U.S. patent application number 14/013947 was filed with the patent office on 2014-07-03 for novel simian t-cell lymphotropic virus.
This patent application is currently assigned to Johns Hopkins University. The applicant listed for this patent is The Government of the U.S.A. as Represented by the Secretary of the Department of He, Johns Hopkins University. Invention is credited to Donald S. Burke, Thomas M. Folks, Walid Heneine, David M. Sintasath, William M. Switzer, Nathan D. Wolfe.
Application Number | 20140186383 14/013947 |
Document ID | / |
Family ID | 39760969 |
Filed Date | 2014-07-03 |
United States Patent
Application |
20140186383 |
Kind Code |
A1 |
Switzer; William M. ; et
al. |
July 3, 2014 |
NOVEL SIMIAN T-CELL LYMPHOTROPIC VIRUS
Abstract
Disclosed are the simian T-cell lymphotropic virus type 3
subtype D (STLV-3 subtype D), isolated nucleic acid molecules
encoding STLV-3 subtype D polypeptides, such as STLV-3 subtype D
envelope, protease, polymerase, tax, rex, and capsid polypeptides,
isolated polypeptides encoded by such nucleic acids. Methods are
also disclosed for detecting STLV-3 subtype D, for example by
detecting a STLV-3 subtype D nucleic acid or polypeptide in the
sample. Accordingly, probes, primers, and antibodies for use in
detecting STLV-3 subtype D nucleic acids or polypeptides are
disclosed. Therapeutic compositions which include isolated nucleic
acid molecules encoding a STLV-3 subtype D polypeptides or isolated
polypeptides encoded by such nucleic acid molecules are also
disclosed.
Inventors: |
Switzer; William M.; (Stone
Mountain, GA) ; Heneine; Walid; (Atlanta, GA)
; Folks; Thomas M.; (Helotes, TX) ; Wolfe; Nathan
D.; (Los Angeles, CA) ; Burke; Donald S.;
(Pittsburgh, PA) ; Sintasath; David M.; (Atlanta,
GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Johns Hopkins University
The Government of the U.S.A. as Represented by the Secretary of the
Department of He |
Baltimore
Atlanta |
MD
GA |
US
US |
|
|
Assignee: |
Johns Hopkins University
Baltimore
MD
The Government of the U.S.A as Represented by the Secretary of
the Department of He
Atlanta
GA
|
Family ID: |
39760969 |
Appl. No.: |
14/013947 |
Filed: |
August 29, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12600995 |
Nov 19, 2009 |
8663968 |
|
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PCT/US08/64270 |
May 20, 2008 |
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14013947 |
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60990138 |
Nov 26, 2007 |
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60939304 |
May 21, 2007 |
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Current U.S.
Class: |
424/187.1 ;
435/188; 435/5; 530/350; 530/387.9; 530/391.3 |
Current CPC
Class: |
C12N 15/86 20130101;
A61P 37/04 20180101; C07K 14/005 20130101; C12N 2740/14022
20130101; C12N 2740/14021 20130101; C12N 2740/14043 20130101; C12N
7/00 20130101; G01N 33/56983 20130101; A61P 31/12 20180101; C07K
16/1036 20130101 |
Class at
Publication: |
424/187.1 ;
530/350; 530/387.9; 530/391.3; 435/188; 435/5 |
International
Class: |
C07K 14/005 20060101
C07K014/005; G01N 33/569 20060101 G01N033/569; C07K 16/10 20060101
C07K016/10 |
Claims
1. An isolated simian T-cell lymphotropic virus type 3 West African
subtype D (STLV-3 subtype D) polypeptide, comprising: an amino acid
sequence encoded by a nucleic acid sequence at least 95% identical
to one of: nucleotides 747-2009 of SEQ ID NO: 1; nucleotides
1961-2494 of SEQ ID NO: 1; nucleotides 2416-5061 of SEQ ID NO: 1;
nucleotides 5054-6535 of SEQ ID NO: 1; SEQ ID NO: 25; or SEQ ID NO:
26; wherein the STLV-3 subtype D (simian T-cell lymphotropic virus
type 3 West African subtype D) polypeptide encoded by the nucleic
acid sequence at least 95% identical to nucleotides 747-2009 of SEQ
ID NO: 1 is a capsid protein; nucleotides 1961-2494 of SEQ ID NO: 1
is a protease; nucleotides 2416-5061 of SEQ ID NO: 1 is a
polymerase; nucleotides 5054-6535 of SEQ ID NO: 1 is an envelope
protein; SEQ ID NO: 25 is a tax polypeptide; and SEQ ID NO: 26 is a
rex polypeptide.
2. An isolated antibody that binds to the isolated polypeptide of
claim 1.
3. The isolated antibody of claim 2, wherein the antibody is
labeled.
4. The antibody according to claim 3, wherein the antibody is
radiolabeled, fluorescently-labeled, biotin-labeled,
enzymatically-labeled, or chemically-labeled.
5. A method for detecting a simian T-cell lymphotropic virus type 3
subtype D (STLV-3 subtype D) in a sample, comprising: amplifying a
STLV-3 subtype D nucleic acid from the sample, using two or more
oligonucleotide primers between 15 and 50 nucleotides in length
that hybridize under very high stringency conditions to a STLV-3
subtype D nucleic acid sequence set forth as SEQ ID NO: 1 to
produce amplified STLV-3 subtype D nucleic acids; and detecting the
amplified STLV-3 subtype D nucleic acids, wherein the presence of
the amplified STLV-3 subtype D nucleic acids indicates the presence
of the STLV-3 subtype D virus in the sample.
6. The method according to claim 5, wherein amplifying the STLV-3
subtype D nucleic acid comprises amplifying with polymerase chain
reaction (PCR), real-time PCR, reverse transcriptase-polymerase
chain reaction (RT-PCR), real-time reverse transcriptase-polymerase
chain reaction (rt RT-PCR), ligase chain reaction, or
transcription-mediated amplification (TMA).
7. The method according to claim 5, further comprising: contacting
the sample with a probe comprising a nucleic acid sequence at least
15 nucleotides in length that hybridizes under very high stringency
conditions to the amplified STLV-3 subtype D nucleic acid sequence
set forth as SEQ ID NO: 1; and detecting hybridization between the
amplified STLV-3 subtype D nucleic acid and the probe, wherein the
detection of hybridization indicates the presence of the STLV-3
subtype D virus in the sample.
8. The method according to claim 5, wherein the two or more
oligonucleotide primers comprise a first and second primer, wherein
the first primer comprises SEQ ID NO: 21 and the second primer
comprises SEQ ID NO: 22.
9. The method according to claim 5, wherein the two or more
oligonucleotide primers comprise a first and second primer, wherein
the first primer comprises SEQ ID NO: 23 and the second primer
comprises SEQ ID NO: 24.
10. The method according to claim 5, wherein the two or more
oligonucleotide primers are two pairs of primers, wherein the first
pair of primers comprises a first primer comprising SEQ ID NO: 21
and a second primer comprising SEQ ID NO: 22, and the second pair
of primers comprises a third primer comprising SEQ ID NO: 23 and a
fourth primer comprising SEQ ID NO: 24.
11. A method for detecting a simian T-cell lymphotropic virus type
3 West African subtype D (STLV-3 subtype D) virus in a sample,
comprising: contacting the sample with at least one probe
comprising a nucleic acid sequence at least 15 nucleotides in
length that hybridizes under very high stringency conditions to a
STLV-3 subtype D nucleic acid sequence set forth as SEQ ID NO: 1;
and detecting hybridization between the STLV-3 subtype D nucleic
acid and the probe, wherein the detection of hybridization
indicates the presence of the STLV-3 subtype D virus in the
sample.
12. The method according to claim 7, wherein the probe is
labeled.
13. The method according to claim 12, wherein the probe is
radiolabeled, fluorescently-labeled, biotin-labeled,
enzymatically-labeled, or chemically-labeled.
14. A method for detecting a STLV-3 subtype D polypeptide in a
sample, comprising: contacting the sample with the antibody of
claim 2; and detecting binding of the antibody to a STLV-3 subtype
D polypeptide in the sample, wherein binding of the antibody to the
polypeptide indicates the presence of the STLV-3 subtype D
polypeptide.
15. The method of claim 5, wherein the sample is a biological
sample.
16. The method of claim 15, wherein the biological sample is
obtained from a primate.
17. The method of claim 16, wherein the primate is a non-human
primate.
18. A method for generating an immune response in a subject to a
STLV-3 subtype D virus, comprising administering to the subject a
therapeutically effective amount of the isolated polypeptide of
claim 1, thereby generating the immune response to the STLV-3
subtype D virus.
19. A method for treating or preventing STLV-3 subtype D viral
infection in a subject; comprising administering to the subject a
therapeutically effective amount of the isolated polypeptide of
claim 1, thereby treating or preventing the STLV-3 subtype D viral
infection in the subject.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 12/600,995, filed Nov. 19, 2009, which is the U.S.
National Stage of International Application No. PCT/US2008/064270,
filed May 20, 2008, which was published in English under PCT
Article 21(2), which in turn claims the benefit of U.S. Provisional
Application 60/990,138, filed Nov. 26, 2007, and U.S. Provisional
Application No. 60/939,304, filed May 21, 2007, all of which are
incorporated by reference herein in their entirety.
FIELD
[0002] This application relates to a highly divergent simian T-cell
lymphotropic virus type 3 subtype, provisionally classified as
simian T-cell lymphotropic virus type 3 West African subtype D
(STLV-3 subtype D), specifically to the nucleic acid sequences from
the virus, open reading frames in this virus, and to amino acid
sequences encoded by these sequences.
BACKGROUND
[0003] Primate T-cell leukemia viruses (PTLVs) are genetically
diverse deltaretroviruses comprised of simian and human T-cell
leukemia viruses (STLVs and HTLVs, respectively). Like human
immunodeficiency virus (HIV), HTLV is a zoonotic simian retrovirus
originating from historical and contemporary contact with
STLV-infected nonhuman primates (NHPs). The genetic diversity of
HTLV is directly related to the genetic diversity of the STLVs from
which the primary zoonotic infection originated, as evidenced by
the clustering of geographically proximal HTLVs and STLVs within
the same phylogenetic lineages. Four PTLV groups have been
identified: PTLV-1, PTLV-2, PTLV-3 and PTLV-4. PTLV-1, PTLV-2 and
PTLV-3 include human (HTLV-1, HTLV-2, and HTLV-3) and simian
(STLV-1, STLV-2, and STLV-3) viruses. PTLV-4 comprises HTLV-4,
which was identified from one individual in Cameroon with known
exposure to primates. A simian counterpart of this virus has not
yet been identified (Wolfe et al. Proc. Natl. Acad. Sci. U.S.A.
102:7994-7999, 2005).
[0004] HTLV-1 and HTLV-2 are known to be transmitted through sexual
contact (Murphy et al. Ann. Intern. Med. 111:555-560, 1989);
mother-to-child transmission through breast-feeding (Hino et al.
Jpn. J. Cancer. Res. 1985, 76:474-480, 1985; Vitek et al. J.
Infect. Dis. 171:1022-1026, 1995); transfusion of blood and/or
blood products (Manns et al. Int. J. Cancer 51:886-891, 1992;
Okochi and Sato Princess Takamatsu Symp. 15:129-135, 1984; Okochi
et al. Vox. Sang. 46:245-253, 1984); and injection drug use (Van
Brussel et al. Rev. Med. Virol. 9:155-170, 1999). The mechanisms of
transmission of PTLVs and other retroviruses between primates and
humans are largely unknown, but it is believed that humans can
become infected with simian retroviruses through direct exposure to
primates via bites or scratches or contact with body fluids from
butchering and handling infected bushmeat (Wolfe et al. Proc. Natl.
Acad. Sci. U.S.A. 102:7994-7999, 2005; Wolfe et al. Lancet
363:932-937, 2004).
[0005] Many of the PTLV strains and subtypes have been described
from human and primate samples derived from central Africa. In
addition to the recent discovery of HTLV-3 (Wolfe et al. Proc.
Natl. Acad. Sci. U.S.A. 102:7994-7999, 2005; Calattini et al.
Retrovirology 2:30, 2005) and HTLV-4 (Wolfe et al. Proc. Natl.
Acad. Sci. U.S.A. 102:7994-7999, 2005) from primate hunters in
southern Cameroon, HTLV-1 subtypes B, D and E (Mahieux et al. J.
Virol. 71:1317-1333, 1997; Salemi et al. Virology 246:277-287,
1998) and HTLV-2 subtypes B and D (Vandamme et al. J. Virol.
72:4327-4340, 1998) have been isolated from inhabitants of this
region. Similarly, STLV-1, found in the HTLV-1 subtype B clade, has
been identified in Cameroonian gorillas (Gorilla gorilla) and
chimpanzees (Pan troglodytes vellerosus) (Nerrienet et al. J. Gen.
Virol. 85:25-29, 2004) and STLV-3 has been found in wild-caught
red-capped mangabeys (Cercocebus torquatus) from Nigeria and
Cameroon (Meertens et al. J. Gen. Virol. 84:2723-2727, 2003).
Furthermore, evidence for dual infections of STLV-1 and STLV-3 in
agile mangabeys (Cercocebus agilis) in Cameroon has also been
reported (Courgnaud et al. J. Virol. 78:4700-4709, 2004). These
studies suggest that humans are exposed to a significant number of
PTLVs in west and central Africa. Therefore, the need exists for
methods of detecting viral infections, for example to monitor the
transmission of such viruses into the human population. In
addition, the need exists for vaccines for such viruses, for
example by producing an immune response to peptides isolated from
such viruses. However, it is not possible to vaccinate populations
against organisms not known to exist, nor can such unknown
organisms be detected and followed in a population at ask of
infection.
SUMMARY
[0006] Widespread exposure to a broad range of non-human primate
body fluids and tissues via hunting and butchering, or keeping
primate pets has been implicated in the emergence of three
different retrovirus genera: HIV, HTLV, and more recently simian
foamy virus (SFV). While very little is known about the public
health implications of SFV infection, HIV and HTLV spread globally
and became pathogenic following cross-species transmission with
enormous social, medical, political, and economic consequences. The
recent discovery of HTLV-3 and HTLV-4 in primate hunters from
Cameroon doubles the number of known deltaretroviruses in humans.
Novel STLV-1-like infections were also identified in primate
hunters in this same study. These discoveries demonstrate that the
diversity of PTLV is far from being understood and that zoonotic
infection of humans with STLV continues to occur in persons exposed
to non-human primates. Thus, understanding the diversity,
prevalence, and geographic range of STLV infection in areas where
frequent contact with wild NHPs is common provides important
information about the origin and emergence of HTLV, and the risks
of exposure to these and possibly other simian viruses.
[0007] As disclosed herein, through analysis of LTR and larger tax
sequences from C. mona and C. nictitans, a divergent STLV-3-like
strain forming a unique PTLV-3 clade provisionally designated
STLV-3 subtype D has been discovered. Given the propensity of STLV
to cross species boundaries, the increased frequency of hunting and
demand for primate bushmeat in Africa, and the apparent broad
diversity of STLV subtypes in Cameroon it is quite possible that
human infection with this unique STLV-3 subtype will or may have
already occurred. The discovery of this novel PTLV-3 subtype in two
different monkey species and an apparent ancient origin of this
lineage suggest a possible wider distribution of this variant.
Therefore, the ease with which STLVs can cross species barriers and
potentially transmit via primate-hunting practices warrants
increased surveillance for human infection with this divergent
subtype. Since both HIV and HTLV have arisen through multiple
introductions from primates to humans, there is an impetus to
expand surveillance for these and other retroviruses in their
natural host reservoirs and in persons exposed to non-human
primatess in order to predict and possibly prevent the next
retrovirus pandemic.
[0008] The present disclosure relates to a highly divergent simian
T-cell lymphotropic virus type 3 subtype, provisionally classified
as simian T-cell lymphotropic virus type 3 West African subtype D
(referred to herein as STLV-3 subtype D) and isolated nucleic acid
molecules from the genome of this virus. In some embodiments,
isolated STLV-3 subtype D nucleic acid molecules encoding STLV-3
subtype D polypeptides are provided. In one embodiment, a nucleic
acid sequence encoding the STLV-3 subtype D genome is at least 95%
identical to the nucleotide sequence according to SEQ ID NO: 1. In
several examples, nucleotides encoding s STLV-3 subtype D envelope
polypeptides, proteases, polymerases, tax polypeptides, rex
polypeptides, and capsid polypeptides and polypeptides expressed
from such nucleic acids are disclosed. In one embodiment, a nucleic
acid sequence encoding a STLV-3 subtype D envelope polypeptide is
disclosed that is at least 95% identical to the nucleotide sequence
according to nucleotides 5054-6535 of SEQ ID NO: 1. In another
embodiment, a nucleic acid sequence encoding a STLV-3 subtype D
capsid polypeptide is disclosed that is at least 95% identical to
the nucleotide sequence according to nucleotides 747-2009 of SEQ ID
NO: 1. In a further embodiment, a nucleic acid sequence encoding a
STLV-3 subtype D protease is disclosed that is at least 95%
identical to the nucleotide sequence according to nucleotides
1961-2494 of SEQ ID NO: 1. In a further embodiment, a nucleic acid
sequence encoding a STLV-3 subtype D polymerase is disclosed that
is at least 95% identical to the nucleotide sequence according to
nucleotides 2416-5061 of SEQ ID NO: 1. In a further embodiment, a
nucleic acid sequence encoding a STLV-3 subtype D tax polypeptide
is disclosed that is at least 95% identical to the nucleotide
sequence according to SEQ ID NO: 25. In a further embodiment, a
nucleic acid sequence encoding a STLV-3 subtype D rex polypeptide
is disclosed that is at least 95% identical to the nucleotide
sequence according to SEQ ID NO: 26. In several examples, a nucleic
acid sequence encoding a STLV-3 subtype D polypeptide is operably
linked to a promoter.
[0009] In some embodiments, a nucleic acid sequence encoding a
STLV-3 subtype D polypeptide is included in a vector, for example a
viral vector, such as a viral vector that can be included in a
viral particle. Also disclosed are isolated and/or purified STLV-3
subtype D viruses, such as such viruses having identifying
sequences disclosed herein. In some embodiments, a disclosed STLV-3
subtype D virus has a nucleotide sequence at least 95% identical to
the nucleotide sequence according to SEQ ID NO: 1.
[0010] Isolated STLV-3 subtype D polypeptides are disclosed. In one
embodiment, an isolated STLV-3 subtype D capsid polypeptide is
disclosed that is encoded by a nucleic acid sequence at least 95%
identical to the nucleotide sequence according to nucleotides
747-2009 of SEQ ID NO: 1. In another embodiment, an isolated STLV-3
subtype D protease polypeptide is disclosed that is encoded by a
nucleic acid sequence at least 95% identical to the nucleotide
sequence according to nucleotides 1961-2494 of SEQ ID NO: 1. In one
embodiment, an isolated STLV-3 subtype D polymerase polypeptide is
disclosed that is encoded by a nucleic acid sequence at least 95%
identical to the nucleotide sequence according to nucleotides
2416-5061 of SEQ ID NO: 1. In another embodiment, an isolated
STLV-3 subtype D envelope polypeptide is disclosed that is encoded
by a nucleic acid sequence at least 95% identical to the nucleotide
sequence according to nucleotides 5054-6535 of SEQ ID NO: 1. In a
further embodiment, an isolated STLV-3 subtype D tax polypeptide is
disclosed that is encoded by a nucleic acid sequence at least 95%
identical to the nucleotide sequence according to SEQ ID NO: 25. In
a further embodiment, an isolated STLV-3 subtype D rex polypeptide
is disclosed that is encoded by a nucleic acid sequence at least
95% identical to the nucleotide sequence according to SEQ ID NO:
26.
