Tyr Trp
Gly Leu His Thr Gly Glu Arg Asp Trp His 65 70 75 80 Leu Gly His Gly
Val Ser Ile Glu Trp Lys Gln Arg Arg Tyr Ser Thr 85 90 95 Gln Ile
Asp Pro Asp Leu Ala Asp Gln Leu Ile His Leu His Tyr Phe 100 105 110
Asn Cys Phe Ser Glu Ser Ala Ile Arg Lys Ala Ile Leu Gly Gln Val 115
120 125 Val Arg Pro Arg Cys Asp Tyr Pro Ala Gly His Ser Lys Val Gly
Ser 130 135 140 Leu Gln Tyr Leu Ala Leu Lys Ala Leu Val Thr Pro Thr
Arg Thr Lys 145 150 155 160 Pro Pro Leu Pro Ser Val Lys Lys Leu Thr
Glu Asp Arg Trp Asn Lys 165 170 175 Pro Gln Lys Thr Arg Gly His Arg
Gly Ser Gly Pro Met Tyr Gly His 180 185 190 111 85 PRT Human 111
Met His His Arg Asp Leu Leu Val Leu Ile Ile Ile Ser Ala Leu Leu 1 5
10 15 Leu Ile Asn Val Ile Ile Trp Met Phe Ile Leu Arg Gln Tyr Leu
Glu 20 25 30 Gln Lys Lys Gln Asp Arg Arg Glu Arg Asp Ile Leu Glu
Arg Leu Arg 35 40 45 Arg Ile Ala Glu Ile Lys Asp Asp Ser Asp Tyr
Glu Ser Asn Glu Glu 50 55 60 Glu Glu Gln Glu Val Arg Asp Leu Ile
His Ser His Gly Phe Asp Asn 65 70 75 80 Pro Met Phe Glu Leu 85 112
86 PRT Human 112 Met Gln Gln Lys Asp Leu Leu Leu Leu Val Ile Ile
Ser Ala Leu Leu 1 5 10 15 Leu Ile Asn Ile Ile Leu Trp Met Phe Asn
Leu Arg Lys Tyr Leu Glu 20 25 30 Gln Lys Lys Gln Asp Arg Arg Glu
Arg Glu Ile Leu Glu Arg Ile Arg 35 40 45 Arg Ile Arg Glu Ile Arg
Asp Asp Ser Asp Tyr Glu Ser Asn Glu Glu 50 55 60 Glu Glu Gln Glu
Val Arg Gly His Leu Val His Met Phe Gly Phe Ala 65 70 75 80 Asn Pro
Val Phe Glu Ile 85 113 85 PRT Human 113 Met His His Lys Asp Leu Leu
Ile Leu Ile Val Ala Ser Ile Leu Leu 1 5 10 15 Phe Thr Asn Ile Val
Ile Trp Thr Phe Ile Leu Lys Lys Tyr Leu Glu 20 25 30 Gln Lys Glu
Gln Asp Arg Arg Glu Arg Glu Leu Leu Lys Arg Ile Lys 35 40 45 Arg
Ile Arg Glu Val Arg Asp Asp Ser Asp Tyr Glu Ser Asn Gly Asp 50 55
60 Gly Gly Gln Glu Val Ile His Leu Val His Thr His Gly Phe Val Asn
65 70 75 80 Pro Met Phe Glu Leu 85 114 85 PRT Human 114 Met Gln Leu
Arg Asp Gln Leu Thr Leu Ile Ile Ile Ser Ala Leu Leu 1 5 10 15 Leu
Val Asn Val Val Leu Trp Thr Phe Ile Leu Arg Gln Tyr Leu Lys 20 25
30 Gln Lys Lys Gln Asp Arg Arg Gly Arg Glu Ile Leu Glu Arg Leu Arg
35 40 45 Arg Ile Arg Gln Ile Glu Asp Asp Ser Asp Tyr Glu Ser Asp
Gly Lys 50 55 60 Glu Glu Gln Glu Val Arg Asp Leu Val His Ser Tyr
Gly Phe Asp Asn 65 70 75 80 Pro Met Phe Glu Pro 85 115 100 PRT
Human 115 Met Glu Arg Ala Pro Glu Asp Gln Gly Pro Ala Arg Glu Pro
Phe Asn 1 5 10 15 Glu Trp Ala Leu Glu Ile Leu Glu Glu Leu Lys Ala
Glu Ala Val Arg 20 25 30 His Phe Pro Arg Gln Trp Leu Gln Ala Leu
Gly Gln Tyr Ile Tyr Glu 35 40 45 Thr Tyr Gly Asp Thr Trp Val Gly
Val Met Ala Ile Thr Arg Ile Leu 50 55 60 Gln Gln Ile Leu Phe Ala
His Phe Arg Ile Gly Cys Gln His Ser Arg 65 70 75 80 Ile Gly Ile Asn
Pro Thr Asn Thr Arg Gly Arg Gly Arg Arg Asn Gly 85 90 95 Ser Ser
Arg Ser 100 116 100 PRT Human 116 Met Glu Gln Ala Pro Glu Asp Gln
Gly Pro Ala Arg Glu Pro Phe Asn 1 5 10 15 Glu Trp Thr Leu Glu Leu
Leu Glu Glu Leu Lys Ala Glu Ala Val Arg 20 25 30 His Phe Pro Arg
Pro Trp Leu Gln Ala Leu Gly Gln Tyr Ile Tyr Asp 35 40 45 Thr Tyr
Gly Asp Thr Trp Val Gly Val Met Ala Ile Ile Arg Leu Leu 50 55 60
Gln Leu Met Ile Phe Ala His Phe Arg Ile Gly Cys Gln His Ser Arg 65
70 75 80 Ile Gly Ile Asn Pro Ser Asn Thr Arg Gly Arg Gly Arg Arg
Asn Gly 85 90 95 Ser Ser Arg Pro 100 117 93 PRT Human 117 Met Glu
Gln Ala Pro Glu Asn Gln Gly Pro Ala Arg Glu Pro Phe Asn 1 5 10 15
Glu Trp Ala Leu Glu Thr Leu Glu Glu Ile Lys Ala Glu Ala Val Arg 20
25 30 His Phe Pro Arg Pro Trp Leu Gln Ser Leu Gly Gln Tyr Ile Tyr
Glu 35 40 45 Thr Tyr Gly Asp Thr Trp Glu Gly Val Met Ala Ile Ile
Arg Ile Leu 50 55 60 Gln Gln Leu Ile Phe Ala His Phe Arg Ile Gly
Cys Gln His Ser Arg 65 70 75 80 Ile Gly Ile Thr Pro Ser Asn Ala Arg
Gly Arg Gly Arg 85 90 118 100 PRT Human 118 Met Glu Gln Ala Pro Glu
Asp Gln Gly Ala Gln Arg Glu Arg Ser Asn 1 5 10 15 Val Trp Thr Leu
Asp Leu Leu Glu Glu Leu Lys His Glu Ala Val Arg 20 25 30 His Phe
Pro Arg Pro Trp Leu Gln Gly Leu Gly Gln Tyr Ile Tyr Glu 35 40 45
Thr Tyr Gly Asp Thr Trp Glu Gly Val Glu Ala Ile Ile Arg Ile Leu 50
55 60 Gln Gln Leu Leu Phe Ala His Phe Arg Ile Gly Cys Gln His Ser
Arg 65 70 75 80 Ile Gly Ile Asn Pro Ser Asn Pro Arg Gly Lys Gly Arg
Arg Asn Gly 85 90 95 Ser Ser Arg Ser 100 119 71 PRT Human 119 Met
Asp Pro Val Asp Pro Glu Met Pro Pro Trp His His Pro Gly Ser 1 5 10
15 Gln Pro Gln Ile Pro Cys Asn Asn Cys Tyr Cys Lys Arg Cys Cys Tyr
20 25 30 His Cys Leu Val Cys Phe Thr Arg Lys Gly Leu Gly Ile Ser
Tyr Gly 35 40 45 Arg Lys Lys Arg Arg Gln Arg Arg Ala Ala Ala Ser
His Pro Asp Asn 50 55 60 Lys Asp Leu Val Pro Glu Gln 65 70 120 71
PRT Human 120 Met Asp Pro Val Asp Pro Glu Met Pro Pro Trp His His
Pro Gly Ser 1 5 10 15 Gln Pro Gln Asn Pro Cys Asn Lys Cys Tyr Cys
Lys Lys Cys Cys Tyr 20 25 30 His Cys Tyr Val Cys Phe Thr Ser Lys
Gly Leu Gly Ile Ser Tyr Gly 35 40 45 Arg Lys Lys Arg Arg Arg Pro
Ala Ala Ala Ala Ser Arg Pro Asp Asn 50 55 60 Lys Asp Leu Val Pro
Glu Gln 65 70 121 71 PRT Human 121 Met Asp Pro Val Asp Pro Glu Val
Pro Pro Trp His His Pro Gly Ser 1 5 10 15 Gln Pro Pro Thr Pro Cys
Asn Ala Cys Tyr Cys Lys Arg Cys Cys Tyr 20 25 30 His Cys Tyr Leu