[0011] In some embodiments, an isolated STLV-3 subtype D capsid
polypeptide is disclosed that is encoded by a nucleic acid sequence
at least 95% identical to a nucleotide sequence encoding the amino
acid sequence according to SEQ ID NO: 16. In another embodiment, an
isolated STLV-3 subtype D protease polypeptide is disclosed that is
encoded by a nucleic acid sequence at least 95% identical to a
nucleotide sequence encoding the amino acid sequence according to
SEQ ID NO: 17. In one embodiment, an isolated STLV-3 subtype D
polymerase polypeptide is disclosed that is encoded by a nucleic
acid sequence at least 95% identical to a nucleotide sequence
encoding the amino acid sequence according to SEQ ID NO: 18. In
another embodiment, an isolated STLV-3 subtype D envelope
polypeptide is disclosed that is encoded by a nucleic acid sequence
at least 95% identical to a nucleotide sequence encoding the amino
acid sequence according to SEQ ID NO: 15. In a further embodiment,
an isolated STLV-3 subtype D rex polypeptide is disclosed that is
encoded by a nucleic acid sequence at least 95% identical to a
nucleotide sequence encoding the amino acid sequence according to
SEQ ID NO: 19. In a further embodiment, an isolated STLV-3 subtype
D tax polypeptide is disclosed that is encoded by a nucleic acid
sequence at least 95% identical to a nucleotide sequence encoding
the amino acid sequence according to SEQ ID NO: 20. Antibodies that
specifically bind isolated STLV-3 subtype D polypeptides are also
disclosed. Methods are also disclosed for detecting STLV-3 subtype
D. These methods can include detecting a STLV-3 subtype D nucleic
acid or polypeptide in the sample. Accordingly, probes, primers,
and antibodies for use in detecting STLV-3 subtype D nucleic acids
or polypeptides are disclosed.
[0012] Methods are disclosed generating an immune response in a
subject to a STLV-3 subtype D virus. In several examples, these
methods include administering to the subject a therapeutically
effective amount, such as a therapeutic composition of an isolated
nucleic acid molecule encoding a STLV-3 subtype D polypeptide or an
isolated polypeptide encoded by such a nucleic acid molecule. In
several embodiments, the methods can be of use for treating or
preventing STLV-3 subtype D viral infection in a subject.
[0013] The foregoing and other features and advantages will become
more apparent from the following detailed description of several
embodiments, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 is a schematic representation of the genomic
sequencing strategy of the STLV-3 subtype D genome.
[0015] FIG. 2 is a phylogenetic tree that depicts PTLV phylogeny
inferred using 161-bp tax sequences. New sequences from nonhuman
primates (NHPs) from Cameroon in this study are boxed. NHPs are
coded using the first letter of the genus followed by the first two
letters of the species name: Cercocebus agilis (Cag), Cercopithecus
nictitans (Cni), Cercopithecus mona (Cmo), and Lophocebus albigena
(Lal). The last two letters in the monkey name indicate the study
site. Support for the branching order was determined by 1,000
bootstrap replicates; only values.gtoreq.60% are shown. Branch
lengths are proportional to the evolutionary distance (scale bar)
between the taxa.
[0016] FIG. 3 is a phylogenetic tree that depicts the
identification of a novel PTLV-3 subtype by phylogenetic inference
of 202-bp tax sequences with PTLV prototypes and partial sequences
from three C. nictitans (Cni217, Cni227, and Cni3038) and those
identified shown in the current application (boxed). GENBANK.RTM.
accession numbers for the previously reported partial STLV-3 tax
sequences included in this analysis are AY039033, AF412120, and
AM746647-AM746673). NHPs are coded using the first letter of the
genus followed by the first two letters of the species name: C.
mona (Cmo), Cercopithecus nictitans (Cni). The last two letters in
the sample name indicate the study site. Support for the branching
order was determined by 1,000 bootstrap replicates; only
values.gtoreq.60% are shown. Branch lengths are proportional to the
evolutionary distance (scale bar) between the taxa.
[0017] FIG. 4 depicts a phylogenetic tree using an alignment of
881-bp sequences from prototypical PTLVs and bovine leukemia virus
(BLV). Sequences were used as an outgroup in the maximum likelihood
analysis. New sequences from this study are boxed. NHPs are coded
using the first letter of the genus followed by the first two
letters of the species name: C. mona (Cmo), Cercopithecus nictitans
(Cni). The last two letters in the sample name indicate the study
site. Support for the branching order was determined by 1,000
bootstrap replicates; only values of 60% or more are shown. Branch
lengths are proportional to the evolutionary distance (scale bar)
between the taxa.
[0018] FIG. 5 is a phylogenetic tree that depicts the
identification of a novel PTLV-3 subtype by phylogenetic analysis
of 275-bp LTR sequences. LTR sequences for PTLV-3 Subtype C were
not available for this analysis. NHPs are coded using the first
letter of the genus followed by the first two letters of the
species name: Cercocebus agilis (Cag), Cercopithecus nictitans
(Cni), C. mona (Cmo), and Lophocebus albigena (Lal). The last two
letters in the specimen name indicate the study site. New sequences
from this study are boxed. Support for the branching order was
determined by 1,000 bootstrap replicates; only values.gtoreq.60%
are shown. Branch lengths are proportional to the evolutionary
distance (scale bar) between the taxa.
[0019] FIG. 6 is a phylogenetic tree depicting the inferred
phylogenetic relationships of PTLV-1 LTR sequences by
neighbor-joining analysis. Sequences from wild nonhuman primates
(NHPs) in Cameroon generated in the current study are boxed. NHPs
are coded using the first letter of the genus followed by the first
two letters of the species name: Cercocebus agilis (Cag) and
Cercopithecus nictitans (Cni). The last two letters in the monkey
name indicate the study site. HTLV-1 sequences are italicized. New
sequences from this study are boxed. Support for the branching
order was determined by 1,000 bootstrap replicates; only
values.gtoreq.60% are shown.
[0020] FIG. 7 is a table showing intrasubtype sequence variation
among STLV3 subtypes.
SEQUENCE LISTING
[0021] The nucleic and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and three letter code for amino
acids, as defined in 37 C.F.R. .sctn.1.822. Only one strand of each
nucleic acid sequence is shown, but the complementary strand is
understood as included by any reference to the displayed strand. In
the accompanying sequence listing:
[0022] SEQ ID NO: 1 is an exemplary genomic sequence of STLV-3
subtype D.
[0023] SEQ ID NO: 2 is the nucleotide sequence of a theoretical
nucleic acid molecule illustrating percent sequence identity.
[0024] SEQ ID NO: 3 is the nucleotide sequence of a theoretical
nucleic acid molecule illustrating percent sequence identity.
[0025] SEQ ID NO: 4 is the nucleotide sequence of PCR primer
8699TF1.
[0026] SEQ ID NO: 5 is the nucleotide sequence of PCR primer
PGTAXR1.
[0027] SEQ ID NO: 6 is the nucleotide sequence of PCR primer
8699TF2.
[0028] SEQ ID NO: 7 is the nucleotide sequence of PCR primer
PGTAXR2.
[0029] SEQ ID NO: 8 is the nucleotide sequence of PCR primer
8699TF6.
[0030] SEQ ID NO: 9 is the nucleotide sequence of PCR primer
8699TF8.
[0031] SEQ ID NO: 10 is the nucleotide sequence of PCR primer
PGTATA1+2R1.
[0032] SEQ ID NO: 11 is the nucleotide sequence of PCR primer
8699TF7.
[0033] SEQ ID NO: 12 is the nucleotide sequence of PCR primer
8699LF3.
[0034] SEQ ID NO: 13 is the nucleotide sequence of PCR primer
PGPBSR1n.
[0035] SEQ ID NO: 14 is the nucleotide sequence of PCR primer
8699LF4.
[0036] SEQ ID NO: 15 is an exemplary amino acid sequence of STLV-3
subtype D envelope polypeptide.
[0037] SEQ ID NO: 16 is an exemplary amino acid sequence of STLV-3
subtype D capsid polypeptide (Gag).
[0038] SEQ ID NO: 17 is an exemplary amino acid sequence of STLV-3
subtype D protease.
[0039] SEQ ID NO: 18 is an exemplary amino acid sequence of STLV-3
subtype D polymerase.
[0040] SEQ ID NO: 19 is an exemplary amino acid sequence of STLV-3
subtype D rex polypeptide.
[0041] SEQ ID NO: 20 is an exemplary amino acid sequence of STLV-3
subtype D tax polypeptide.
[0042] SEQ ID NO: 21 is the nucleotide sequence of PCR primer
P5TAXF3.
[0043] SEQ ID NO: 22 is the nucleotide sequence of PCR primer
P5TAXR3.
[0044] SEQ ID NO: 23 is the nucleotide sequence of PCR primer
P5TAXF2.
[0045] SEQ ID NO: 24 is the nucleotide sequence of PCR primer
P5TAXR1.
[0046] SEQ ID NO: 25 is the nucleotide sequence of a STLV-3 subtype
D tax gene formed from the spice of nucleotides 5054-5057 and
7232-8280 of SEQ ID NO: 1.
[0047] SEQ ID NO: 26 is the nucleotide sequence of a STLV-3 subtype
D rex gene formed from the spice of nucleotides 4995-5057 and
7232-7717 of SEQ ID NO: 1.
DETAILED DESCRIPTION
I. Abbreviations
[0048] CTL Cytotoxic T lymphocyte [0049] DBS Dried blood spots
[0050] DNA Deoxyribonucleic acid [0051] HIV Human immunodeficiency
virus [0052] HTLV Human T-cell lymphotropic virus, human T-cell
leukemia virus or human T-lymphotropic virus [0053] LTR Long
terminal repeat [0054] NHP Non-human primate [0055] PCR Polymerase
chain reaction [0056] PTLV Primate T-cell lymphotropic virus,
primate T-cell leukemia virus or primate T-lymphotropic virus
[0057] STLV Simian T-cell lymphotropic virus, simian T-cell
leukemia virus, or simian T-lymphotropic virus
II. Terms
[0058] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology can be found in Benjamin Lewin, Genes VII, published by
Oxford University Press, 1999; Kendrew et al. (eds.), The
Encyclopedia of Molecular Biology, published by Blackwell Science
Ltd., 1994; and Robert A. Meyers (ed.), Molecular Biology and
Biotechnology: a Comprehensive Desk Reference, published by VCH
Publishers, Inc., 1995; and other similar references.
[0059] As used herein, the singular forms "a," "an," and "the,"
refer to both the singular as well as plural, unless the context
clearly indicates otherwise. For example, the term "a probe"
includes single or plural probes and can be considered equivalent
to the phrase "at least one probe."
[0060] As used herein, the term "comprises" means "includes." Thus,
"comprising a probe" means "including a probe" without excluding
other elements.
[0061] It is further to be understood that all base sizes or amino
acid sizes, and all molecular weight or molecular mass values,
given for nucleic acids or polypeptides are approximate, and are
provided for descriptive purposes, unless otherwise indicated.
Although many methods and materials similar or equivalent to those
described herein can be used, particular suitable methods and
materials are described below. In case of conflict, the present
specification, including explanations of terms, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
[0062] To facilitate review of the various embodiments of the
invention, the following explanations of terms are provided:
[0063] Adjuvant: A vehicle used to enhance antigenicity; such as a
suspension of minerals (alum, aluminum hydroxide, aluminum
phosphate) on which antigen is adsorbed; or water-in-oil emulsion
in which antigen solution is emulsified in oil (MF-59, Freund's
incomplete adjuvant), sometimes with the inclusion of killed
mycobacteria (Freund's complete adjuvant) to further enhance
antigenicity (inhibits degradation of antigen and/or causes influx
of macrophages). Adjuvants also include immunostimulatory
molecules, such as cytokines, costimulatory molecules, and for
example, immunostimulatory DNA or RNA molecules, such as CpG
oligonucleotides.
[0064] Administration: The introduction of a composition into a
subject by a chosen route. For example, if the chosen route is
intravenous, the composition is administered by introducing the
composition into a vein of the subject.
[0065] Amplification: To increase the number of copies of a nucleic
acid molecule. The resulting amplification products are called
"amplicons." Amplification of a nucleic acid molecule (such as a
DNA or RNA molecule) refers to use of a technique that increases
the number of copies of a nucleic acid molecule in a sample, for
example the number of copies of a STLV-3 subtype D nucleic acid,
such as a STLV-3 subtype D env nucleic acid or fragment thereof. An
example of amplification is the polymerase chain reaction (PCR), in
which a sample is contacted with a pair of oligonucleotide primers
under conditions that allow for the hybridization of the primers to
a nucleic acid template in the sample. The primers are extended
under suitable conditions, dissociated from the template,
re-annealed, extended, and dissociated to amplify the number of
copies of the nucleic acid. This cycle can be repeated. The product
of amplification can be characterized by such techniques as
electrophoresis, restriction endonuclease cleavage patterns,
oligonucleotide hybridization or ligation, and/or nucleic acid
sequencing.
[0066] Other examples of in vitro amplification techniques include
quantitative real-time PCR; reverse transcriptase PCR (RT-PCR);
real-time PCR (rt PCR); real-time reverse transcriptase PCR (rt
RT-PCR); nested PCR; strand displacement amplification (see U.S.
Pat. No. 5,744,311); transcription-free isothermal amplification
(see U.S. Pat. No. 6,033,881, repair chain reaction amplification
(see WO 90/01069); ligase chain reaction amplification (see
European patent publication EP-A-320 308); gap filling ligase chain
reaction amplification (see U.S. Pat. No. 5,427,930); coupled
ligase detection and PCR (see U.S. Pat. No. 6,027,889); and
NASBA.TM. RNA transcription-free amplification (see U.S. Pat. No.
6,025,134) amongst others.
[0067] Animal: A living multi-cellular vertebrate or invertebrate
organism, a category that includes, for example, mammals and birds.
The term mammal includes both human and non-human mammals. The term
"primate" includes both human and non-human primates. "Non-human
primates" are simian primates such as monkeys, chimpanzees,
orangutans, baboons, and macaques. Similarly, the term "subject"
includes both human and veterinary subjects, such as non-human
primates.
[0068] Antibody: Immunoglobulin molecules and immunologically
active portions of immunoglobulin molecules, for instance,
molecules that contain an antigen binding site that specifically
binds (immunoreacts with) an antigen.
[0069] A naturally occurring antibody (for example, IgG, IgM, IgD)
includes four polypeptide chains, two heavy (H) chains and two
light (L) chains inter-connected by disulfide bonds. However, it
has been shown that the antigen-binding function of an antibody can
be performed by fragments of a naturally occurring antibody. Thus,
these antigen-binding fragments are also intended to be designated
by the term "antibody." Specific, non-limiting examples of binding
fragments encompassed within the term antibody include (i) an Fab
fragment consisting of the V.sub.L, V.sub.H, CL, and CH1 domains;
(ii) an Fd fragment consisting of the V.sub.H and CH1 domains;
(iii) an Fv fragment consisting of the V.sub.L and V.sub.H domains
of a single arm of an antibody, (iv) a dAb fragment (Ward et al.,
Nature 341:544-546, 1989) which consists of a V.sub.H domain; (v)
an isolated complementarity determining region (CDR); and (vi) an
F(ab').sub.2 fragment, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region.
[0070] Immunoglobulins and certain variants thereof are known and
many have been prepared in recombinant cell culture (for example,
see U.S. Pat. No. 4,745,055; U.S. Pat. No. 4,444,487; WO 88/03565;
EP 0256654; EP 0120694; EP 0125023; Faoulkner et al., Nature
298:286, 1982; Morrison, J. Immunol. 123:793, 1979; Morrison et
al., Ann Rev. Immunol 2:239, 1984).
[0071] Capsid: An outer shell of a virus, such as a STLV-3 subtype
D virus, comprising monomeric subunits of protein, such as capsid
monomers. Capsid polypeptides are encoded by a viral gag gene.
[0072] Cell: A plant, animal, insect, bacterial, or fungal
cell.
[0073] cDNA (complementary DNA): A piece of DNA lacking internal,
non-coding segments (introns) and transcriptional regulatory
sequences. cDNA also can contain untranslated regions (UTRs) that
are responsible for translational control in the corresponding RNA
molecule. cDNA can be synthesized in the laboratory by reverse
transcription from RNA, for example an RNA from STLV-3 subtype D,
such as an RNA encoding STLV-3 subtype D env.
[0074] Complementary: A double-stranded DNA or RNA strand consists
of two complementary strands of base pairs. Complementary binding
occurs when the base of one nucleic acid molecule forms a hydrogen
bond to the base of another nucleic acid molecule. Normally, the
base adenine (A) is complementary to thymidine (T) and uracil (U),
while cytosine (C) is complementary to guanine (G). For example,
the sequence 5'-ATCG-3' of one ssDNA molecule can bond to
3'-TAGC-5' of another ssDNA to form a dsDNA. In this example, the
sequence 5'-ATCG-3' is the reverse complement of 3'-TAGC-5'.
[0075] Nucleic acid molecules can be complementary to each other
even without complete hydrogen-bonding of all bases of each
molecule. For example, hybridization with a complementary nucleic
acid sequence can occur under conditions of differing stringency in
which a complement will bind at some but not all nucleotide
positions. In some examples, a nucleic acid molecule, such as
probes and primers specific for STLV-3 subtype D nucleic acid
disclosed herein, are complementary to a STLV-3 subtype D nucleic
acid molecule or the amplification products of such a nucleic acid
molecule.
[0076] Detect: To determine if an agent (such as a signal,
particular nucleotide, amino acid, nucleic acid molecule, and/or
organism, for example a virus) is present or absent, such as a
STLV-3 subtype D virus. In some examples, this can further include
quantification. The detection of a STLV-3 subtype D nucleic acid
molecule indicates the presence of STLV-3 subtype D virus in the
sample.
[0077] Degenerate variant and conservative variant: A
polynucleotide encoding a polypeptide or an antibody that includes
a sequence that is degenerate as a result of the genetic code. For
example, a polynucleotide encoding a STLV-3 subtype D polypeptide,
such as a STLV-3 subtype D envelope polypeptide, includes a
sequence that is degenerate as a result of the genetic code. There
are 20 natural amino acids, most of which are specified by more
than one codon. Therefore, all degenerate nucleotide sequences are
included as long as the amino acid sequence of the STLV-3 subtype D
polypeptide encoded by the nucleotide sequence is unchanged.
Because of the degeneracy of the genetic code, a large number of
functionally identical nucleic acids encode any given polypeptide.
For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all
encode the amino acid arginine. Thus, at every position where an
arginine is specified within a protein encoding sequence, the codon
can be altered to any of the corresponding codons described without
altering the encoded protein. Such nucleic acid variations are
"silent variations," which are one species of conservative
variations. Each nucleic acid sequence herein that encodes a
polypeptide also describes every possible silent variation. One of
skill will recognize that each codon in a nucleic acid (except AUG,
which is ordinarily the only codon for methionine) can be modified
to yield a functionally identical molecule by standard techniques.
Accordingly, each "silent variation" of a nucleic acid which
encodes a polypeptide is implicit in each described sequence.
[0078] Furthermore, one of ordinary skill will recognize that
individual substitutions, deletions or additions which alter, add
or delete a single amino acid or a small percentage of amino acids
(for instance less than 5%, such as less than 4%, less than 3%,
less than 2%, or even less than 1%) in an encoded sequence are
conservative variations where the alterations result in the
substitution of an amino acid with a chemically similar amino
acid.
[0079] Conservative amino acid substitutions providing functionally
similar amino acids are well known in the art. The following six
groups each contain amino acids that are conservative substitutions
for one another:
[0080] 1) Alanine (A), Serine (S), Threonine (T);
[0081] 2) Aspartic acid (D), Glutamic acid (E);
[0082] 3) Asparagine (N), Glutamine (Q);
[0083] 4) Arginine (R), Lysine (K);
[0084] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
and
[0085] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0086] Not all residue positions within a protein will tolerate an
otherwise "conservative" substitution. For instance, if an amino
acid residue is essential for a function of the protein, even an
otherwise conservative substitution may disrupt that activity.
[0087] Emission or emission signal: The light of a particular
wavelength generated from a source. In particular examples, an
emission signal is emitted from a fluorophore after the fluorophore
absorbs light at its excitation wavelengths.
[0088] Envelope or Envelope proteins: Viral envelopes typically
include some viral glycoproteins (envelope proteins). Functionally,
viral envelopes, such as STLV-3 subtype D envelopes are used to
help viruses enter host cells. Glycoproteins on the surface of the
envelope serve to identify and bind to receptor sites on the host's
membrane. The viral envelope then fuses with the host cell's
membrane, allowing the capsid and viral genome to enter and infect
the host cell.