Cys Phe Thr Lys Lys Gly Leu Gly Ile Ser His Gly 35 40 45 Arg Lys
Lys Arg Arg Arg Pro Ala Ala Ala Ala Ser Ser Ser Asn Asn 50 55 60
Lys Asp Leu Val Pro Glu Gln 65 70 122 71 PRT Human 122 Met Asp Pro
Val Asp Pro Glu Ile Pro Pro Trp His His Pro Gly Ser 1 5 10 15 Gln
Pro Gln Thr Pro Cys Asn Asn Cys Ser Cys Lys Lys Cys Cys Tyr 20 25
30 His Cys Tyr Val Cys Phe Thr Arg Lys Gly Leu Glu Ile Ser Tyr Gly
35 40 45 Arg Lys Lys Arg Arg Arg Ser Ala Ala Glu Thr Arg His Pro
Asp Asn 50 55 60 Gln Asp Ile Val Pro Glu Gln 65 70 123 13 PRT Human
123 Ala Ile Arg Ile Ile Lys Ile Leu Tyr Gln Ser Ser Lys 1 5 10 124
26 PRT Human 124 Met Ala Gly Arg Ser Asp Asp Asp Gln Gln Leu Leu
Gln Ala Ala Arg 1 5 10 15 Ile Ile Lys Ile Leu Tyr Gln Ser Ser Lys
20 25 125 26 PRT Human 125 Met Ala Gly Arg Ser Asp Asp Asp Gln Gln
Leu Leu Gln Ala Leu Arg 1 5 10 15 Ile Ile Lys Ile Leu Tyr Gln Ser
Ser Lys 20 25 126 26 PRT Human 126 Met Ala Gly Arg Ser Asp Glu Asp
Gln Pro Leu Lys Arg Val Ile Gln 1 5 10 15 Ile Ile Lys Ile Leu Tyr
Gln Ser Ser Lys 20 25 127 42 PRT Human 127 Ile Lys Val Val Pro Arg
Arg Lys Ala Lys Ile Ile Arg His Tyr Gly 1 5 10 15 Lys Gln Met Ala
Gly Ala Asp Ser Met Ala Ser Gly Gln Thr Glu Ser 20 25 30 Glu Ser
Val Glu Gln Pro Gly Glu Ile Pro 35 40 128 42 PRT Human 128 Ile Lys
Val Val Pro Arg Arg Lys Ala Lys Ile Ile Arg Asp Tyr Gly 1 5 10 15
Lys Gln Met Ala Gly Thr Asp Ser Met Ala Ser Arg Gln Thr Glu Ser 20
25 30 Glu Asn Val Glu Gln Leu Gly Glu Ile Pro 35 40 129 36 PRT
Human 129 Ile Lys Val Val Pro Arg Arg Lys Ala Lys Ile Leu Arg Asp
Tyr Gly 1 5 10 15 Lys Gln Met Ala Gly Ala Asp Ser Met Ala Ser Gly
Gln Thr Glu Ser 20 25 30 Glu Ser Met Glu 35 130 106 PRT Human 130
His Asn Phe Lys Arg Lys Gly Gly Ile Gly Gly Tyr Ser Ala Gly Glu 1 5
10 15 Arg Ile Ile Asp Ile Ile Ala Ser Asp Ile Gln Thr Lys Glu Leu
Gln 20 25 30 Lys Gln Ile Thr Lys Ile Gln Asn Phe Arg Val Tyr Tyr
Arg Asp Ser 35 40 45 Arg Asp Pro Ile Trp Lys Gly Pro Ala Lys Leu
Leu Trp Lys Gly Glu 50 55 60 Gly Ala Val Val Ile Gln Asp Asn Ser
Asp Ile Lys Val Val Pro Arg 65 70 75 80 Arg Lys Ala Lys Ile Ile Lys
Asp Tyr Gly Lys Gln Met Ala Gly Asp 85 90 95 Asp Cys Val Ala Ser
Arg Gln Asn Glu Asp 100 105 131 35 PRT Human 131 Met Ile Val Thr
Met Lys Ala Met Lys Arg Arg Asn Arg Lys Leu Glu 1 5 10 15 Ile Leu
Tyr Ile Val Met Ala Leu Ile Ile Pro Cys Leu Ser Ser Asp 20 25 30
Gln Lys Tyr 35 132 36 PRT Human 132 Met Ile Val Thr Met Lys Ala Met
Lys Arg Lys Asn Lys Lys Leu Gly 1 5 10 15 Val Ile Leu Cys Ile Cys
Leu Ala Leu Leu Ile Pro Cys Leu Arg Ser 20 25 30 Asn Asp Leu Tyr 35
133 35 PRT Human 133 Met Ile Val Ile Met Lys Ala Met Glu Met Glu
Asp Lys Lys Leu Tyr 1 5 10 15 Ile Leu Cys Ile Leu Met Val Leu Leu
Thr Pro Cys Leu Ser Ser Asp 20 25 30 Lys Leu Tyr 35 134 715 PRT
Human 134 Met Thr Val Thr Met Lys Ala Met Glu Lys Arg Asn Arg Lys
Leu Gly 1 5 10 15 Ile Leu Cys Ile Val Met Ala Leu Ile Thr Pro Cys
Leu Ser His Asp 20 25 30 Gln Arg Tyr Ala Thr Val Tyr Ala Gly Val
Pro Val Trp Glu Glu Ala 35 40 45 Asn Pro Val Leu Phe Cys Ala Ser
Asp Ala Asn Leu Thr Ser Thr Glu 50 55 60 Lys His Asn Ile Trp Ala
Ser Gln Ala Cys Val Pro Thr Asp Pro Thr 65 70 75 80 Pro His Glu Tyr
Pro Leu His Asn Val Thr Asp Asn Phe Asn Ile Trp 85 90 95 Lys Asn
Tyr Met Val Glu Gln Met Gln Asp Asp Ile Ile Ser Leu Trp 100 105 110
Glu Gln Ser Leu Lys Pro Cys Val Gln Met Thr Phe Leu Cys Val Gln 115
120 125 Met Asn Cys Thr Ser Val Ser Asn Ser Ser Val Ser Asn Ser Ser
Val 130 135 140 Ser Asn Ser Ser Val Ser Asn Ser Ser Val Ser Asp Ser
Thr Ile Pro 145 150 155 160 Lys Lys Lys Asn Asn Ser Ser Ser Glu Asp
Leu Leu Lys Gln Cys Asp 165 170 175 Phe Asn Ala Thr Thr Val Leu Lys
Asp Lys Lys Glu Lys Lys Gln Thr 180 185 190 Leu Phe Tyr Val Ser Asp
Leu Met Lys Leu Thr Asn Val Thr Asn Asp 195 200 205 Thr Met Tyr Thr
Leu Ile Asn Cys Asn Ser Thr Thr Ile Lys Gln Ala 210 215 220 Cys Pro
Lys Val Thr Phe Glu Pro Ile Pro Ile His Tyr Cys Ala Pro 225 230 235
240 Ala Gly Tyr Ala Ile Phe Lys Cys Asn Asn Thr Glu Phe Asn Gly Thr
245 250 255 Gly Pro Cys Asn Asn Ile Thr Val Val Thr Cys Thr His Gly
Ile Arg 260 265 270 Pro Thr Val Ser Thr Gln Leu Ile Leu Asn Gly Thr
Leu Ser Glu Gly 275 280 285 Lys Ile Arg Ile Met Gly Arg Asn Ile Thr
Asp Ser Gly Lys Asn Ile 290 295 300 Ile Val Thr Leu Asn Tyr Thr Ile
Asn Ile Thr Cys Glu Arg Thr Trp 305 310 315 320 Asn Gln Ser Val Gln
Glu Ile Pro Ile Gly Pro Met Ala Trp Tyr Ser 325 330 335 Met Ser Val
Glu Lys Asp Lys Asn Thr Thr Gly Ser Arg Ser Ala Asp 340 345 350 Cys
Gln Tyr Asn Thr Ser Glu Trp Thr Arg Ala Leu Glu Gln Thr Ala 355 360
365 Glu Arg Tyr Leu Glu Leu Met Asn Asn Thr Gly Asn Thr Asp Asn Thr
370 375 380 Thr Val Ile Phe Asn His Ser Thr Gly Gly Asp Pro Glu Val
Ser Phe 385 390 395 400 Leu His Phe Asn Cys His Gly Glu Phe Phe Tyr
Cys Asn Thr Ser Gly 405 410 415 Met Phe Asn Tyr Thr Phe Ser Cys Lys
Gly Thr Asn Cys Thr Gln Val 420 425 430 Gly Ser Gln Asn Glu Tyr Asn
Asn His Thr Thr Lys Ile Pro Cys Arg 435 440 445 Ile Lys Gln Val Val
Arg Ser Trp Ile Arg Gly Gly Ser Gly Leu Tyr 450 455 460 Ala Pro Pro
Arg Gln Gly Pro Leu Lys Cys Ser Ser Asn Ile Thr Gly 465 470 475 480