[0089] Epitope: An antigenic determinant. These are particular
chemical groups or peptide sequences on a molecule that are
antigenic, such that they elicit a specific immune response. An
antibody binds a particular antigenic epitope, such as an epitope
of a STLV-3 subtype D polypeptide, for example an epitope of a
STLV-3 subtype D envelope polypeptide.
[0090] Excitation or excitation signal: The light of a particular
wavelength necessary and/or sufficient to excite an electron
transition to a higher energy level. In particular examples, an
excitation is the light of a particular wavelength necessary and/or
sufficient to excite a fluorophore to a state such that the
fluorophore will emit a different (such as a longer) wavelength of
light then the wavelength of light from the excitation signal.
[0091] Expression: Translation of a nucleic acid into a protein,
for example the translation of a STLV-3 subtype D mRNA into a
protein. This includes the translation of the nucleic acid set
forth as nucleotides 747-2009 of SEQ ID NO: 1, 1961-2494 of SEQ ID
NO: 1, 2416-5061 of SEQ ID NO: 1, 5054-6535 of SEQ ID NO: 1.
5054-5057 and 7232-8280 of SEQ ID NO: 1, or 4995-5057 and 7232-7717
of SEQ ID NO: 1.
[0092] Expression Control Sequences: Nucleic acid sequences that
regulate the expression of a heterologous nucleic acid sequence to
which it is operatively linked, for example the expression of a
STLV-3 subtype D nucleic acid encoding a protein operably linked to
expression control sequences. Expression control sequences are
operatively linked to a nucleic acid sequence when the expression
control sequences control and regulate the transcription and, as
appropriate, translation of the nucleic acid sequence. Thus
expression control sequences can include appropriate promoters,
enhancers, transcription terminators, a start codon (ATG) in front
of a protein-encoding gene, splicing signal for introns,
maintenance of the correct reading frame of that gene to permit
proper translation of mRNA, and stop codons. The term "control
sequences" is intended to include, at a minimum, components whose
presence can influence expression, and can also include additional
components whose presence is advantageous, for example, leader
sequences and fusion partner sequences. Expression control
sequences can include a promoter.
[0093] A promoter is a minimal sequence sufficient to direct
transcription. Also included are those promoter elements which are
sufficient to render promoter-dependent gene expression
controllable for cell-type specific, tissue-specific, or inducible
by external signals or agents; such elements may be located in the
5' or 3' regions of the gene. Both constitutive and inducible
promoters are included (see for example, Bitter et al., Methods in
Enzymology 153:516-544, 1987). For example, when cloning in
bacterial systems, inducible promoters such as pL of bacteriophage
lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like
may be used. In one embodiment, when cloning in mammalian cell
systems, promoters derived from the genome of mammalian cells (such
as metallothionein promoter) or from mammalian viruses (such as the
retrovirus long terminal repeat; the adenovirus late promoter; the
vaccinia virus 7.5K promoter) can be used. Promoters produced by
recombinant DNA or synthetic techniques may also be used to provide
for transcription of the nucleic acid sequences.
[0094] A polynucleotide can be inserted into an expression vector
that contains a promoter sequence, which facilitates the efficient
transcription of the inserted genetic sequence of the host. The
expression vector typically contains an origin of replication, a
promoter, as well as specific nucleic acid sequences that allow
phenotypic selection of the transformed cells.
[0095] Fluorophore: A chemical compound, which when excited by
exposure to a particular stimulus, such as a defined wavelength of
light, emits light (fluoresces), for example at a different
wavelength (such as a longer wavelength of light).
[0096] Fluorophores are part of the larger class of luminescent
compounds. Luminescent compounds include chemiluminescent
molecules, which do not require a particular wavelength of light to
luminesce, but rather use a chemical source of energy. Therefore,
the use of chemiluminescent molecules (such as aequorin) can
eliminate the need for an external source of electromagnetic
radiation, such as a laser.
[0097] Examples of particular fluorophores that can be used in the
probes and primers disclosed herein are provided in U.S. Pat. No.
5,866,366 to Nazarenko et al., such as
4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid, acridine
and derivatives such as acridine and acridine isothiocyanate,
5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),
4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate
(Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide,
anthranilamide, Brilliant Yellow, coumarin and derivatives such as
coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),
7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine;
4',6-diaminidino-2-phenylindole (DAPI);
5',5''-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin;
diethylenetriamine pentaacetate;
4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid;
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid;
5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl
chloride); 4-dimethylaminophenylazophenyl-4'-isothiocyanate
(DABITC); eosin and derivatives such as eosin and eosin
isothiocyanate; erythrosin and derivatives such as erythrosin B and
erythrosin isothiocyanate; ethidium; fluorescein and derivatives
such as 5-carboxyfluorescein (FAM),
5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),
2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE),
fluorescein, fluorescein isothiocyanate (FITC), and QFITC (XRITC);
fluorescamine; IR144; IR1446; Malachite Green isothiocyanate;
4-methylumbelliferone; ortho cresolphthalein; nitrotyro sine;
pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde;
pyrene and derivatives such as pyrene, pyrene butyrate and
succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron.TM.
Brilliant Red 3B-A); rhodamine and derivatives such as
6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine
rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B,
rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B,
sulforhodamine 101 and sulfonyl chloride derivative of
sulforhodamine 101 (Texas Red);
N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl
rhodamine; tetramethyl rhodamine isothiocyanate (TRITC);
riboflavin; rosolic acid and terbium chelate derivatives;
LightCycler Red 640; Cy5.5; and Cy56-carboxyfluorescein;
5-carboxyfluorescein (5-FAM); boron dipyrromethene difluoride
(BODIPY); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA);
acridine, stilbene, -6-carboxy-fluorescein (HEX), TET (Tetramethyl
fluorescein), 6-carboxy-X-rhodamine (ROX), Texas Red,
2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), Cy3,
Cy5, VIC.RTM. (Applied Biosystems), LC Red 640, LC Red 705, Yakima
yellow amongst others.
[0098] Other suitable fluorophores include those known to those
skilled in the art, for example those available from Molecular
Probes (Eugene, Oreg.). In particular examples, a fluorophore is
used as a donor fluorophore or as an acceptor fluorophore.
[0099] "Acceptor fluorophores" are fluorophores which absorb energy
from a donor fluorophore, for example in the range of about 400 to
900 nm (such as in the range of about 500 to 800 nm). Acceptor
fluorophores generally absorb light at a wavelength which is
usually at least 10 nm higher (such as at least 20 nm higher), than
the maximum absorbance wavelength of the donor fluorophore, and
have a fluorescence emission maximum at a wavelength ranging from
about 400 to 900 nm. Acceptor fluorophores have an excitation
spectrum overlapping with the emission of the donor fluorophore,
such that energy emitted by the donor can excite the acceptor.
Ideally, an acceptor fluorophore is capable of being attached to a
nucleic acid molecule.
[0100] In a particular example, an acceptor fluorophore is a dark
quencher, such as, Dabcyl, QSY7 (Molecular Probes), QSY33
(Molecular Probes), BLACK HOLE QUENCHERS.TM. (Glen Research),
ECLIPSE.TM. Dark Quencher (Epoch Biosciences), IOWA BLACK.TM.
(Integrated DNA Technologies). A quencher can reduce or quench the
emission of a donor fluorophore. In such an example, instead of
detecting an increase in emission signal from the acceptor
fluorophore when in sufficient proximity to the donor fluorophore
(or detecting a decrease in emission signal from the acceptor
fluorophore when a significant distance from the donor
fluorophore), an increase in the emission signal from the donor
fluorophore can be detected when the quencher is a significant
distance from the donor fluorophore (or a decrease in emission
signal from the donor fluorophore when in sufficient proximity to
the quencher acceptor fluorophore).
[0101] "Donor Fluorophores" are fluorophores or luminescent
molecules capable of transferring energy to an acceptor
fluorophore, thereby generating a detectable fluorescent signal
from the acceptor. Donor fluorophores are generally compounds that
absorb in the range of about 300 to 900 nm, for example about 350
to 800 nm. Donor fluorophores have a strong molar absorbance
coefficient at the desired excitation wavelength, for example
greater than about 10.sup.3 M.sup.-1 cm.sup.-1.
[0102] Fluorescence Resonance Energy Transfer (FRET): A
spectroscopic process by which energy is passed between an
initially excited donor to an acceptor molecule separated by 10-100
.ANG.. The donor molecules typically emit at shorter wavelengths
that overlap with the absorption of the acceptor molecule. The
efficiency of energy transfer is proportional to the inverse sixth
power of the distance (R) between the donor and acceptor
(1/R.sup.6) fluorophores and occurs without emission of a photon.
In applications using FRET, the donor and acceptor dyes are
different, in which case FRET can be detected either by the
appearance of sensitized fluorescence of the acceptor or by
quenching of donor fluorescence. For example, if the donor's
fluorescence is quenched it indicates the donor and acceptor
molecules are within the Forster radius (the distance where FRET
has 50% efficiency, about 20-60 .ANG.), whereas if the donor
fluoresces at its characteristic wavelength, it denotes that the
distance between the donor and acceptor molecules has increased
beyond the Forster radius. In another example, energy is
transferred via FRET between two different fluorophores such that
the acceptor molecule can emit light at its characteristic
wavelength, which is always longer than the emission wavelength of
the donor molecule.
[0103] Host cells: Cells in which a vector can be propagated and
its nucleic acids expressed. The cell may be prokaryotic or
eukaryotic. The term also includes any progeny of the subject host
cell. It is understood that all progeny may not be identical to the
parental cell since there may be mutations that occur during
replication. However, such progeny are included when the term "host
cell" is used.
[0104] Hybridization: The ability of complementary single-stranded
DNA or RNA to form a duplex molecule (also referred to as a
hybridization complex). Nucleic acid hybridization techniques can
be used to form hybridization complexes between a probe or primer
and a nucleic acid, such as a STLV-3 subtype D nucleic acid
molecule. For example, a probe or primer having some homology to a
STLV-3 subtype D nucleic acid molecule will form a hybridization
complex with a STLV-3 subtype D nucleic acid molecule (such as any
of the nucleic acids set forth as nucleotides 747-2009 of SEQ ID
NO: 1, 1961-2494 of SEQ ID NO: 1, 2416-5061 of SEQ ID NO: 1, or
5054-6535 of SEQ ID NO: 1, 5054-5057 of SEQ ID NO: 1, 7232-8280 of
SEQ ID NO: 1, 4995-5057 of SEQ ID NO: 1, or 7232-7717 of SEQ ID NO:
1).
[0105] Hybridization conditions resulting in particular degrees of
stringency will vary depending upon the nature of the hybridization
method and the composition and length of the hybridizing nucleic
acid sequences. Generally, the temperature of hybridization and the
ionic strength (such as the Na+ concentration) of the hybridization
buffer will determine the stringency of hybridization. Calculations
regarding hybridization conditions for attaining particular degrees
of stringency are discussed in Sambrook et al., (1989) Molecular
Cloning, second edition, Cold Spring Harbor Laboratory, Plainview,
N.Y. (chapters 9 and 11). The following is an exemplary set of
hybridization conditions and is not limiting:
[0106] Very High Stringency (Detects Sequences that Share at Least
90% Identity)
[0107] Hybridization: 5.times.SSC at 65.degree. C. for 16 hours
[0108] Wash twice: 2.times.SSC at room temperature (RT) for 15
minutes each
[0109] Wash twice: 0.5.times.SSC at 65.degree. C. for 20 minutes
each
[0110] High Stringency (Detects Sequences that Share at Least 80%
Identity)
[0111] Hybridization: 5.times.-6.times.SSC at 65.degree.
C.-70.degree. C. for 16-20 hours
[0112] Wash twice: 2.times.SSC at RT for 5-20 minutes each
[0113] Wash twice: 1.times.SSC at 55.degree. C.-70.degree. C. for
30 minutes each
[0114] Low Stringency (Detects Sequences that Share at Least 50%
Identity)
[0115] Hybridization: 6.times.SSC at RT to 55.degree. C. for 16-20
hours
[0116] Wash at least twice: 2.times.-3.times.SSC at RT to
55.degree. C. for 20-30 minutes each.
[0117] Immune response: A response of a cell of the immune system,
such as a B cell, T cell, or monocyte, to a stimulus. In one
embodiment, the response is specific for a particular antigen (an
"antigen-specific response"), such as an antigen from a STLV-3
subtype D virus. In one embodiment, an immune response is a T cell
response, such as a CD4+ response or a CD8+ response. In another
embodiment, the response is a B cell response, and results in the
production of specific antibodies, for example antibodies specific
for the antigen, such as a STLV-3 subtype D viral antigen.
[0118] Immunogenic peptide: A peptide which comprises an
allele-specific motif or other sequence, such as an N-terminal
repeat, such that the peptide will bind an MHC molecule and induce
a cytotoxic T lymphocyte ("CTL") response, or a B cell response
(for example antibody production) against the antigen from which
the immunogenic peptide is derived.
[0119] In one embodiment, immunogenic peptides are identified using
sequence motifs or other methods, such as neural net or polynomial
determinations known in the art. Typically, algorithms are used to
determine the "binding threshold" of peptides to select those with
scores that give them a high probability of binding at a certain
affinity and will be immunogenic. The algorithms are based either
on the effects on MHC binding of a particular amino acid at a
particular position, the effects on antibody binding of a
particular amino acid at a particular position, or the effects on
binding of a particular substitution in a motif-containing peptide.
Within the context of an immunogenic peptide, a "conserved residue"
is one which appears in a significantly higher frequency than would
be expected by random distribution at a particular position in a
peptide. In one embodiment, a conserved residue is one where the
MHC structure may provide a contact point with the immunogenic
peptide. In some specific non-limiting examples, an immunogenic
polypeptide includes a region of STLV-3 subtype D polypeptide, such
as a STLV-3 subtype D envelope polypeptide, a STLV-3 subtype D
capsid polypeptide, a STLV-3 subtype D polymerase, a STLV-3 subtype
D rex polypeptide, a STLV-3 subtype D tax polypeptide, a STLV-3
subtype D protease, or a fragment thereof.
[0120] Immunogenic composition: A composition comprising an
immunogenic peptide that induces a measurable CTL response against
virus expressing the immunogenic peptide, or induces a measurable B
cell response (such as production of antibodies) against the
immunogenic peptide. In one example, an "immunogenic composition"
is composition comprising a STLV-3 subtype D polypeptide that
induces a measurable CTL or B cell response against virus
expressing STLV-3 subtype D polypeptide, such as a STLV-3 subtype D
envelope polypeptide, or induces a measurable B cell response (such
as production of antibodies) against a STLV-3 subtype D
polypeptide, such as a STLV-3 subtype D envelope polypeptide. It
further refers to isolated nucleic acids encoding an immunogenic
peptide, such as a nucleic acid that can be used to express the
STLV-3 subtype D polypeptide, such as a STLV-3 subtype D envelope
polypeptide (and thus be used to elicit an immune response against
this polypeptide).
[0121] For in vitro use, an immunogenic composition may consist of
the isolated protein, peptide epitope, or nucleic acid encoding the
protein, or peptide epitope. For in vivo use, the immunogenic
composition will typically comprise the protein or immunogenic
peptide in pharmaceutically acceptable carriers, and/or other
agents. Any particular peptide, such as a STLV-3 subtype D
polypeptide, such as a STLV-3 subtype D envelope polypeptide, or
nucleic acid encoding the polypeptide, can be readily tested for
its ability to induce a CTL or B cell response by art-recognized
assays. Immunogenic compositions can include adjuvants, which are
well known to one of skill in the art.
[0122] Immunologically reactive conditions: Includes reference to
conditions which allow an antibody raised against a particular
STLV-3 subtype D epitope, to bind to that epitope to a detectably
greater degree than, and/or to the substantial exclusion of,
binding to substantially all other epitopes. Immunologically
reactive conditions are dependent upon the format of the antibody
binding reaction and typically are those utilized in immunoassay
protocols or those conditions encountered in vivo. The
immunologically reactive conditions employed in the methods are
"physiological conditions" which include reference to conditions
(such as temperature, osmolarity, pH) that are typical inside a
living mammal or a mammalian cell. While it is recognized that some
organs are subject to extreme conditions, the intra-organismal and
intracellular environment is normally about pH 7 (such as from pH
6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the
predominant solvent, and exists at a temperature above 0.degree. C.
and below 50.degree. C. Osmolarity is within the range that is
supportive of cell viability and proliferation.
[0123] Immunotherapy: A method of evoking an immune response
against on their production of target antigens. Immunotherapy based
on cell-mediated immune responses involves generating a
cell-mediated response to cells that produce particular antigenic
determinants, while immunotherapy based on humoral immune responses
involves generating specific antibodies to virus that produce
particular antigenic determinants.
[0124] Inhibiting or treating a disease: Inhibiting the full
development of a disease or condition, for example, in a subject
who is at risk for a disease such as T-cell leukemia, STLV-3
subtype D viral infection, or combinations thereof. "Treatment"
refers to a therapeutic intervention that ameliorates a sign or
symptom of a disease or pathological condition after it has begun
to develop. The term "ameliorating," with reference to a disease or
pathological condition, refers to any observable beneficial effect
of the treatment. The beneficial effect can be evidenced, for
example, by a delayed onset of clinical symptoms of the disease in
a susceptible subject, a reduction in severity of some or all
clinical symptoms of the disease, a slower progression of the
disease, a reduction in the number of metastases, an improvement in
the overall health or well-being of the subject, or by other
parameters well known in the art that are specific to the
particular disease. A "prophylactic" treatment is a treatment
administered to a subject who does not exhibit signs of a disease
or exhibits only early signs for the purpose of decreasing the risk
of developing pathology.
[0125] Isolated: An "isolated" biological component (such as a
protein or a nucleic acid) has been substantially separated or
purified away from other biological components in which the
component naturally occurs, such as other chromosomal and
extrachromosomal DNA, RNA, and proteins. Nucleic acids or proteins
that have been "isolated" include nucleic acids or proteins
purified by standard purification methods. The term also embraces
nucleic acids or proteins prepared by recombinant expression in a
host cell as well as chemically synthesized nucleic acids or
proteins. Isolated does not require absolute purity, and can
include nucleic acid or protein molecules that are at least 50%
isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even
100% isolated.
[0126] Label: An agent capable of detection, for example by
spectrophotometry, flow cytometry, or microscopy. For example, a
label can be attached to a nucleotide, thereby permitting detection
of the nucleotide, such as detection of the nucleic acid molecule
of which the nucleotide is a part, such as a STLV-3 subtype D
specific probe or primer. Labels can also be attached to
antibodies. Examples of labels include, but are not limited to,
radioactive isotopes, enzyme substrates, co-factors, ligands,
chemiluminescent agents, fluorophores, haptens, enzymes, and
combinations thereof. Methods for labeling and guidance in the
choice of labels appropriate for various purposes are discussed for
example in Sambrook et al. (Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor, N.Y., 1989) and Ausubel et al. (In Current
Protocols in Molecular Biology, John Wiley & Sons, New York,
1998).
[0127] Nucleic acid (molecule or sequence): A deoxyribonucleotide
or ribonucleotide polymer including without limitation, cDNA, mRNA,
genomic DNA, and synthetic (such as chemically synthesized) DNA or
RNA. The nucleic acid can be double stranded (ds) or single
stranded (ss). Where single stranded, the nucleic acid can be the
sense strand or the antisense strand. Nucleic acids can include
natural nucleotides (such as A, T/U, C, and G), and can include
analogs of natural nucleotides, such as labeled nucleotides. In
some examples, a nucleic acid is a STLV-3 subtype D nucleic acid,
which can include nucleic acids purified from a STLV-3 subtype D as
well as the amplification products of such nucleic acids.
[0128] Nucleotide: The fundamental unit of nucleic acid molecules.
A nucleotide includes a nitrogen-containing base attached to a
pentose monosaccharide with one, two, or three phosphate groups
attached by ester linkages to the saccharide moiety.
[0129] The major nucleotides of DNA are deoxyadenosine
5'-triphosphate (dATP or A), deoxyguanosine 5'-triphosphate (dGTP
or G), deoxycytidine 5'-triphosphate (dCTP or C) and deoxythymidine
5'-triphosphate (dTTP or T). The major nucleotides of RNA are
adenosine 5'-triphosphate (ATP or A), guanosine 5'-triphosphate
(GTP or G), cytidine 5'-triphosphate (CTP or C) and uridine
5'-triphosphate (UTP or U).
[0130] Nucleotides include those nucleotides containing modified
bases, modified sugar moieties and modified phosphate backbones,
for example as described in U.S. Pat. No. 5,866,336 to Nazarenko et
al.
[0131] Examples of modified base moieties which can be used to
modify nucleotides at any position on its structure include, but
are not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, acetylcytosine,
5-(carboxyhydroxylmethyl)uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N.about.6-sopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N.about.6-isopentenyladenine,
uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, and
2,6-diaminopurine amongst others.
[0132] Examples of modified sugar moieties, which may be used to
modify nucleotides at any position on its structure, include, but
are not limited to arabinose, 2-fluoroarabinose, xylose, and
hexose, or a modified component of the phosphate backbone, such as
phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a
phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl
phosphotriester, or a formacetal or analog thereof.
[0133] Oligonucleotide: A linear polynucleotide sequence of up to
about 200 nucleotide bases in length, for example a polynucleotide
(such as DNA or RNA) which is at least 6 nucleotides, for example
at least 15, 25, 50, 100 or even 200 nucleotides long.
[0134] Operably linked: A first nucleic acid sequence is operably
linked with a second nucleic acid sequence when the first nucleic
acid sequence is placed in a functional relationship with the
second nucleic acid sequence. For instance, a promoter is operably
linked to a coding sequence if the promoter affects the
transcription or expression of the coding sequence. Generally,
operably linked DNA sequences are contiguous and, where necessary
to join two protein coding regions, in the same reading frame. In
some examples disclosed herein, a promoter is operably linked to a
STLV-3 subtype D nucleic acid.
[0135] ORF: Open reading frame. Contains a series of nucleotide
triplets (codons) coding for amino acids without any termination
codons. These sequences are usually translatable into protein.
[0136] Pharmaceutical agent: A chemical compound or composition
capable of inducing a desired therapeutic or prophylactic effect
when properly administered to a subject or a cell. "Incubating"
includes a sufficient amount of time for a drug to interact with a
cell. An "anti-viral agent" or "anti-viral drug" is an agent that
specifically inhibits a virus from replicating or infecting cells.
Similarly, an "anti-retroviral agent" is an agent that specifically
inhibits a retrovirus from replicating or infecting cells.
[0137] A "therapeutically effective amount" is a quantity of a
chemical composition or an anti-viral agent sufficient to achieve a
desired effect in a subject being treated. For instance, this can
be the amount necessary to inhibit viral replication or to
measurably alter outward symptoms of the viral infection. In
general, this amount will be sufficient to measurably inhibit virus
(for example STLV-3 subtype D) replication or infectivity. When
administered to a subject, a dosage will generally be used that
will achieve target tissue concentrations that has been shown to
achieve in vitro inhibition of viral replication.
[0138] Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers of use are conventional. Remington's
Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co.,
Easton, Pa., 15th Edition, 1975, describes compositions and
formulations suitable for pharmaceutical delivery of the fusion
proteins herein disclosed.
[0139] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol, or the like as a vehicle. For solid
compositions (such as powder, pill, tablet, or capsule forms),
conventional non-toxic solid carriers can include, for example,
pharmaceutical grades of mannitol, lactose, starch, or magnesium
stearate. In addition to biologically neutral carriers,
pharmaceutical compositions to be administered can contain minor
amounts of non-toxic auxiliary substances, such as wetting or
emulsifying agents, preservatives, and pH buffering agents and the
like, for example sodium acetate or sorbitan monolaurate.
[0140] Polypeptide: Any chain of amino acids, regardless of length
or post-translational modification (such as glycosylation or
phosphorylation). "Polypeptide" applies to amino acid polymers to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer as well as in which one or more amino acid
residue is a non-natural amino acid, for example a artificial
chemical mimetic of a corresponding naturally occurring amino acid.
In some embodiments, the polypeptide is a STLV-3 subtype D
polypeptide. A "residue" refers to an amino acid or amino acid
mimetic incorporated in a polypeptide by an amide bond or amide
bond mimetic. A polypeptide has an amino terminal (N-terminal) end
and a carboxy terminal (C-terminal) end. "Polypeptide" is used
interchangeably with peptide or protein, and is used
interchangeably herein to refer to a polymer of amino acid
residues.
[0141] Primer: A short nucleic acid molecule, such as a DNA
oligonucleotide, for example sequences of at least 15 nucleotides,
which can be annealed to a complementary target nucleic acid
molecule by nucleic acid hybridization to form a hybrid between the
primer and the target nucleic acid strand. A primer can be extended
along the target nucleic acid molecule by a polymerase enzyme.
Therefore, primers can be used to amplify a target nucleic acid
molecule (such as a portion of a STLV-3 subtype D nucleic acid
molecule). In some examples, the primers amplify a portion of a
STLV-3 subtype D nucleic acid molecule as set forth as nucleotides
747-2009 of SEQ ID NO: 1, 1961-2494 of SEQ ID NO: 1, 2416-5061 of
SEQ ID NO: 1, or 5054-6535 of SEQ ID NO: 1, 5054-5057 of SEQ ID NO:
1, 7232-8280 of SEQ ID NO: 1, 4995-5057 of SEQ ID NO: 1, or
7232-7717 of SEQ ID NO: 1, wherein the sequence of the primer is
specific for the target nucleic acid molecule, for example so that
the primer will hybridize to the target nucleic acid molecule under
very high stringency hybridization conditions.
[0142] The specificity of a primer increases with its length. Thus,
for example, a primer that includes 30 consecutive nucleotides will
anneal to a target sequence with a higher specificity than a
corresponding primer of only 15 nucleotides. Thus, to obtain
greater specificity, probes and primers can be selected that
include at least 15, 20, 25, 30, 35, 40, 45, 50 or more consecutive
nucleotides.
[0143] In particular examples, a primer is at least 15 nucleotides
in length, such as at least 15 contiguous nucleotides complementary
to a target nucleic acid molecule. Particular lengths of primers
that can be used to practice the methods of the present disclosure
(for example, to amplify a region of a STLV-3 subtype D nucleic
acid molecule) include primers having at least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, at least 21, at
least 22, at least 23, at least 24, at least 25, at least 26, at
least 27, at least 28, at least 29, at least 30, at least 31, at
least 32, at least 33, at least 34, at least 35, at least 36, at
least 37, at least 38, at least 39, at least 40, at least 45, at
least 50, or more contiguous nucleotides complementary to the
target nucleic acid molecule to be amplified, such as a primer of
15-60 nucleotides, 15-50 nucleotides, or 15-30 nucleotides.
[0144] Primer pairs can be used for amplification of a nucleic acid
sequence, for example, by PCR, real-time PCR, or other nucleic-acid
amplification methods known in the art. An "upstream" or "forward"
primer is a primer 5' to a reference point on a nucleic acid
sequence. A "downstream" or "reverse" primer is a primer 3' to a
reference point on a nucleic acid sequence. In general, at least
one forward and one reverse primer are included in an amplification
reaction. PCR primer pairs can be derived from a known sequence
(such as the STLV-3 subtype D nucleic acid molecules as set forth
as nucleotides 747-2009 of SEQ ID NO: 1, 1961-2494 of SEQ ID NO: 1,
2416-5061 of SEQ ID NO: 1, or 5054-6535 of SEQ ID NO: 1, 5054-5057
of SEQ ID NO: 1, 7232-8280 of SEQ ID NO: 1, 4995-5057 of SEQ ID NO:
1, or 7232-7717 of SEQ ID NO: 1), for example, by using computer
programs intended for that purpose such as Primer (Version 0.5,
.COPYRGT. 1991, Whitehead Institute for Biomedical Research,
Cambridge, Mass.) or PRIMER EXPRESS.RTM. Software (Applied
Biosystems, AB, Foster City, Calif.).
[0145] Methods for preparing and using primers are described in,
for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor, N.Y.; Ausubel et al. (1987) Current
Protocols in Molecular Biology, Greene Publ. Assoc. &
Wiley-Intersciences. In one example, a primer includes a label.
[0146] Probe: A probe comprises an isolated nucleic acid capable of
hybridizing to a target nucleic acid (such as a STLV-3 subtype D
nucleic acid molecule). A detectable label or reporter molecule can
be attached to a probe. Typical labels include radioactive
isotopes, enzyme substrates, co-factors, ligands, chemiluminescent
or fluorescent agents, haptens, and enzymes.
[0147] Methods for labeling and guidance in the choice of labels
appropriate for various purposes are discussed, for example, in
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press (1989) and Ausubel et al., Current
Protocols in Molecular Biology, Greene Publishing Associates and
Wiley-Intersciences (1987).
[0148] In a particular example, a probe includes at least one
fluorophore, such as an acceptor fluorophore or donor fluorophore.
For example, a fluorophore can be attached at the 5'- or 3'-end of
the probe. In specific examples, the fluorophore is attached to the
base at the 5'-end of the probe, the base at its 3'-end, the
phosphate group at its 5'-end or a modified base, such as a T
internal to the probe.
[0149] Probes are generally at least 15 nucleotides in length, such
as at least 15, at least 16, at least 17, at least 18, at least 19,
least 20, at least 21, at least 22, at least 23, at least 24, at
least 25, at least 26, at least 27, at least 28, at least 29, at
least 30, at least 31, at least 32, at least 33, at least 34, at
least 35, at least 36, at least 37, at least 38, at least 39, at
least 40, at least 41, at least 42, at least 43, at least 44, at
least 45, at least 46, at least 47, at least 48, at least 49, at
least 50 at least 51, at least 52, at least 53, at least 54, at
least 55, at least 56, at least 57, at least 58, at least 59, at
least 60, or more contiguous nucleotides complementary to the
target nucleic acid molecule, such as 20-60 nucleotides, 20-50
nucleotides, 20-40 nucleotides, or 20-30 nucleotides.
[0150] Protease: A protease is any enzyme that hydrolyses the
peptide bonds that link amino acids together in a polypeptide
chain. Viral proteases, such as a STLV-3 subtype D protease,
hydrolyses the peptide bonds that individual viral polypeptides
together.
[0151] Polymerizing agent: A compound capable of reacting monomer
molecules (such as nucleotides) together in a chemical reaction to
form linear chains or a three-dimensional network of polymer
chains. A particular example of a polymerizing agent is polymerase,
an enzyme, which catalyzes the 5' to 3' elongation of a primer
strand complementary to a nucleic acid template. Examples of
polymerases that can be used to amplify a nucleic acid molecule
include, but are not limited to the E. coli DNA polymerase I,
specifically the Klenow fragment which has 3' to 5' exonuclease
activity, Taq polymerase, reverse transcriptase (such as HIV-1 RT),
E. coli RNA polymerase, and wheat germ RNA polymerase II.
[0152] The choice of polymerase is dependent on the nucleic acid to
be amplified. If the template is a single-stranded DNA molecule, a
DNA-directed DNA or RNA polymerase can be used; if the template is
a single-stranded RNA molecule, then a reverse transcriptase (such
as an RNA-directed DNA polymerase) can be used.
[0153] Quantitating a nucleic acid molecule: Determining or
measuring a quantity (such as a relative quantity) of nucleic acid
molecules present, such as the number of amplicons or the number of
nucleic acid molecules present in a sample. In particular examples,
it is determining the relative amount or actual number of nucleic
acid molecules present in a sample, such as STLV-3 subtype D
nucleic acid molecules present in a sample.
[0154] Recombinant: A recombinant nucleic acid is one that has a
sequence that is not naturally occurring or has a sequence that is
made by an artificial combination of two otherwise separated
segments of sequence. This artificial combination is often
accomplished by chemical synthesis or, more commonly, by the
artificial manipulation of isolated segments of nucleic acids, for
example, by genetic engineering techniques.
[0155] Sample: A sample, such as a biological sample, is a sample
obtained from a plant or animal subject. As used herein, biological
samples include all clinical samples useful for detection STLV-3
subtype D viral infection in subjects, including, but not limited
to, cells, tissues, and bodily fluids, such as: blood; derivatives
and fractions of blood, such as serum; extracted galls; biopsied or
surgically removed tissue, including tissues that are, for example,
unfixed, frozen, fixed in formalin and/or embedded in paraffin;
tears; milk; skin scrapes; surface washings; urine; sputum;
cerebrospinal fluid; prostate fluid; pus; or bone marrow aspirates.
In particular, embodiments, the biological sample is obtained from
a subject, such as in the form of blood.
[0156] Sequence identity/similarity: The identity/similarity
between two or more nucleic acid sequences, or two or more amino
acid sequences, is expressed in terms of the identity or similarity
between the sequences. Sequence identity can be measured in terms
of percentage identity; the higher the percentage, the more
identical the sequences are. Homologs or orthologs of nucleic acid
or amino acid sequences possess a relatively high degree of
sequence identity/similarity when aligned using standard
methods.
[0157] Methods of alignment of sequences for comparison are well
known in the art. Various programs and alignment algorithms are
described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981;
Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson &
Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins &
Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3,
1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et
al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson
et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol.
Biol. 215:403-10, 1990, presents a detailed consideration of
sequence alignment methods and homology calculations.
[0158] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul
et al., J. Mol. Biol. 215:403-10, 1990) is available from several
sources, including the National Center for Biological Information
(NCBI, National Library of Medicine, Building 38A, Room 8N805,
Bethesda, Md. 20894) and on the Internet, for use in connection
with the sequence analysis programs blastp, blastn, blastx,
tblastn, and tblastx. Blastn is used to compare nucleic acid
sequences, while blastp is used to compare amino acid sequences.
Additional information can be found at the NCBI web site.
[0159] Once aligned, the number of matches is determined by
counting the number of positions where an identical nucleotide or
amino acid residue is present in both sequences. The percent
sequence identity is determined by dividing the number of matches
either by the length of the sequence set forth in the identified
sequence, or by an articulated length (such as 100 consecutive
nucleotides or amino acid residues from a sequence set forth in an
identified sequence), followed by multiplying the resulting value
by 100. For example, a nucleic acid sequence that has 1166 matches
when aligned with a test sequence having 1554 nucleotides is 75.0
percent identical to the test sequence (1166/1554*100=75.0). The
percent sequence identity value is rounded to the nearest tenth.
For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to
75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to
75.2. The length value will always be an integer. In another
example, a target sequence containing a 20-nucleotide region that
aligns with 20 consecutive nucleotides from an identified sequence
as follows contains a region that shares 75 percent sequence
identity to that identified sequence (i.e., 15/20*100=75).
##STR00001##
[0160] One indication that two nucleic acid molecules are closely
related is that the two molecules hybridize to each other under
stringent conditions. Stringent conditions are sequence-dependent
and are different under different environmental parameters.
[0161] Simian T-cell lymphotropic virus (STLV): Simian
single-stranded RNA delataretroviruses known to infect primates.
Closely related viruses include Human T-cell lymphotropic virus
type (HTLV) and bovine leukemia virus (BLV). Simian and human T
cell leukemia virus (STLV and HTLV) are important pathogens causing
life-long chronic infections that may lead to T-cell
leukemia/lymphoma (ATLL) and a variety of neuromuscular
diseases.
[0162] Target nucleic acid molecule: A nucleic acid molecule whose
detection, quantitation, qualitative detection, or a combination
thereof, is intended. The nucleic acid molecule need not be in a
purified form. Various other nucleic acid molecules can also be
present with the target nucleic acid molecule. For example, the
target nucleic acid molecule can be a specific nucleic acid
molecule (which can include RNA such as STLV-3 subtype D viral RNA,
or DNA, such as STLV-3 subtype D viral DNA, for example STLV-3
subtype D viral DNA integrated into a host genome and/or
replicated, such as amplified STLV-3 subtype D viral DNA), the
amplification and/or detection of which is intended. Purification
or isolation of the target nucleic acid molecule, if needed, can be
conducted by methods known to those in the art, such as by using a
commercially available purification kit or the like. In one
example, a target nucleic acid molecule is a STLV-3 subtype D
nucleic acid sequence.
[0163] Virus: Microscopic infectious organism that reproduces
inside living cells. A virus consists essentially of a core of a
single nucleic acid surrounded by a protein coat, and has the
ability to replicate only inside a living cell. "Viral replication"
is the production of additional virus by the occurrence of at least
one viral life cycle. A virus may subvert the host cells' normal
functions, causing the cell to behave in a manner determined by the
virus. For example, a viral infection may result in a cell
producing a cytokine, or responding to a cytokine, when the
uninfected cell does not normally do so.
[0164] "Retroviruses," such as deltaretroviruses, for example
simian T-cell lymphotropic viruses, such as STLV-3 subtype D
viruses, are RNA viruses wherein the viral genome is RNA. When a
host cell is infected with a retrovirus, the genomic RNA is reverse
transcribed into a DNA intermediate which is integrated very
efficiently into the chromosomal DNA of infected cells. The
integrated DNA intermediate is referred to as a provirus.
[0165] Virion: A complete viral particle including envelope,
capsid, and nucleic acid elements.
III. Overview of Several Embodiments
[0166] Nonhuman primates can be naturally infected with a plethora
of viruses with zoonotic potential, including retroviruses. These
simian viruses present risks to both captive nonhuman primate
populations and persons exposed to nonhuman primates. Simian
retroviruses, including simian immunodeficiency virus, simian type
D retrovirus, simian T-lymphotropic virus, and gibbon ape leukemia
virus, have been shown to cause clinical disease in nonhuman
primates. Disclosed herein is the characterization of a new simian
T-cell lymphotropic virus (STLV) designated simian T-cell
lymphotropic virus type 6 (STLV-3 subtype D) isolated from
non-human primates in Cameroon. When this virus was originally
isolated it was initially given the designation T-cell lymphotropic
virus type 5 (STLV-5) or T-cell lymphotropic virus type 6, however
the disclosed virus has been provisionally redesignated as simian
T-cell lymphotropic virus type 3 West African subtype D (referred
to herein as STLV-3 subtype D). Thus, the reference to STLV-5 in
U.S. Provisional Application No. 60/939,304, filed May 21, 2007
refers to the STLV-3 subtype D virus disclosed herein. Similarly,
the reference to STLV-6 in U.S. Provisional Application No.
60/990,138, filed Nov. 26, 2007 refers to the STLV-3 subtype D
virus disclosed herein.
[0167] It has been shown that human retrovirus infections with
human T-lymphotropic virus and human immunodeficiency virus
originated through multiple independent introductions of simian
retroviruses into human populations that then spread globally, but
little is known about the frequency of such zoonotic events. Thus,
monitoring STLV-3 subtype D, for example in non-human primate
populations, limits the possibility of this virus making the cross
species jump into the human population.