Met Ile Leu Gln Leu Asp Lys Pro Trp Asn Arg Ser Gly His Asn Asn 485
490 495 Asp Thr Thr Phe Arg Pro Ile Gly Gly Glu Met Lys Asp Ile Trp
Arg 500 505 510 Thr Glu Leu Phe Lys Tyr Lys Val Val Lys Val Lys Pro
Phe Ser Val 515 520 525 Ala Pro Thr Lys Ile Ala Arg Pro Val Ile Gly
Thr Gly Thr Gln Arg 530 535 540 Glu Lys Arg Ala Val Gly Leu Gly Met
Leu Phe Leu
Gly Val Leu Ser 545 550 555 560 Ala Ala Gly Ser Thr Met Gly Ala Ala
Ala Thr Thr Leu Ala Val Gln 565 570 575 Thr His Thr Leu Met Lys Gly
Ile Val Gln Gln Gln Asp Asn Leu Leu 580 585 590 Arg Ala Ile Gln Ala
Gln Gln Gln Leu Leu Arg Leu Ser Val Trp Gly 595 600 605 Ile Arg Gln
Leu Arg Ala Arg Leu Leu Ala Leu Glu Thr Leu Ile Gln 610 615 620 Asn
Gln Gln Leu Leu Asn Leu Trp Gly Cys Lys Gly Arg Leu Val Cys 625 630
635 640 Tyr Thr Ser Val Gln Trp Asn Arg Thr Trp Thr Asn Asn Thr Asn
Leu 645 650 655 Asp Ser Ile Trp Glu Asn Leu Thr Trp Gln Glu Trp Asp
Gln Gln Ile 660 665 670 Ser Asn Ile Ser Ser Thr Ile Tyr Glu Glu Ile
Gln Lys Ala Gln Ile 675 680 685 Gln Gln Glu Tyr Asn Glu Lys Lys Leu
Leu Glu Leu Asp Glu Trp Ala 690 695 700 Ser Ile Trp Asn Trp Leu Asp
Ile Thr Lys Cys 705 710 715 135 38 PRT Human PEPTIDE (1)..(38) 135
Cys Glu Arg Thr Trp Asn Gln Ser Val Gln Glu Ile Pro Ile Gly Pro 1 5
10 15 Met Ala Trp Tyr Ser Met Ser Val Glu Leu Asp Leu Asn Thr Thr
Gly 20 25 30 Ser Arg Ser Ala Asp Cys 35 136 7 PRT Human PEPTIDE
(1)..(7) 136 Ser Val Gln Glu Ile Pro Ile 1 5 137 23 PRT Human
PEPTIDE (1)..(23) 137 Asn Gln Gln Leu Leu Asn Leu Trp Gly Cys Lys
Gly Arg Leu Val Cys 1 5 10 15 Tyr Thr Ser Val Gln Trp Asn 20 138 24
PRT Human PEPTIDE (1)..(24) 138 Asn Gln Gln Leu Leu Asn Leu Trp Gly
Cys Lys Gly Arg Leu Val Cys 1 5 10 15 Tyr Thr Ser Val Lys Trp Asn
Asn 20 139 34 DNA Human 139 gggttcttgg gagcagcagg aagcactatg ggcg
34 140 33 DNA Human 140 tctgaaacga cagaggtgag tatccctgcc taa 33 141
33 DNA Human 141 tggatcccac agtgtactga agggtatagt gca 33 142 28 DNA
Human 142 catttagtta tgtcaagcca attccaaa 28 143 31 DNA Human 143
gttctccata tatctttcat atctccccct a 31 144 31 DNA Human 144
ttgtacacat ggcattaggc caacagtaag t 31 145 31 DNA Human 145
tgaattccta atattgaatg ggacactctc t 31 146 32 DNA Human 146
tggatcctac aataaaagaa ttctccatga ca 32 147 24 DNA Human 147
gggtttatta cagggacagc agag 24 148 24 DNA Human 148 ggttggggtc
tgtgggtaca cagg 24 149 22 DNA Human 149 gcaaaactac tctggaaagg tg 22
150 23 DNA Human 150 gcwtctttcc acacaggtac ccc 23 151 21 DNA Human
151 catattgggg attgatgcca g 21 152 20 DNA Human 152 gcatyagcgt
tacttactgc 20
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