STLV-3 Subtype D Nucleic Acids
[0168] This disclosure provides STLV-3 subtype D nucleic acid
sequences. In one embodiment, the genomic nucleic acid sequence of
STLV-3 subtype D as set forth as SEQ ID NO: 1 is provided. Nucleic
acid sequences are also provided that are at least 95% identical,
such as at least 96% identical, at least 97% identical, at least
98% identical, or at least 99% identical, to the nucleic acid
sequence set forth as SEQ ID NO: 1. An exemplary genomic sequence
of STLV-3 subtype D is set forth below as SEQ ID NO: 1:
TABLE-US-00001 (SEQ ID NO: 1).
tgacagtgacagcaagccccaaggcgagccacaactactagccaaagggc
atacagttgaatcatctgtctaggggacgtctcgcacccagagtatgtcc
aaagaacaccagggctctgacgtctctccctgccttgtctcccggaaaaa
accttaaaccacccatttcctcatgtttgcccaaggctctgacgataacc
ctgaaaaatttgactaacaaataaaggaacctggaccctataaaagggga
gagcgacctaaaaatgggatcaaccttttctccccaacgccctttcgcgc
cccgcggacagccactgtccgggctactcctggcctacctagatcattgc
tccgcgcccgagccattcttctgcagccaagcggcaccttgcaccttcgc
ttctcctgtcctggtaagatcccactgggtagagctaggccgttactccc
tggccgctcccctggagctcctttgcttagctcttaaggtcgctctctcc
ttctcgttagggtccaaggactaactttacttccgtgtctcggtctcctt
tctttggcggtctcgtctaaagtcgaaagtaacacctcaaactgtcagca
gcgaggcctggcccggggccagcgcctgtgagctttactcggctcggagc
caggggctcagaaagtaaaggctgtagctgccagcctttgaggggaacca
aaaacaggtgggggctcgtccgggattgatcaccctcctattaaacatgg
gaaattcatacagccgtgccgccaaccccatccccaaggccccaaaaggg
ctagcaattcaccactggttaaactttctacaagctgcctatcggctgca
accggggccctcagagtttgatttccatcagttacgaaattttcttaaat
tagctataaaaacccctgtttggctaaaccccatcaattattccgtccta
gctgaactcgttcctaaaaattatccaggcagaatccaagaaattatagc
catcctaatccaagaaacctctacgcaggaggttcccccatccgccccac
cggccagcgaaccccaaaatcccccgccttatccagaaccagggcaagcc
ataccccagtgcctacctgttctgcacccccatggtgcccctgccgccca
tcgcccttggcagatgaaagatctccaagctataaaacaggaagttacct
cttccgcaccagggagccctcagttcatgcaaaccgtgcgcctggcagtc
caacaatttgacccgactgccaaagacctccatgacctcttacaatacct
gtgctcctcactagttgcctccctgcaccaccagcagctcgagaccctca
tcgctcaggctgaaacccaagggataaccggatataatcccctggccggc
cccctgcgagtacaggccaacaacccaactcagcaagggctccggcgaga
ataccaaaacttatggctgtcggccttttctgccctcccaggaaatacta
aagaccccacctgggcggcaatcctccagggccccgaggaaccgttttgc
acattcgtagaaagacttaatgtggccctagacaacggcctccctgaagg
aacccccaaagagcctattcttcggtccttagcatattctaatgccaaca
aagaatgccagaaactcctacaagcccgagggcagacaaacggtccctta
ggggacatgctcagagcttgccaggcgtggacgccccgggacaaaaacaa
agtactaatggtccaacctaaaaagacacctcccccaaatcaaccatgct
tccggtgcgggcaggcgggccactggagcagagactgtaaacaacctcgt
ccccccccaggcccatgtccgctctgtcaagaccccacccactggaagcg
agattgcccgcagctaaaaccagatcctgaagaaggcatgttgttagatc
tgccttgtgaagacccagcggccagagaccaaaaaaacttcatagggggg
gaggactagcctccccccaaacagtgctgccttttataccattatcccag
caaaaacaaccagtcctacacgtccgagtatccttcccaggtaccccccc
agtaagcatccaggcgcttttagacacaggggcagatgtaaccgtcctcc
cagcccgtctatgcccccctgacctaaaattacaagacaccactgtcctt
ggagccagcgggccaagcaccgacaagtttaaagttctaccctgttttac
gtatgtccatctgcccttccgaggacgaccagtaaccttaccatcatgct
taattgatattaataatcaatgggccattctaggccgagatgtcctccag
caatgccaaagttccctttaccttgcagaccaaccctctcgcgttctacc
aatccagacacctagtgtcattgggctggaacatctccccccgcccccag
aagttccacaatttccgttaaaccagagcgcctccaggccttgactgacc
tggtatccaaggcgctggaggccaaatacatagaaccttatcaaggacca
ggcaataatccaattttcccggtcaaaaaaccgaatggaaaatggcgctt
catccatgatctccgggccaccaactgcctcactaaaaccctaacttccc
cgtctcccggcccccccgaccttaccagtctgccccaaggcctcccacat
cttcgaaccattgacctgactgacgccttttttcaaatcccactgcctgt
tgccttccagccctattttgcatttaccctccctcagcccaacaaccatg
gccccggggctcggtattcctggaaagtactaccccaagggtttaaaaat
agcccaactctatttgaacaacaactctctcatatactcacacctgtaag
acaggcctttccaaaatctatagtcattcagtacatggatgacatactct
tggccagccctacccttgaagagtccatcgttctcgcccaggaaataacc
aatgctctagcccaggagggcttgcccatgtccacagaaaaaacccaatc
cactcctggtcccatacactttctcggacaaaccatatccaaaaaataca
taacttatgaaaccctccctaccatacatgtcaagcctaattggacctta
acagaattacagtccaccttaggggaattgcaatgggtatccaaagggac
tcctacactccgctcatccctccatcaattatatacggccctccgaggtc
atcatgacccccgcgataccatacaacttaccccaccacaactacaagcg
ctcaacacgcttcaaaaggctctgacccacaattgcagaagcagaatagt
cagtaatctgcctatcctggccctcataatgctccgccccacaggcacta
cagcagttctttttcaaacaaaacaaaagtggccacttgtctggctgcac
accccccacccggccactagtctgcgcctttggggacaattattggccaa
tgccatcattactctagataagtactcactacaacactatggccaggtat
gcaaatcctttcatcataacatatctaatcaggcccttacccactaccta
cacacgtcagaccagtcaagtgttgccattctcctacagcactcgcatag
gttccataatctcggggcccaaccatcgggaccatggaaaggcctcctac
aagtaccccaaatcttccaaaatgttgccacacttagccctccattcact
atttcacctgtggttatcaaccacgccccttgcctcttttccgatggatc
caactctcaggctgccttcactatctgggataaaaaaataattcaccaac
aagtccttcctcttcctaccgccagctcggctcaagcaggggaacttttt
gccctattagcggccctacgagaatgcaaaccctggtcatcactaaacat
attcttagactcaaagtttcttgttggccagctccggcgcctggcccttg
gggctttcataggtccatccacccaatgtgacttacactcgcaactcctg
ccgctcttgtataacaaaaccatttatgttcatcatgtaagaagccacac
cttattacaggaccctatatcccgcctcaatgaggctaccgatgccctca
tgctcgcaccccttctgcccctcagtccagcgacccttcatgaaatcacc
cactgcaacccccctgcactgtgcaaccatggggctacagcaactgagac
taaggctattgtccgggcatgtcacacctgtaagataaccaatccccaag
ggagactgccccagggtcacattcgcagagggcacgccccaaacactatc
tggcaaggagatgtcactcacctacaatacaaaaaatataaatactgcct
tttagtctgggtcgatacttactcaggagcagtagctgtgtcgtgccggc
gtaaagaaaccagctcagaatgtgtggcctcgctgctagcagccatttcc
atcctaggaaaaccacacaccattaatacagacaatggggcagcatattt
gtcccaggaattccaacaattttgtacctcactctccataaaacacacca
ctcatgtcccctacaatcccaccagttccggattagtggaaagaactaat
ggaatcctaaaaaccttaatctccaaatacctcctagatgaccaccactt
gcccctggacacagccatttccaaaactttgtggaccataaaccatctca
atgtcctctcttcctgccaaaagacacgatggcagttacatcaagctcaa
cccctgccccccgttcctgagaatttgccccttcctgaaccagtgccaaa
atggtattattataaaatcccaggtcttaccagttcaaggtggagtgggc
ctgtacaatctgttaaagaagcagccggagcggccctcatcccggtaggt
actaggcacatctggattccgtggcgtctcctgaaacgaggtgcatgccc
aagacccggagacagcgtaaccaccgaatcaaaacacaaagaccttcaac
tccatgggtaagtctagtctctttatttgcctcttttgctcatacatggc
tagtctctttgtccctggcgaccccagtcggtgcacactttttataggag
cctcctcctaccactccagtccctgcgggtctaactaccctcaatgtact
tggacactcgacctagtgtcacttaccagggatcaaagtctaaaccctcc
atgcccagatctagtcacctactcccagtatcacagaccttattccttgt
atctttttccccattggattactaaaccgaatcgtcaaggccttggttat
tactctgcctcctactcagatccctgtgctatcaagtgcccctacctagg
atgtcaatcttggacatgtccctatacaggacctatgtccagcccatact
ggaagtacacctcagacctaaatttcacccaaaaggtgtcctctgtcacc
ctccatctacatttctcaaaatgcggatcctccttctctcttttactcga
cgcacccggttatgaccccgtatggttcctttcctcccaaactacacagg
ccccacctacacccgcccctctgacacaagactccgacttccaacatatc
ttggagccctctgtgccctggagctccaaaatcctcaaccttatcctctt
aactcttaaaagcactaactactcctgcatggtttgcgttgaccgctcca
gcctctcctcatggcatgtcttgtatgacccactaaaagttcccaagcaa
cacgaaccccgtgcccgggccctcttgcggccctctctggccattccaat
aactaataccacacccccctttccttggtcccattgctactgcccccttc
tacaggctgtcatctccaataactgcaacaactcagttatactgcccccc
ttctctctgtcccctgtcctcgatctctccaagcctcgtcagcgccgagc
cgtccccatcgccgtttggctggtgtccgccctagcggtcggtacaggta
tagccggcggcaccaccgggtccctatccttggcatccagcaggagcctg
ctacatgaagtagaccaagatataagccatctcactcaagccatagttaa
gaaccataacaatatccttcgggttgctcaatacgctgcacaaaaccgac
gaggcctagatttactcttctgggaacaaggaggtctatgcaaggctatc
agggaacaatgttgttttctcaatatcagcaatacccacgtgtctgtgct
ccaagagagaccccccttagaaaaaagggtgattaccggttggggactca
attgggacctcggcctatcccaatgggcccgtgaagccctccagaccggt
attaccctgttagccctcttcctcctacttatcatggtaggcccttgtgt
cctgcgccagctacaggccctcctgttccgcctacagcaccgtagccacc
catactccctcctcaatcgcgaaaccaacctataacacctctgcaacctc
ctgtagcaatgagccatagtcctcgcccctaccagaaacccacatacagc
ataggcccgaagaatctccccaaatatccatgccttgactccagtaatcc
atgtacccaaagtattcccctaatgcctcctcacaatccacgcgaagttg
gaaattctctcgttccaaaaagtctatataacccgtcaacaaattgcaaa
acccctcaaaccccagtaagtctatacaatccaactgctgccgccgctcc
ttttttctcctctttctctcctctttttcctcgtgacacctcctccggcg
ctcttctcttcttttccgaccccgccagtagcttagcaattgcttctgct
cctgagcaaggtcttctaagcgacccttccaatatcctgaatcctttgta
ctagatcccagaggacgccctcggggtcgcctaccacccccctgcagcat
gtccacttgatcttttcccgattgatcacacaactccaataaagcttcca
ccggtgtgagaggatcttcggccgccagtatcggtggtcccacactccta
gaccgagaggtcaagctgcccccggaagtagagacgcaggaatacaccac
aggcatagtccccgcagttgtggtctctggagtcagtaaaggcatcttcc
taaaataccctgtaaaataatctcctgtcagcccactttccaggtttcgg
gcagagcctgctctacgggtaccctgtctacgttttcggcgattgtgtgc
aggccgattggtgccccatttccggggggctttgttccgcccggctacat
cggcacgccttactggccacctgtcctgaacaccagatcacctgggaccc
catcgatggacgcgttgtcagctcgcctctacaataccttatccctcgcc
tcccctccttccccacccaaagaacttcccgcaccctcaaggtcctcacc
ccgccgcccactgctacaacccccaaagttcctccctccttcttccatgc
agtcaggaaacacacccctttccgaaacaactgcctcgagctcaccttgg
gagagcaactacccgccatgtctttccccgaccccggcctccgaccccaa
aatgtctataccatgtggggaagcaccatcgtgtgcttatacctctacca
actcacacctccaatgacctggccgttaatcccacatgtcattttttgcc
atccggaccaactaggggccttcctaacaaaaatccctaccaaacgcttg
gaagaactcttatacaaactattcttaagtacaggggccatacttatcct
acctgaaaattgcttcccaactaccctgtttcagcccacccgcgcaccag
taattcaagccccctggcactcaggcctactcccatacctaaaggaaatt
gtcacccccgggctgatttgggtgtttactgacggtagttctatgatttc
cggaccctgccccaaggaagggcagccatctttggtggtccaatcatcta
cattcattttccaaaaatttcaaaccaaagcctatcacccagccttcctc
ctgtcccataaattaatccaatactcctcgttccattccctccatctact
ttttgaagaatacaccactgtccccttttctttattgtttaacgaaaaag
aggcaaatgacagtgacagcaagccccaaggcgagccacaactactagcc
aaagggcatacagttgaatcatctgtctaggggacgtctcgcacccagag
tatgtccaaagaacaccagggctctgacgtctctccctgccttgtctccc
ggaaaaaaccttaaaccacccatttcctcatgtttgcccaaggctctgac
gataaccctgaaaaatttgactaacaaataaaggaacctggaccctataa
aaggggagagcgacctaaaaatgggatcaaccttttctccccaacgccct
ttcgcgccccgcggacagccactgtccgggctactcctggcctacctaga
tcattgctccgcgcccgagccattcttctgcagccaagcggcaccttgca
ccttcgcttctcctgtcctggtaagatcccactgggtagagctaggccgt
tactccctggccgctcccctggagctcctttgcttagctcttaaggtcgc
tctctccttctcgttagggtccaaggactaactttacttccgtgtctcgg
tctcctttctttggcggtctcgtctaaagtcgaaagtaacacctcaaact
gtcagcagcgaggcctggcccggggccagcgcctgtgagctttactcggc
tcggagccaggggctcagaaagtaaaggctgtagctgccagcctttgagg
ggaaccaaaaaca
[0169] In several embodiments, the nucleic acid sequences of
several STLV-3 subtype D open reading frames (ORFs) are disclosed
(see Table 4), such as ORFs for a STLV-3 subtype D capsid
polypeptide, a STLV-3 subtype D protease, a STLV-3 subtype D
polymerase, a STLV-3 subtype D tax polypeptide, a STLV 5 rex
polypeptide, or a STLV-3 subtype D envelope polypeptide. Specific
non-limiting examples of STLV-3 subtype D nucleic acid sequences
encoding a STLV-3 subtype D polypeptide include, but are not
limited, to nucleotides 747-2009 of SEQ ID NO: 1, which encodes a
STLV-3 subtype D capsid polypeptide, nucleotides 1961-2494 of SEQ
ID NO: 1, which encodes a STLV-3 subtype D protease polypeptide,
nucleotides 2416-5061 of SEQ ID NO: 1, which encodes a STLV-3
subtype D polymerase polypeptide, nucleotides 5054-6535 of SEQ ID
NO: 1, which encodes a STLV-3 subtype D envelope polypeptide, SEQ
ID NO: 25, which encodes a STLV-3 subtype D tax polypeptide, and
SEQ ID NO: 26, which encodes a STLV-3 subtype D rex
polypeptide.
[0170] As shown in FIG. 1, the nucleotide sequence encoding the
STLV-3 subtype D tax polypeptide is composed of two portions of
non-contiguous nucleic acid sequence that is spliced together to
form the entire coding region of the STLV-3 subtype D tax gene,
such as a the STLV-3 subtype D tax gene depicted below as SEQ ID
NO: 25. The sequence set forth as SEQ ID NO: 25 is composed of
nucleotides 5054-5057 of SEQ ID NO: 1 spliced (at the three prime
end) to the five prime end of nucleotides 7232-8280 of SEQ ID NO:
1.
An Exemplary STLV-3 Subtype D Tax Gene:
TABLE-US-00002 [0171] (SEQ ID NO: 25)
atggcccactttccaggtttcgggcagagcctgctctacgggtaccctgt
ctacgttttcggcgattgtgtgcaggccgattggtgccccatttccgggg
ggctttgttccgcccggctacatcggcacgccttactggccacctgtcct
gaacaccagatcacctgggaccccatcgatggacgcgttgtcagctcgcc
tctacaataccttatccctcgcctcccctccttccccacccaaagaactt
cccgcaccctcaaggtcctcaccccgccgcccactgctacaacccccaaa
gttcctccctccttcttccatgcagtcaggaaacacacccctttccgaaa
caactgcctcgagctcaccttgggagagcaactacccgccatgtctttcc
ccgaccccggcctccgaccccaaaatgtctataccatgtggggaagcacc
atcgtgtgcttatacctctaccaactcacacctccaatgacctggccgtt
aatcccacatgtcattttttgccatccggaccaactaggggccttcctaa
caaaaatccctaccaaacgcttggaagaactcttatacaaactattctta
agtacaggggccatacttatcctacctgaaaattgcttcccaactaccct
gtttcagcccacccgcgcaccagtaattcaagccccctggcactcaggcc
tactcccatacctaaaggaaattgtcacccccgggctgatttgggtgttt
actgacggtagttctatgatttccggaccctgccccaaggaagggcagcc
atctttggtggtccaatcatctacattcattttccaaaaatttcaaacca
aagcctatcacccagccttcctcctgtcccataaattaatccaatactcc
tcgttccattccctccatctactttttgaagaatacaccactgtcccctt
ttctttattgtttaacgaaaaagaggcaaatgacagtgacagcaagcccc
aaggcgagccacaactactagccaaagggcatacagttgaatcatctgtc tag
[0172] As shown in FIG. 1, the nucleotide sequence encoding the
STLV-3 subtype D tax polypeptide is composed of two portions of
non-contiguous nucleic acid sequence that is spliced together to
form the entire coding region of the STLV-3 subtype D rex gene,
such as a the STLV-3 subtype D rex gene depicted below as SEQ ID
NO: 25. The sequence set forth as SEQ ID NO: 25 nucleotides
4995-5057 of SEQ ID NO: 1 spliced (at the three prime end) to the
five prime end of nucleotides to the five prime end of nucleotides
7232-7717 of SEQ ID NO: 1.
TABLE-US-00003 An exemplary STLV-3 subtype D rex gene: (SEQ ID NO:
26) atgcccaagacccggagacagcgtaaccaccgaatcaaaacacaaagacc
ttcaactccatggcccactttccaggtttcgggcagagcctgctctacgg
gtaccctgtctacgttttcggcgattgtgtgcaggccgattggtgcccca
tttccggggggctttgttccgcccggctacatcggcacgccttactggcc
acctgtcctgaacaccagatcacctgggaccccatcgatggacgcgttgt
cagctcgcctctacaataccttatccctcgcctcccctccttccccaccc
aaagaacttcccgcaccctcaaggtcctcaccccgccgcccactgctaca
acccccaaagttcctccctccttcttccatgcagtcaggaaacacacccc
tttccgaaacaactgcctcgagctcaccttgggagagcaactacccgcca
tgtctttccccgaccccggcctccgaccccaaaatgtctataccatgtgg
ggaagcaccatcgtgtgcttatacctctaccaactcacacctccaatga
[0173] These polynucleotides include DNA, cDNA, and RNA sequences
that encode a STLV-3 subtype D polypeptide. In specific
embodiments, these sequences are used for generating
oligonucleotide primers and probes for the detection of STLV-3
subtype D in samples, for example probes and primers for the
detection of STLV-3 subtype D ORFs, such as ORFs for a STLV-3
subtype D capsid polypeptide, a STLV-3 subtype D protease
polypeptide a STLV-3 subtype D polymerase polypeptide, a STLV-3
subtype D tax polypeptide a STLV-3 subtype D rex polypeptide, or a
STLV-3 subtype D envelope polypeptide. In other embodiments, these
sequences are used for generating polypeptides corresponding to a
STLV-3 subtype D capsid polypeptide, a STLV-3 subtype D protease, a
STLV-3 subtype D envelope polypeptide, STLV-3 subtype D polymerase,
a STLV-3 subtype D tax polypeptide, a STLV-3 subtype D rex
polypeptide, or fragments thereof.
[0174] All polynucleotides encoding a STLV-3 subtype D polypeptide,
such as a STLV-3 subtype D polypeptide fragment, are also included
herein. In one embodiment, a STLV-3 subtype D nucleic acid sequence
that encodes a polypeptide that functions as a STLV-3 subtype D
capsid polypeptide is at least 95% identical, such as at least 96%
identical, at least 97% identical, at least 98% identical, at least
99% identical, or even 100% identical to nucleotides 747-2009 of
SEQ ID NO: 1. In one embodiment, a STLV-3 subtype D nucleic acid
sequence that encodes a polypeptide that functions as a STLV-3
subtype D protease is at least 95% identical, such as at least 96%
identical, at least 97% identical, at least 98% identical, at least
99% identical, or even 100% identical to nucleotides 1961-2494 of
SEQ ID NO: 1. In one embodiment, a STLV-3 subtype D nucleic acid
sequence that encodes a polypeptide that functions as a STLV-3
subtype D envelope polypeptide is at least 95% identical, such as
at least 96% identical, at least 97% identical, at least 98%
identical, at least 99% identical, or even 100% identical to
nucleotides 5054-6535 of SEQ ID NO: 1. In one embodiment, a STLV-3
subtype D nucleic acid sequence that encodes a polypeptide that
functions as a STLV-3 subtype D polymerase is at least 95%
identical, such as at least 96% identical, at least 97% identical,
at least 98% identical, at least 99% identical, or even 100%
identical to nucleotides 2416-5061 of SEQ ID NO: 1. In one
embodiment, a STLV-3 subtype D nucleic acid sequence that encodes a
polypeptide that functions as a STLV-3 subtype D tax polypeptide is
at least 95% identical, such as at least 96% identical, at least
97% identical, at least 98% identical, at least 99% identical, or
even 100% identical to SEQ ID NO: 25. In one embodiment, a STLV-3
subtype D nucleic acid sequence that encodes a polypeptide that
functions as a STLV-3 subtype D rex polypeptide is at least 95%
identical, such as at least 96% identical, at least 97% identical,
at least 98% identical, at least 99% identical, or even 100%
identical to SEQ ID NO: 26.
[0175] The polynucleotides of this disclosure include sequences
that are degenerate as a result of the genetic code. For example,
there are 20 natural amino acids, most of which are specified by
more than one codon. Therefore, all degenerate nucleotide sequences
are included in the disclosure as long as the amino acid sequence
of the STLV-3 subtype D polypeptide encoded by the nucleotide
sequence is functionally unchanged.
[0176] Also disclosed herein are STLV-3 subtype D oligonucleotides
that specifically hybridize to STLV-3 subtype D nucleic acids, such
as probes and primers, for example probes and primers that
hybridize to the STLV-3 subtype D nucleic acid sequence set for as
SEQ ID NO: 1. In some embodiments, the disclosed STLV-3 subtype D
oligonucleotides specifically hybridize to a nucleic acid sequence
encoding a STLV-3 subtype D capsid polypeptide (for example
nucleotides 747-2009 of SEQ ID NO: 1), a nucleic acid sequence
encoding a STLV-3 subtype D protease (for example nucleotides
1961-2494 of SEQ ID NO: 1), a nucleic acid sequence encoding a
STLV-3 subtype D envelope polypeptide (for example nucleotides
5054-6535 of SEQ ID NO: 1), a nucleic acid sequence encoding a
STLV-3 subtype D polymerase (for example nucleotides 2416-5061 of
SEQ ID NO: 1), a nucleic acid sequence encoding a STLV-3 subtype D
tax polypeptide (for example SEQ ID NO: 25), or a nucleic acid
sequence encoding a STLV-3 subtype D rex polypeptide (for example
SEQ ID NO: 26), or a nucleic acid sequence encoding a STLV-3
subtype D LTR (for example nucleotides 7-706 of SEQ ID NO: 1).
Exemplary primers that specifically hybridize to the nucleic acid
sequence set forth as SEQ ID NO: 1 are given in Table 1. The
methods disclosed herein take advantage of the fact that under
appropriate conditions oligonucleotides, such as probes and
primers, form base-paired duplexes with oligonucleotides, which
have a complementary base sequence. The stability of the duplex is
dependent on a number of factors, including the length of the
oligonucleotides, the base composition, and the composition of the
solution in which hybridization is effected. The effects of base
composition on duplex stability may be reduced by carrying out the
hybridization in particular solutions, for example in the presence
of high concentrations of tertiary or quaternary amines.
[0177] The thermal stability of the duplex is also dependent on the
degree of sequence similarity between the sequences. By carrying
out the hybridization at temperatures close to the anticipated
T.sub.m's of the type of duplexes expected to be formed between the
target sequence(s) and the oligonucleotides, for example
amplification primers, real-time PCR primers and probes, or
oligonucleotides bound to an array, the rate of formation of
mis-matched duplexes may be substantially reduced.
[0178] The length of each oligonucleotide sequence can be selected
to optimize binding of target STLV-3 subtype D nucleic acid
sequence, for example a STLV-3 subtype D capsid, protease, rex,
tax, polymerase, LTR, or envelope nucleic acid sequence. An optimum
length for use with a particular STLV-3 subtype D nucleic acid
sequence under specific screening conditions can be determined
empirically. Oligonucleotides, for example probes or primers, of
the disclosed STLV-3 subtype D nucleic acid sequences may be
comprised of at least 15 consecutive nucleic acids, which is
sufficient to permit the oligonucleotide to selectively hybridize,
for example under conditions of very high stringency, to a STLV-3
subtype D nucleic acid, for example a STLV-3 subtype D nucleic acid
that encodes a STLV-3 subtype D capsid polypeptide, protease,
polymerase, or envelope polypeptide, or a STLV-3 subtype D nucleic
acid sequence that encodes a rex, or tax, polypeptide or even a
nucleic acid that encodes a STLV-3 subtype D LTR.
[0179] In some embodiments, the disclosed STLV-3 subtype D
oligonucleotides specifically hybridize to a nucleic acid sequence
encoding a STLV-3 subtype D capsid polypeptide (for example
nucleotides 747-2009 of SEQ ID NO: 1), a nucleic acid sequence
encoding a STLV-3 subtype D protease (for example nucleotides
1961-2494 of SEQ ID NO: 1), a nucleic acid sequence encoding a
STLV-3 subtype D envelope polypeptide (for example nucleotides
5054-6535 of SEQ ID NO: 1), a nucleic acid sequence encoding a
STLV-3 subtype D polymerase polypeptide (for example 2416-5061 of
SEQ ID NO: 1), a nucleic acid sequence encoding a STLV-3 subtype D
tax polypeptide (for example SEQ ID NO: 25), or a nucleic acid
sequence encoding a STLV-3 subtype D rex polypeptide (for example
SEQ ID NO: 26).
[0180] In some embodiments, an oligonucleotide sequence is selected
such that it hybridizes under high stringency conditions to a
nucleic acid sequence at least 95% identical to, such as at least
96% identical, at least 97% identical, at least 98% identical, at
least 99% identical, or even 100% identical to nucleotides 747-2009
of SEQ ID NO: 1. In some embodiments, an oligonucleotide sequence
is selected such that it hybridizes under high stringency
conditions to a nucleic acid sequence at least 95% identical to,
such as at least 96% identical, at least 97% identical, at least
98% identical, at least 99% identical, or even 100% identical to
nucleotides 1961-2494 of SEQ ID NO: 1. In some embodiments, an
oligonucleotide sequence is selected such that it hybridizes under
high stringency conditions to a nucleic acid sequence at least 95%
identical to, such as at least 96% identical, at least 97%
identical, at least 98% identical, at least 99% identical, or even
100% identical to nucleotides 2416-5061 of SEQ ID NO: 1. In some
embodiments, an oligonucleotide sequence is selected such that it
hybridizes under high stringency conditions to a nucleic acid
sequence at least 95% identical to, such as at least 96% identical,
at least 97% identical, at least 98% identical, at least 99%
identical, or even 100% identical to nucleotides 5054-6535 of SEQ
ID NO: 1. In some embodiments, an oligonucleotide sequence is
selected such that it hybridizes under high stringency conditions
to a nucleic acid sequence at least 95% identical to, such as at
least 96% identical, at least 97% identical, at least 98%
identical, at least 99% identical, or even 100% identical to SEQ ID
NO: 25. In some embodiments, an oligonucleotide sequence is
selected such that it hybridizes under high stringency conditions
to a nucleic acid sequence at least 95% identical to, such as at
least 96% identical, at least 97% identical, at least 98%
identical, at least 99% identical, or even 100% identical to SEQ ID
NO: 26.
[0181] In some embodiments, the disclosed STLV-3 subtype D
oligonucleotides comprise at least 15, 20, 25, 30, 35, 40, or more
consecutive nucleotides of an STLV-3 subtype D nucleic acid
sequence, such as 15, 20, 25, 30, 35, 40, or more consecutive
nucleotides of STLV-3 subtype D a nucleic acid sequence set forth
as SEQ ID NO: 1. In specific non-limiting examples, the disclosed
STLV-3 subtype D oligonucleotide includes nucleotides 1-15, 16-30,
or 31-45, etc., 2-16, 17-31, or 32-46, etc., or 3-17, 18-32, or
33-47, etc. of a STLV-3 subtype D nucleic acid sequence, such as
nucleotides 1-15, 16-30, or 31-45, etc., 2-16, 17-31, or 32-46,
etc., or 3-17, 18-32, or 33-47, etc. of the nucleic acid sequence
set forth as SEQ ID NO: 1. In other specific non-limiting examples,
the oligonucleotide includes nucleotides 1-20, 21-40, or 41-60,
etc., 2-21, 22-41, or 42-61, etc., or 3-22, 23-42, or 43-62, etc.
of a STLV-3 subtype D nucleic acid sequence, such as nucleotides
1-20, 21-40, or 41-60, etc., 2-21, 22-41, or 42-61, etc., or 3-22,
23-42, or 43-62, etc. of the nucleic acid sequence set forth as SEQ
ID NO: 1. In further specific non-limiting examples, the
oligonucleotide includes nucleotides 1-25, 26-50, 51-75, etc.,
2-26, 27-51, or 52-76, or 2-27, 28-52, or 53-77, etc. of a STLV-3
subtype D nucleic acid sequence, such as nucleotides 1-25, 26-50,
51-75, etc., 2-26, 27-51, or 52-76, or 2-27, 28-52, or 53-77, etc.
of the nucleic acid sequence set forth as nucleotides SEQ ID NO: 1.
In one embodiment, the STLV-3 subtype D capsid, polymerase,
protease, or envelope nucleic acid sequence is a target sequence
for amplification.
[0182] A STLV-3 subtype D primer can be used to sequence a STLV-3
subtype D nucleic acid in order to identify a STLV-3 subtype D
nucleic acid, for example to identify the presence of a STLV-3
subtype D nucleic acid in a sample, for example to detect the
presence of STLV-3 subtype D in the sample. Alternatively, two, or
more, STLV-3 subtype D primers can be used to amplify a region
within a STLV-3 subtype D gene, for example by polymerase chain
reaction (PCR) or more specifically by real-time PCR. In some
examples, a primer comprises the nucleotide sequence as set forth
as P5TAXF3 (SEQ ID NO: 21), P5TAXR3 (SEQ ID NO: 22), P5TAXF2 (SEQ
ID NO: 23), or P5TAXR1 (SEQ ID NO: 24).
[0183] STLV-3 subtype D oligonucleotides, such as primers and
probes, can be used to identify STLV-3 subtype D. Thus, these
STLV-3 subtype D probes and primers specifically hybridize to a
region in a STLV-3 subtype D nucleic acid which is unique to STLV-3
subtype D and not present in other viruses.
[0184] The primers and probes disclosed herein can be end-labeled
(for example, radiolabeled, enzymatically-labeled,
fluorescently-labeled, or biotinylated). One specific, non-limiting
example of a primer label is a fluoresceinated STLV-3 subtype D
primer. The probes disclosed herein can be fluorescently-labeled,
such as for use in real-time PCR. In one embodiment, the
oligonucleotide probes in the sample are labeled to render them
readily detectable. Detectable labels may be any species or moiety
that may be detected either visually or with the aid of an
instrument. Detectable labels can be radioisotopes,
chemiluminescent tags, haptens, or fluorescent markers. Specific,
non-limiting examples of fluorescent markers include FITC,
LIGHTCYCLER.TM. Red 640, LIGHTCYCLER.TM. Red 705,
6-carboxy-X-rhodamine (ROX), 5-carboxyfluorescein (FAM),
2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), and
6-carboxy-2',4,7,7'-tetrachlorofluorescein (TET). In one
embodiment, the fluorescent markers coupled to the oligonucleotide
probes have spectrally distinct emission spectra such that the
amplified DNA sequences to which they specifically hybridize can be
distinguished within the same reaction tube. In some embodiments,
four, five, six, seven, or more probes that are sufficiently
complementary to a STLV-3 subtype D nucleic acid sequence, such as
a STLV-3 subtype D nucleic acid sequence set forth as SEQ ID NO: 1
may be used in a single reaction tube. Several examples of probes
that can be used in the disclosed methods include HybProbes,
Molecular beacon probes, TAQMAN.RTM. probes, amongst others.
[0185] HybProbes include an upstream probe labeled with a 3' donor
fluorophore, such as FITC, and a downstream probe labeled with an
acceptor fluorophore, such as LIGHTCYCLER.TM. Red 640 or Red 705,
at the 5' terminus. The nucleic acid sequence of a HybProbe
includes a nucleic acid sequence that detects the amplified product
from the target nucleic acid sequence of interest, such as a STLV-3
subtype D sequence, for example, a STLV-3 subtype D capsid,
protease, or envelope nucleic acid sequence. When the HybProbes are
not hybridized to the target sequence, the donor fluorophore is
excited by a filtered light source, such as by a LIGHTCYCLER.TM.'s
light emitting diode (LED), and a green fluorescent light is
emitted at a slightly longer wavelength. However, when the pair of
HybProbes hybridize to the target sequence, the two fluorophores
are in close proximity to each other, for example within 1-10
nucleotides of each other, and the energy emitted by the excitation
of the donor fluorophore excited the acceptor fluorophore, for
example a LIGHTCYCLER.TM. Red 640 attached to the probe. The
resultant energy transfer via FRET results in the emission of a red
fluorescent light at an even longer wavelength. The intensity of
the light emitted by the acceptor fluorophore is measured by the
apparatus, such as a LIGHTCYCLER.TM.. The increasing amount of
measured fluorescence is proportional to the increasing amount of
the amplified target nucleic acid generated during the ongoing PCR
process. Since the acceptor fluorophore only emits a signal when
both labeled probes are hybridized to the target nucleic acid
sequence, the fluorescence measurement is performed after the
annealing step in the PCR process.
[0186] Molecular beacon probes include probes coupled to a
fluorescent marker in combination with a quencher molecule. The
nucleic acid sequence of a molecular beacon probe includes a
nucleic acid that detects the amplified product from the DNA
sequence of interest, and sequences that permit the molecular
beacon probe to form a hairpin structure. Attached to opposite ends
(the 5' and the 3' end of the molecular beacon) are a fluorescent
reporter molecule and a quencher molecule. When the molecular
beacon is in the hairpin conformation (not hybridized to product)
any fluorescence emitted by the fluorescent label is absorbed
(quenched) by the quencher molecule via FRET and no fluorescence is
detected. When a molecular beacon hybridizes to an amplified target
nucleic acid with a complementary nucleic acid sequence, the
fluorescent label and the quencher molecule are separated, and
fluorescence is detected and can be measured during each PCR cycle.
These probes are known to one of skill in the art (see the
Molecular-Probes, Eugene, Oreg. website).
[0187] TAQMAN.RTM. probes include linear oligonucleotide probes
with a 5' reporter fluorophore and a 3' quencher fluorophore, such
as TAMRA. In the intact TAQMAN.RTM. probe, energy is transferred
(via FRET) from the short-wavelength fluorophore to the
long-wavelength fluorophore on the other end, quenching the
short-wavelength fluorescence. After hybridization, the probe is
susceptible to degradation by the endonuclease activity of a
processing Taq polymerase. Upon degradation, FRET is interrupted,
increasing the fluorescence from the short-wavelength fluorophore
and decreasing fluorescence from the long-wavelength
fluorophore.
[0188] Specific, non-limiting examples of quencher molecules
include N,N,N',N-tetramethyl-6-carboxyrhodamine (TAMRA) and
4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL). Many suitable
forms of these fluorescent markers and quenchers are widely
available commercially with substituents on their phenyl moieties,
which can be used as the site for coupling or as the coupling
functionality for attachment to an oligonucleotide.
Expression of STLV-3 Subtype D Nucleic Aid Sequences
[0189] The STLV-3 subtype D polynucleotides disclosed herein
include recombinant DNA which is incorporated into a vector; into
an autonomously replicating plasmid or virus; or into the genomic
DNA of a prokaryote or eukaryote, or which exists as a separate
molecule (for example, a cDNA) independent of other sequences. DNA
sequences encoding STLV-3 subtype D polypeptides, such as STLV-3
subtype D polypeptides, can be expressed in vitro by DNA transfer
into a suitable host cell. The cell may be prokaryotic or
eukaryotic. The term also includes any progeny of the subject host
cell. It is understood that all progeny may not be identical to the
parental cell since there may be mutations that occur during
replication. Methods of stable transfer, meaning that the foreign
DNA is continuously maintained in the host, are known in the
art.
[0190] STLV-3 subtype D polynucleotide sequences, such as STLV-3
subtype D capsid, protease, polymerase, tax polypeptide, rex
polypeptide, and envelope polynucleotide sequences, can be
operatively linked to expression control sequences. An expression
control sequence operatively linked to a coding sequence is ligated
such that expression of the coding sequence is achieved under
conditions compatible with the expression control sequences. The
expression control sequences include, but are not limited to
appropriate promoters, enhancers, transcription terminators, a
start codon (for instance, ATG) in front of a protein-encoding
gene, splicing signal for introns, maintenance of the correct
reading frame of that gene to permit proper translation of mRNA,
and stop codons.
[0191] Transformation of a host cell with recombinant DNA may be
carried out by conventional techniques as are well known to those
skilled in the art. Where the host is prokaryotic, such as E. coli,
competent cells, which are capable of DNA uptake can be prepared
from cells harvested after exponential growth phase and
subsequently treated by the CaCl.sub.2 method using procedures well
known in the art. Alternatively, MgCl.sub.2, or RbCl can be used.
Transformation can also be performed after forming a protoplast of
the host cell if desired, or by electroporation.
[0192] When the host is a eukaryote, such methods of transfection
of DNA as calcium phosphate coprecipitates, conventional mechanical
procedures such as microinjection, electroporation, insertion of a
plasmid encased in liposomes, or virus vectors may be used.
Eukaryotic cells can also be cotransformed with a STLV-3 subtype D
polynucleotide sequences, and a second foreign DNA molecule
encoding a selectable phenotype, such as the herpes simplex
thymidine kinase gene. Another method is to use a eukaryotic viral
vector, such as simian virus 40 (SV40) or bovine papilloma virus,
to transiently infect or transform eukaryotic cells and express the
protein (see for example, Eukaryotic Viral Vectors, Cold Spring
Harbor Laboratory, Gluzman ed., 1982).
[0193] Provided herein are the nucleic acid sequences that encode
polypeptides, such as a STLV-3 subtype D capsid polypeptide, an
STLV-3 subtype D protease, and an STLV-3 subtype D envelope
polypeptide. In some embodiments, an isolated STLV-3 subtype D
capsid polypeptide is provided that is at least 95% identical, such
as at least 96%, at least 97%, at least 98%, at least 99%, or even
100% identical to the STLV-3 subtype D capsid polypeptide encoded
by the nucleic acid sequence set forth as nucleotides 747-2009 of
SEQ ID NO: 1. In some embodiments, an isolated STLV-3 subtype D
envelope polypeptide is provided that is at least 95% identical,
such as at least 96%, at least 97%, at least 98%, at least 99%, or
even 100% identical to the STLV-3 subtype D envelope polypeptide
encoded by the nucleic acid sequence set forth as nucleotides
5054-6535 of SEQ ID NO: 1. In some embodiments, an isolated STLV-3
subtype D protease is provided that is at least 95% identical, such
as at least 96%, at least 97%, at least 98%, at least 99%, or even
100% identical to the STLV-3 subtype D protease encoded by the
nucleic acid sequence set forth as nucleotides 1961-2494 of SEQ ID
NO: 1. In some embodiments, an isolated STLV-3 subtype D polymerase
is provided that is at least 95% identical, such as at least 96%,
at least 97%, at least 98%, at least 99%, or even 100% identical to
the STLV-3 subtype D polymerase encoded by the nucleic acid
sequence set forth as nucleotides 2416-5061 of SEQ ID NO: 1. In
some embodiments, an isolated STLV-3 subtype D tax polypeptide is
provided that is at least 95% identical, such as at least 96%, at
least 97%, at least 98%, at least 99%, or even 100% identical to
the tax polypeptide encoded by SEQ ID NO: 25. In some embodiments,
an isolated STLV-3 subtype D rex polypeptide is provided that is at
least 95% identical, such as at least 96% identical, at least 97%
identical, at least 98% identical, at least 99% identical, or even
100% identical to the rex polypeptide encoded by SEQ ID NO: 26.
[0194] The expression and purification of any of these STLV-3
subtype D proteins, by standard laboratory techniques, is now
enabled. Fragments amplified as described herein can be cloned into
standard cloning vectors and expressed in commonly used expression
systems consisting of a cloning vector and a cell system in which
the vector is replicated and expressed. Purified proteins may be
used for functional analyses, antibody production, diagnosis, and
subject therapy. Furthermore, the DNA sequences of the STLV-3
subtype D cDNAs can be manipulated in studies to understand the
expression of STLV-3 subtype D genes and the function of their
products. Partial or full-length cDNA sequences, which encode for
the protein, may be ligated into bacterial expression vectors.
Methods for expressing large amounts of protein from a cloned gene
introduced into E. coli may be utilized for the purification,
localization and functional analysis of proteins. For example,
fusion proteins consisting of amino terminal peptides encoded by a
portion of the E. coli lacZ or trpE gene linked to STLV-3 subtype D
protein, such as a STLV-3 subtype D protease, capsid, or envelope
protein, may be used to prepare polyclonal and monoclonal
antibodies against this protein. Thereafter, these antibodies may
be used to purify proteins by immunoaffinity chromatography, in
diagnostic assays to quantitate the levels of protein and to
localize proteins in tissues and individual cells by
immunofluorescence and microscopy.
[0195] Intact native protein may also be produced in E. coli in
large amounts for functional studies. Standard prokaryotic cloning
vectors may also be used, for example, pBR322, pUC18, or pUC19 as
described in Sambrook et al. (Molecular Cloning: A Laboratory
Manual, 2.sup.nd ed., vol. 1-3, Cold Spring Harbor, N.Y. 1989).
Nucleic acids of STLV-3 subtype D may be cloned into such vectors,
which may then be transformed into bacteria such as E. coli, which
may then be cultured to express the protein of interest. Other
prokaryotic expression systems include, for instance, the
arabinose-induced pBAD expression system that allows tightly
controlled regulation of expression, the IPTG-induced pRSET system
that facilitates rapid purification of recombinant proteins and the
IPTG-induced pSE402 system that has been constructed for optimal
translation of eukaryotic genes. These three systems are available
commercially from INVITROGEN.TM. and, when used according to the
manufacturer's instructions, allow routine expression and
purification of proteins.
[0196] Methods and plasmid vectors for producing fusion proteins
and intact native proteins in bacteria are described in Sambrook et
al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,
N.Y., 1989, Chapter 17). Such fusion proteins may be made in large
amounts, are easy to purify, and can be used to elicit antibody
response. Native proteins can be produced in bacteria by placing a
strong, regulated promoter and an efficient ribosome binding site
upstream of the cloned gene. If low levels of protein are produced,
additional steps may be taken to increase protein production; if
high levels of protein are produced, purification is relatively
easy. Suitable methods are presented in Sambrook et al. (Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989) and
are well known in the art. Often, proteins expressed at high levels
are found in insoluble inclusion bodies. Methods for extracting
proteins from these aggregates are described by Sambrook et al.
(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.,
1989, Chapter 17).
[0197] Vector systems suitable for the expression of lacZ fusion
genes include the pUR series of vectors (Ruther and Muller-Hill,
EMBO J. 2:1791, 1983), pEX1-3 (Stanley and Luzio, EMBO J. 3:1429,
1984) and pMR100 (Gray et al., Proc. Natl. Acad. Sci. USA 79:6598,
1982). Vectors suitable for the production of intact native
proteins include pKC30 (Shimatake and Rosenberg, Nature 292:128,
1981), pKK177-3 (Amann and Brosius, Gene 40:183, 1985) and pET-3
(Studiar and Moffatt, J. Mol. Biol. 189:113, 1986). The STLV-3
subtype D fusion protein may be isolated from protein gels,
lyophilized, ground into a powder, and used as an antigen. The DNA
sequence can also be transferred to other cloning vehicles, such as
other plasmids, bacteriophages, cosmids, animal viruses, and yeast
artificial chromosomes (YACs) (Burke et al., Science 236:806-12,
1987). These vectors may then be introduced into a variety of hosts
including somatic cells, and simple or complex organisms, such as
bacteria, fungi (Timberlake and Marshall, Science 244:1313-7,
1989), invertebrates, plants (Gasser and Fraley, Science 244:1293,
1989), and mammals (Pursel et al., Science 244:1281-8, 1989), which
cell or organisms are rendered transgenic by the introduction of
one or more heterologous STLV-3 subtype D DNAs.
[0198] Various yeast strains and yeast-derived vectors are commonly
used for expressing and purifying proteins, for example, Pichia
pastoris expression systems are available from INVITROGEN.TM.
(Carlsbad, Calif.). Such systems include suitable Pichia pastoris
strains, vectors, reagents, transformants, sequencing primers and
media.
[0199] Non-yeast eukaryotic vectors can also be used for expression
of the STLV-3 subtype D proteins. Examples of such systems are the
well known Baculovirus system, the Ecdysone-inducible mammalian
expression system that uses regulatory elements from Drosophila
melanogaster to allow control of gene expression, and the Sindbis
viral expression system that allows high level expression in a
variety of mammalian cell lines. These expression systems are
available from INVITROGEN.TM..
[0200] For expression in mammalian cells, the cDNA sequence may be
ligated to heterologous promoters, such as the simian virus SV40,
promoter in the pSV2 vector (Mulligan and Berg, 1981, Proc. Natl.
Acad. Sci. USA 78:2072-6), and introduced into cells, such as
monkey COS-1 cells (Gluzman, Cell 23:175-82, 1981), to achieve
transient or long-term expression. The stable integration of the
chimeric gene construct may be maintained in mammalian cells by
biochemical selection, such as neomycin (Southern and Berg, J. Mol.
Appl. Genet. 1:327-41, 1982) and mycophoenolic acid (Mulligan and
Berg, Proc. Natl. Acad. Sci. USA 78:2072-6, 1981).
[0201] DNA sequences can be manipulated with standard procedures
such as restriction enzyme digestion, fill-in with DNA polymerase,
deletion by exonuclease, extension by terminal deoxynucleotide
transferase, ligation of synthetic or cloned DNA sequences,
site-directed sequence-alteration via single-stranded bacteriophage
intermediate or with the use of specific oligonucleotides in
combination with PCR.
[0202] The cDNA sequence (or portions derived from it) or a mini
gene (a cDNA with an intron and its own promoter) may be introduced
into eukaryotic expression vectors by conventional techniques.
These vectors are designed to permit the transcription of the cDNA
eukaryotic cells by providing regulatory sequences that initiate
and enhance the transcription of the cDNA and ensure its proper
splicing and polyadenylation. Vectors containing the promoter and
enhancer regions of the SV40 or long terminal repeat (LTR) of the
Rous Sarcoma virus and polyadenylation and splicing signal from
SV40 are readily available (Mulligan and Berg, Proc. Natl. Acad.
Sci. USA 78:2072-6, 1981; Gorman et al., Proc. Natl. Acad. Sci USA
78:6777-81, 1982). The level of expression of the cDNA can be
manipulated with this type of vector, either by using promoters
that have different activities (for example, the baculovirus pAC373
can express cDNAs at high levels in S. frugiperda cells (Summers
and Smith, 1985, Genetically Altered Viruses and the Environment,
Fields et al. (Eds.) 22:319-328, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.) or by using vectors that contain
promoters amenable to modulation, for example, the
glucocorticoid-responsive promoter from the mouse mammary tumor
virus (Lee et al., Nature 294:228, 1982). The expression of the
cDNA can be monitored in the recipient cells 24 to 72 hours after
introduction (transient expression).
[0203] In addition, some vectors contain selectable markers such as
the gpt (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-6,
1981) or neo (Southern and Berg, J. Mol. Appl. Genet. 1:327-41,
1982) bacterial genes. These selectable markers permit selection of
transfected cells that exhibit stable, long-term expression of the
vectors (and therefore the cDNA). The vectors can be maintained in
the cells as episomal, freely replicating entities by using
regulatory elements of viruses such as papilloma (Sarver et al.,
Mol. Cell Biol. 1:486, 1981) or Epstein-Barr (Sugden et al., Mol.
Cell Biol. 5:410, 1985). Alternatively, one can also produce cell
lines that have integrated the vector into genomic DNA. Both of
these types of cell lines produce the gene product on a continuous
basis. One can also produce cell lines that have amplified the
number of copies of the vector (and therefore of the cDNA as well)
to create cell lines that can produce high levels of the gene
product (Alt et al., J. Biol. Chem. 253:1357, 1978).
[0204] The transfer of DNA into eukaryotic, in particular human, or
other mammalian cells, is now a conventional technique. The vectors
are introduced into the recipient cells as pure DNA (transfection)
by, for example, precipitation with calcium phosphate (Graham and
vander Eb, 1973, Virology 52:466) or strontium phosphate (Brash et
al., Mol. Cell Biol. 7:2013, 1987), electroporation (Neumann et
al., EMBO J. 1:841, 1982), lipofection (Felgner et al., Proc. Natl.
Acad. Sci USA 84:7413, 1987), DEAE dextran (McCuthan et al., J.
Natl. Cancer Inst. 41:351, 1968), microinjection (Mueller et al.,
Cell 15:579, 1978), protoplast fusion (Schafner, Proc. Natl. Acad.
Sci. USA 77:2163-7, 1980), or pellet guns (Klein et al, Nature
327:70., 1987). Alternatively, the cDNA can be introduced by
infection with virus vectors. Systems are developed that use, for
example, retroviruses (Bernstein et al., Gen. Engrg. 7:235, 1985),
adenoviruses (Ahmad et al., J. Virol. 57:267, 1986), or Herpes
virus (Spaete et al., Cell 30:295, 1982).
[0205] Using the above techniques, the expression vectors
containing STLV-3 subtype D genes or cDNA sequence or fragments or
variants or mutants thereof can be introduced into human cells,
primate cells, mammalian cells from other species, or non-mammalian
cells as desired. The choice of cell is determined by the purpose
of the treatment. For example, monkey COS cells (Gluzman, Cell
23:175-82, 1981) that produce high levels of the SV40 T antigen and
permit the replication of vectors containing the SV40 origin of
replication may be used. Similarly, Chinese hamster ovary (CHO),
mouse NIH 3T3 fibroblasts or human fibroblasts or lymphoblasts may
be used.
[0206] One method that can be used to express STLV-3 subtype D
polypeptides from the cloned STLV-3 subtype D cDNA sequence in
mammalian cells is to use the cloning vector, pXT1. This vector is
commercially available from STRATAGENE.TM., contains the Long
Terminal Repeats (LTRs) and a portion of the GAG gene from Moloney
Murine Leukemia Virus. The position of the viral LTRs allows highly
efficient, stable transfection of the region within the LTRs. The
vector also contains the Herpes Simplex Thymidine Kinase promoter
(TK), active in embryonal cells and in a wide variety of tissues in
mice, and a selectable neomycin gene conferring G418 resistance.
Two unique restriction sites BglII and XhoI are directly downstream
from the TK promoter. STLV-3 subtype D cDNA, including the entire
open reading frame for an STLV-3 subtype D protein such as such as
a STLV-3 subtype D protease, capsid, or envelope protein is cloned
into one of the two unique restriction sites downstream from the
promoter.
[0207] The ligated product is transfected into mouse NIH 3T3 cells
using LIPOFECTIN.TM. (Life Technologies, Inc.) under conditions
outlined in the product specification. Positive transfectants are
selected after growing the transfected cells in 600 .mu.g/ml G418
(Sigma, St. Louis, Mo.). The protein is released into the
supernatant and may be purified by standard immunoaffinity
chromatography techniques using antibodies raised against STLV-3
subtype D proteins.
[0208] Expression of STLV-3 subtype D proteins in eukaryotic cells
can be used as a source of proteins to raise antibodies. The STLV-3
subtype D proteins may be extracted following release of the
protein into the supernatant as described above, or, the cDNA
sequence may be incorporated into a eukaryotic expression vector
and expressed as a chimeric protein with, for example,
.beta.-globin. Antibody to .beta.-globin is thereafter used to
purify the chimeric protein. Corresponding protease cleavage sites
engineered between the .beta.-globin gene and the cDNA are then
used to separate the two polypeptide fragments from one another
after translation. One useful expression vector for generating
.beta.-globin chimeric proteins is pSG5 (STRATAGENE.TM.). This
vector encodes rabbit .beta.-globin.
Methods of Detecting a STLV-3 Subtype D Nucleic Acid
[0209] A major application of the STLV-3 subtype D nucleic acid
sequences disclosed herein is for the detection of STLV-3 subtype D
virus in a sample, such as a biological sample obtained from a
subject that has or is suspected of having a STLV-3 subtype D
infection. Accordingly, methods for the detection of STLV-3 subtype
D are disclosed, for example to determine if a subject is infected
with STLV-3 subtype D. The methods described herein may be used for
any purpose where the detection of STLV-3 subtype D is desirable,
including diagnostic and prognostic applications, such as in
laboratory and clinical settings. A method for screening a subject
to determine if the subject has been infected with STLV-3 subtype D
is disclosed herein. In some examples, detection is performed for a
nucleic acid sequence of STLV-3 subtype D ORFs or the polypeptides
encoded by such ORFs, such as nucleotides 747-2009 of SEQ ID NO: 1,
nucleotides 1961-2494 of SEQ ID NO: 1, nucleotides 2416-5061 of SEQ
ID NO: 1 or nucleotides 5054-6535 of SEQ ID NO: 1. Any STLV-3
subtype D nucleic acid disclosed herein can be used in the
disclosed methods. In some examples, a nucleic acid sequence
encoding a STLV-3 subtype D envelope polypeptide is detected, such
as the nucleic acid sequence set forth as nucleotides 5054-6535 of
SEQ ID NO: 1 or a portion thereof. In some examples, a nucleic acid
sequence encoding a STLV-3 subtype D protease is detected, such as
nucleotides 1961-2494 of SEQ ID NO: 1 or a portion thereof. In some
examples, a nucleic acid sequence encoding a STLV-3 subtype D
capsid polypeptide is detected, such as nucleotides 747-2009 of SEQ
ID NO: 1 or a portion thereof. In some examples, the nucleic acid
sequence of a STLV-3 subtype D polymerase is detected, such as
nucleotides 2416-5061 of SEQ ID NO: 1 or a portion thereof. In some
examples, a nucleic acid sequence of a STLV-3 subtype D tax gene is
detected, such as SEQ ID NO: 25 or a portion thereof. In some
examples, a nucleic acid sequence of a STLV-3 subtype D rex gene is
detected, such as SEQ ID NO: 26 or a portion thereof.
[0210] In some embodiments, the disclosed methods include providing
a biological sample obtained from the subject, in which sample
includes DNA or RNA, and providing an assay for detecting in the
biological sample the presence of any of the STLV-3 subtype D
nucleic acids or proteins. Appropriate samples include any
conventional environmental or biological samples, including
clinical samples obtained from a human or veterinary subject, such
as a non-human primate. Suitable samples include all biological
samples useful for detection of viral infection in subjects,
including, but not limited to, cells, tissues, and bodily fluids,
such as: blood; derivatives and fractions of blood, such as serum;
extracted galls; biopsied or surgically removed tissue, including
tissues that are, for example, unfixed, frozen, fixed in formalin
and/or embedded in paraffin; tears; milk; skin scrapes; surface
washings; urine; sputum; cerebrospinal fluid; prostate fluid; pus;
bone marrow aspirates. In particular embodiments, the biological
sample is obtained from an animal subject, such as in the form of
blood.
[0211] In some embodiments, methods for the detection of STLV-3
subtype D nucleic acids in a sample, and thus STLV-3 subtype D in a
sample, include amplifying a STLV-3 subtype D nucleic acid from the
sample, for example using two or more oligonucleotide primers at
least 15 nucleotides in length that hybridize under very high
stringency conditions to a STLV-3 subtype D nucleic acid sequence
to produce amplified STLV-3 subtype D nucleic acids; and detecting
the amplified STLV-3 subtype D nucleic acid, wherein the presence
of an amplified STLV-3 subtype D nucleic acid indicates the
presence of the STLV-3 subtype D virus in the sample. These
include, but are not limited to, the nucleic acids sequences set
forth as SEQ ID NO: 1 or a portion thereof. In some examples, a
primer used to amplify a STLV-3 subtype D nucleic acid sequence
comprises P5TAXF3 (SEQ ID NO: 21), P5TAXR3 (SEQ ID NO: 22), P5TAXF2
(SEQ ID NO: 23), or P5TAXR1 (SEQ ID NO: 24). In specific examples,
a primer pair is used to amplify a STLV-3 subtype D nucleic acid
sequence. In some embodiments, the primer pair includes a first
primer containing the nucleic acid sequence set forth an SEQ ID NO:
21 and a second primer set forth as SEQ ID NO: 22. In some
embodiments, the primer pair includes a first primer containing the
nucleic acid sequence set forth an SEQ ID NO: 23 and a second
primer set forth as SEQ ID NO: 24.
[0212] In specific examples, amplification of the STLV-3 subtype D
nucleic acid includes the use of polymerase chain reaction (PCR),
real-time PCR, reverse transcriptase-polymerase chain reaction
(RT-PCR), real-time reverse transcriptase-polymerase chain reaction
(rt RT-PCR), ligase chain reaction, or transcription-mediated
amplification (TMA).
[0213] In some embodiments, methods for the detection of STLV-3
subtype D nucleic acids in a sample, and thus STLV-3 subtype D in a
sample, include contacting the sample with a probe including a
nucleic acid sequence at least 15 nucleotides in length that
hybridizes under very high stringency conditions to an STLV-3
subtype D nucleic acid sequence, such as an amplified STLV-3
subtype D nucleic acid sequence; and detecting hybridization
between the STLV-3 subtype D nucleic acid and the probe, wherein
the detection of hybridization indicates the presence of the STLV-3
subtype D virus in the sample. In specific non-limiting examples a
probe is selected such that it hybridizes under very high
stringency conditions to an STLV-3 subtype D nucleic acid, such as
but not limited to the sequence set forth as SEQ ID NO: 1 or a
portion thereof.
[0214] One embodiment of such detection techniques is the
polymerase chain reaction amplification of reverse transcribed RNA
(RT-PCR) of RNA isolated from cells (for example lymphocytes)
followed by direct DNA sequence determination of the products. The
presence of one or more STLV-3 subtype D nucleic acids is taken as
indicative of potential STLV-3 subtype D infection.
[0215] Oligonucleotides specific to normal, mutant or alternative
sequences can be chemically synthesized using commercially
available machines, labeled radioactively with isotopes (such as
.sup.32P) or non-radioactively, with tags such as biotin (Ward and
Langer et al., Proc. Natl. Acad. Sci. USA 78:6633-57, 1981), and
hybridized to individual DNA samples immobilized on membranes or
other solid supports by dot-blot or transfer from gels after
electrophoresis. The presence of these specific sequences are
visualized by methods such as autoradiography or fluorometric
(Landegren et al., Science 242:229-37, 1989) or colorimetric
reactions (Gebeyehu et al., Nucleic Acids Res. 15:4513-34, 1987).
The absence of hybridization would indicate that the subject is not
infected with STLV-3 subtype D.
[0216] Cloned DNA segments may be used as probes to detect specific
DNA segments. The sensitivity of this method is greatly enhanced
when combined with PCR (Wrichnik et al., Nucleic Acids Res.
15:529-42, 1987; Wong et al., Nature 330:384-6, 1987; Stoflet et
al., Science 239:491-4, 1988). In this approach, a sequencing
primer which lies within the amplified sequence is used with
double-stranded PCR product or single-stranded template generated
by a modified PCR. The sequence determination is performed by
conventional procedures with radiolabelled nucleotides or by
automatic sequencing procedures with fluorescent tags.
[0217] Sequence alterations may occasionally generate fortuitous
restriction enzyme recognition sites or may eliminate existing
restriction sites. Changes in restriction sites are revealed by the
use of appropriate enzyme digestion followed by conventional
gel-blot hybridization (Southern, J. Mol. Biol. 98:503, 1975). DNA
fragments carrying the site (either normal, mutant, or alternative)
are detected by their reduction in size or increase of
corresponding restriction fragment numbers. Genomic DNA samples may
also be amplified by PCR prior to treatment with the appropriate
restriction enzyme; fragments of different sizes are then
visualized under UV light in the presence of ethidium bromide after
gel electrophoresis.
[0218] In addition to conventional gel-electrophoresis and
blot-hybridization methods, DNA fragments may also be visualized by
methods where the individual DNA samples are not immobilized on
membranes. The probe and target sequences may be both in solution,
or the probe sequence may be immobilized (Saiki et al., Proc. Nat.
Acad. Sci. USA 86:6230-4, 1989). A variety of detection methods,
such as autoradiography involving radioisotopes, direct detection
of radioactive decay (in the presence or absence of scintillant),
spectrophotometry involving calorigenic reactions and fluorometry
involved fluorogenic reactions, may be used to identify STLV-3
subtype D.
[0219] In another embodiment, a melting curve analysis of the
amplified target nucleic acid can be performed subsequent to the
amplification process. The T.sub.m of a nucleic acid sequence
depends on the length of the sequence and its G/C content. Thus,
the identification of the T.sub.m for a nucleic acid sequence can
be used to identify the amplified nucleic acid, for example by
using double-stranded DNA binding dye chemistry, which quantitates
the amplicon production by the use of a non-sequence specific
fluorescent intercalating agent (such as SYBR-green or ethidium
bromide). SYBR green is a fluorogenic minor groove binding dye that
exhibits little fluorescence when in solution but emits a strong
fluorescent signal upon binding to double-stranded DNA. Typically,
SYBR green is used in singleplex reactions, however when coupled
with melting point analysis, it can be used for multiplex
reactions.
[0220] Any type of thermal cycler apparatus can be used for the
amplification of the STLV-3 subtype D nucleic acid, such as a
STLV-3 subtype D capsid, protease, or envelope nucleic acid, and/or
the determination of hybridization. Examples of suitable
apparatuses include a PTC-100.RTM. Peltier Thermal Cycler (MJ
Research, Inc.; San Francisco, Calif.), a ROBOCYCLER.RTM. 40
Temperature Cycler (STRATAGENE.TM.; La Jolla, Calif.), or a
GENEAMP.RTM. PCR System 9700 (Applied Biosystems; Foster City,
Calif.). For real-time PCR, any type of real-time thermocycler
apparatus can be used. For example, a BioRad iCycler iQ.TM.,
LIGHTCYCLER.TM. (Roche; Mannheim, Germany), a 7700 Sequence
Detector (Perkin Elmer/Applied Biosystems; Foster City, Calif.),
ABI.TM. systems such as the 7000, 7500, 7700, or 7900 systems
(Applied Biosystems; Foster City, Calif.), or an MX4000.TM.,
MX3000.TM. or MX3005.TM. (STRATAGENE.TM.; La Jolla, Calif.); DNA
Engine Opticon Continuous Fluorescence Detection System (MJ
Research); and Cepheid SMARTCYCLER.TM. can by used to amplify
nucleic acid sequences in real-time.
[0221] In some embodiments, detecting the presence of a STLV-3
subtype D nucleic acid sequence in a sample includes the extraction
of STLV-3 subtype D DNA. DNA extraction relates to releasing DNA
from a latent or inaccessible form in a cell or sample and allowing
the DNA to become freely available. In such a state, it is suitable
for effective detection and/or amplification of the STLV-3 subtype
D nucleic acid. Releasing DNA may include steps that achieve the
disruption of cells. Additionally, extraction of RNA may include
steps that achieve at least a partial separation of the RNA
dissolved in an aqueous medium from other cellular components,
wherein such components may be either particulate or dissolved.
[0222] In some embodiments, detecting the presence of a STLV-3
subtype D nucleic acid sequence in a sample includes the extraction
of STLV-3 subtype D RNA. RNA extraction relates to releasing RNA
from a latent or inaccessible form in a cell or sample and allowing
the RNA to become freely available. In such a state, it is suitable
for effective detection and/or amplification of the STLV-3 subtype
D nucleic acid. Releasing RNA may include steps that achieve the
disruption of cells. Extraction of RNA is generally carried out
under conditions that effectively exclude or inhibit any
ribonuclease activity that may be present. Additionally, extraction
of RNA may include steps that achieve at least a partial separation
of the RNA dissolved in an aqueous medium from other cellular
components, wherein such components may be either particulate or
dissolved.
[0223] One of ordinary skill in the art will know suitable methods
for extracting nucleic acids such as RNA and/or DNA from a sample;
such methods will depend upon, for example, the type of sample in
which the STLV-3 subtype D nucleic acid is found. For example, the
nucleic acids may be extracted using guanidinium isothiocyanate,
such as the single-step isolation by acid guanidinium
isothiocyanate-phenol-chloroform extraction of Chomczynski et al.
(Anal. Biochem. 162:156-59, 1987). The sample can be used directly
or can be processed, such as by adding solvents, preservatives,
buffers, or other compounds or substances. Nucleic acids can be
extracted using standard methods. For instance, rapid nucleic acid
preparation can be performed using a commercially available kit
(such as the QIAGEN.RTM. DNA Mini kit (QIAGEN.RTM.) Roche MagNA
Pure Compact Nucleic Acid Isolation Kit I or RNEASY.RTM. Mini Kit
(QIAGEN.RTM.); NUCLISENS.RTM. NASBA Diagnostics (bioMerieux); or
the MASTERPURE.TM. Complete DNA and RNA Purification Kit
(EPICENTRE.RTM.)).
[0224] In some embodiments, the probe is detectably labeled, either
with an isotopic or non-isotopic label; in alternative embodiments,
the STLV-3 subtype D nucleic acid is labeled. Non-isotopic labels
can, for instance, include a fluorescent or luminescent molecule,
or an enzyme, co-factor, enzyme substrate, or hapten. The probe is
incubated with a single-stranded or double-stranded preparation of
RNA, DNA, or a mixture of both, and hybridization determined. In
some examples, the hybridization results in a detectable change in
signal such as in increase or decrease in signal, for example from
the labeled probe. Thus, detecting hybridization can include
detecting a change in signal from the labeled probe during or after
hybridization relative to signal from the label before
hybridization.
STLV-3 Subtype D Proteins
[0225] Disclosed are STLV-3 subtype D polypeptides, such as STLV-3
subtype D polymerase polypeptides, STLV-3 subtype D envelope
polypeptides, STLV-3 subtype D protease polypeptides, STLV-3
subtype D capsid polypeptides, STLV-3 subtype D rex polypeptides,
and STLV-3 subtype D tax polypeptides.
[0226] In some embodiments, a STLV-3 subtype D Env polypeptide is
at least 95% identical, such as at least 96%, at least 97%, at
least 98%, at least 99%, or even 100% identical to the sequence set
forth as below as SEQ ID NO: 15:
TABLE-US-00004 (SEQ ID NO: 15).
MGKSSLFICLFCSYMASLFVPGDPSRCTLFIGASSYHSSPCGSNYPQCTW
TLDLVSLTRDQSLNPPCPDLVTYSQYHRPYSLYLFPHWITKPNRQGLGYY
SASYSDPCAIKCPYLGCQSWTCPYTGPMSSPYWKYTSDLNFTQKVSSVTL
HLHFSKCGSSFSLLLDAPGYDPVWFLSSQTTQAPPTPAPLTQDSDFQHIL
EPSVPWSSKILNLILLTLKSTNYSCMVCVDRSSLSSWHVLYDPLKVPKQH
EPRARALLRPSLAIPITNTTPPFPWSHCYCPLLQAVISNNCNNSVILPPF
SLSPVLDLSKPRQRRAVPIAVWLVSALAVGTGIAGGTTGSLSLASSRSLL
HEVDQDISHLTQAIVKNHNNILRVAQYAAQNRRGLDLLFWEQGGLCKAIR
EQCCFLNISNTHVSVLQERPPLEKRVITGWGLNWDLGLSQWAREALQTGI
TLLALFLLLIMVGPCVLRQLQALLFRLQHRSHPYSLLNRETNL
[0227] In some embodiments, a STLV-3 subtype D capsid polypeptide
is at least 95% identical, such as at least 96%, at least 97%, at
least 98%, at least 99%, or even 100% identical to the sequence set
forth as below as SEQ ID NO: 16:
TABLE-US-00005 (SEQ ID NO: 16).
MGNSYSRAANPIPKAPKGLAIHHWLNFLQAAYRLQPGPSEFDFHQLRNFL
KLAIKTPVWLNPINYSVLAELVPKNYPGRIQEITAILIQETSTQEVPPSA
PPASEPQNPPPYPEPGQAIPQCLPVLHPHGAPAAHRPWQMKDLQAIKQEV
TSSAPGSPQFMQTVRLAVQQFDPTAKDLHDLLQYLCSSLVASLHHQQLET
LIAQAETQGITGYNPLAGPLRVQANNPTQQGLRREYQNLWLSAFSALPGN
TKDPTWAAILQGPEEPFCTFVERLNVALDNGLPEGTPKEPILRSLAYSNA
NKECQKLLQARGQTNGPLGDMLRACQAWTPRDKNKVLMVQPKKTPPPNQP
CFRCGQAGHWSRDCKQPRPPPGPCPLCQDPTHWKRDCPQLKPDPEEGMLL
DLPCEDPAARDQKNFIGGED
[0228] In some embodiments, a STLV-3 subtype D protease polypeptide
is at least 95% identical, such as at least 96%, at least 97%, at
least 98%, at least 99%, or even 100% identical to the sequence set
forth as below as SEQ ID NO: 17;
TABLE-US-00006 (SEQ ID NO: 17).
PSGQRPKKLHRGGGLASPQTVLPFIPLSQQKQPVLHVRVSFPGTPPVSIQ
ALLDTGADVTVLPARLCPPDLKLQDTTVLGASGPSTDKFKVLPCFTYVHL
PFRGRPVTLPSCLIDINNQWAILGRDVLQQCQSSLYLADQPSRVLPIQTP
SVIGLEHLPPPPEVPQFPLNQSASRP
[0229] In some embodiments, a STLV-3 subtype D polymerase
polypeptide is at least 95% identical, such as at least 96%, at
least 97%, at least 98%, at least 99%, or even 100% identical to
the sequence set forth as below as SEQ ID NO: 18:
TABLE-US-00007 (SEQ ID NO: 18).
HWAGTSPPAPRSSTISVKPERLQALTDLVSKALEAKYIEPYQGPGNNPIF
PVKKPNGKWRFIHDLRATNCLTKTLTSPSPGPPDLTSLPQGLPHLRTIDL
TDAFFQIPLPVAFQPYFAFTLPQPNNHGPGARYSWKVLPQGFKNSPTLFE
QQLSHILTPVRQAFPKSIVIQYMDDILLASPTLEESIVLAQEITNALAQE
GLPMSTEKTQSTPGPIHFLGQTISKKYITYETLPTIHVKPNWTLTELQST
LGELQWVSKGTPTLRSSLHQLYTALRGHHDPRDTIQLTPPQLQALNTLQK
ALTHNCRSRIVSNLPILALIMLRPTGTTAVLFQTKQKWPLVWLHTPHPAT
SLRLWGQLLANAIITLDKYSLQHYGQVCKSFHHNISNQALTHYLHTSDQS
SVAILLQHSHRFHNLGAQPSGPWKGLLQVPQIFQNVATLSPPFTISPVVI
NHAPCLFSDGSNSQAAFTIWDKKIIHQQVLPLPTASSAQAGELFALLAAL
RECKPWSSLNIFLDSKFLVGQLRRLALGAFIGPSTQCDLHSQLLPLLYNK
TIYVHHVRSHTLLQDPISRLNEATDALMLAPLLPLSPATLHEITHCNPPA
LCNHGATATETKAIVRACHTCKITNPQGRLPQGHIRRGHAPNTIWQGDVT
HLQYKKYKYCLLVWVDTYSGAVAVSCRRKETSSECVASLLAAISILGKPH
TINTDNGAAYLSQEFQQFCTSLSIKHTTHVPYNPTSSGLVERTNGILKTL
ISKYLLDDHHLPLDTAISKTLWTINHLNVLSSCQKTRWQLHQAQPLPPVP
ENLPLPEPVPKWYYYKIPGLTSSRWSGPVQSVKEAAGAALIPVGTRHIWI
PWRLLKRGACPRPGDSVTTESKHKDLQLHG
[0230] In some embodiments, a STLV-3 subtype D rex polypeptide is
at least 95% identical, such as at least 96%, at least 97%, at
least 98%, at least 99%, or even 100% identical to the sequence set
forth as below as SEQ ID NO: 19:
TABLE-US-00008 (SEQ ID NO: 19).
MPKTRRQRNHRIKTQRPSTPWPTFQVSGRACSTGTLSTFSAIVCRPIGAP
FPGGFVPPGYIGTPYWPPVLNTRSPGTPSMDALSARLYNTLSLASPPSPP
KELPAPSRSSPRRPLLQPPKFLPPSSMQSGNTPLSETTASSSPWESNYPP
CLSPTPASDPKMSIPCGEAPSCAYTSTNSHLQ
[0231] In some embodiments, a STLV-3 subtype D tax polypeptide is
at least 95% identical, such as at least 96%, at least 97%, at
least 98%, at least 99%, or even 100% identical to the sequence set
forth as below as SEQ ID NO: 20:
TABLE-US-00009 (SEQ ID NO: 20).
MAHFPGFGQSLLYGYPVYVFGDCVQADWCPISGGLCSARLHRHALLATCP
EHQITWDPIDGRVVSSPLQYLIPRLPSFPTQRTSRTLKVLTPPPTATTPK
VPPSFFHAVRKHTPFRNNCLELTLGEQLPAMSFPDPGLRPQNVYTMWGST
IVCLYLYQLTPPMTWPLIPHVIFCHPDQLGAFLTKIPTKRLEELLYKLFL
STGAILILPENCFPTTLFQPTRAPVIQAPWHSGLLPYLKEIVTPGLIWVF
TDGSSMISGPCPKEGQPSLVVQSSTFIFQKFQTKAYHPAFLLSHKLIQYS
SFHSLHLLFEEYTTVPFSLLFNEKEANDSDSKPQGEPQLLAKGHTVESSV
Quantitation of STLV-3 Subtype D Proteins
[0232] An alternative method of detecting a STLV-3 subtype D virus
in a sample is to detect a STLV-3 subtype D protein in a sample,
for example a sample obtained form a subject to determine if the
subject has a STLV-3 subtype D infection, for example detecting a
STLV-3 subtype D viral protein. These include, but are not limited
to, the proteins encoded by the nucleic acid sequence set forth as
nucleotides 747-2009 of SEQ ID NO: 1, nucleotides 1961-2494 of SEQ
ID NO: 1, nucleotides 2416-5061 of SEQ ID NO: 1, nucleotides
5054-6535 of SEQ ID NO: 1, nucleotide 5054-5057 and 7232-8280 of
SEQ ID NO: 1, or nucleotides 4995-5057 and 7232-7717 of SEQ ID NO:
1. The methods typically include contacting a sample with an
antibody that specifically binds a STLV-3 subtype D polypeptide
(such as a monoclonal or a polyclonal antibody that specifically
binds a STLV-3 subtype D polypeptide), such as a STLV-3 subtype D
polypeptide encoded by the nucleotide sequence according to
nucleotides 747-2009 of SEQ ID NO: 1, nucleotides 1961-2494 of SEQ
ID NO: 1, nucleotides 2416-5061 of SEQ ID NO: 1, nucleotides
5054-6535 of SEQ ID NO: 1, SEQ ID NO: 25, or SEQ ID NO: 26, and
detecting binding of the antibody to a STLV-3 subtype D polypeptide
in the sample, wherein binding of the antibody to the polypeptide
indicates the presence of the STLV-3 subtype D polypeptide.
[0233] In some examples, the antibody is immobilized on a support
surface, such as in the wells of a microtiter plate or on a column.
The biological sample is then introduced onto the support surface
and allowed to interact with the antibody to form complexes. Excess
biological sample is then removed by washing, and the complexes are
detected with a reagent, such as a second anti-STLV-3 subtype D
polypeptide antibody, that is conjugated with a detectable
marker.
[0234] In some examples, the cellular proteins are isolated and
subjected to SDS-PAGE followed by Western blotting. After resolving
the proteins, the proteins are transferred to a membrane, which is
probed with a specific antibody that specifically binds a STLV-3
subtype D polypeptide. The STLV-3 subtype D polypeptide is
detected, for example with labeled (such as horseradish peroxidase,
HRP)-conjugated secondary antibodies, and quantitated.
[0235] In yet other examples, the level of one or more STLV-3
subtype D polypeptides in a cell is analyzed using microscopy. For
example, using an antibody that specifically binds a STLV-3 subtype
D polypeptide, such as but not limited to, a STLV-3 subtype D
polypeptide encoded by the nucleotide sequence according to
nucleotides 747-2009 of SEQ ID NO: 1, nucleotides 1961-2494 of SEQ
ID NO: 1, nucleotides 2416-5061 of SEQ ID NO: 1, nucleotides
5054-6535 of SEQ ID NO: 1, SEQ ID NO: 25, or SEQ ID NO: 26, samples
can be analyzed for the presence of one or more STLV-3 subtype D
polypeptides. For example, frozen biopsied tissue sections are
thawed at room temperature and fixed with acetone at -200.degree.
C. for 5 minutes. Slides are washed twice in cold PBS for 5 minutes
each, then air-dried. Sections are covered with 20-30 .mu.l of
antibody solution (15-45 .mu.g/ml) (diluted in PBS, 2% BSA at 15-50
.mu.g/ml) and incubated at room temperature in humidified chamber
for 30 minutes. Slides are washed three times with cold PBS 5
minutes each, allowed to air-dry briefly (5 minutes) before
applying 20-30 .mu.l of the second antibody solution (diluted in
PBS, 2% BSA at 15-50 .mu.g/ml) and incubated at room temperature in
humidified chamber for 30 minutes. The label on the second antibody
may contain a fluorescent probe, enzyme, radiolabel, biotin, or
other detectable marker. The slides are washed three times with
cold PBS 5 minutes each then quickly dipped in distilled water,
air-dried, and mounted with PBS containing 30% glycerol. Slides can
be stored at 4.degree. C. prior to viewing.
[0236] The foregoing methods of detecting STLV-3 subtype D may be
assembled in the form of a diagnostic kit and preferably comprises
either: hybridization with oligonucleotides; PCR amplification of
the gene or a part thereof using oligonucleotide primers; RT-PCR
amplification of the RNA or a part thereof using oligonucleotide
primers; or direct sequencing of any of the STLV-3 subtype D genes
present in a subject using oligonucleotide primers. The efficiency
of these molecular genetic methods should permit the rapid
identification of subjects infected with STLV-3 subtype D. Thus,
kits can include containers with STLV-3 subtype D nucleic acid
sequences (such as probes or primers) and/or containers including
an antibody that specifically binds STLV-3 subtype D.
STLV-3 Subtype D Virus Antibodies
[0237] A STLV-3 subtype D polypeptide or a fragment or conservative
variant thereof can be used to produce antibodies which are
immunoreactive or bind to an epitope of the STLV-3 subtype D
polypeptide. Accordingly, antibodies are disclosed (such as
monoclonal or polyclonal antibodies) that specifically bind a
STLV-3 subtype D polypeptide. In several example non limiting
examples, the antibody binds a STLV-3 subtype D polypeptide encoded
by the nucleotide sequence according nucleotides 747-2009 of SEQ ID
NO: 1, nucleotides 1961-2494 of SEQ ID NO: 1, nucleotides 2416-5061
of SEQ ID NO: 1, or nucleotides 5054-6535 of SEQ ID NO: 1, SEQ ID
NO: 25, or SEQ ID NO: 26. Polyclonal antibodies, antibodies which
consist essentially of pooled monoclonal antibodies with different
epitopic specificities, as well as distinct monoclonal antibody
preparations are included.
[0238] The preparation of polyclonal antibodies is well-known to
those skilled in the art. See, for example, Green et al.,
"Production of Polyclonal Antisera," in Immunochemical Protocols
pages 1-5, Manson, ed., Humana Press 1992; Coligan et al.,
"Production of Polyclonal Antisera in Rabbits, Rats, Mice and
Hamsters," in: Current Protocols in Immunology, section 2.4.1,
1992.
[0239] The preparation of monoclonal antibodies likewise is
conventional. See, for example, Kohler & Milstein, Nature
256:495, 1975; Coligan et al., sections 2.5.1-2.6.7; and Harlow et
al., in: Antibodies: a Laboratory Manual, page 726, Cold Spring
Harbor Pub., 1988. Briefly, monoclonal antibodies can be obtained
by injecting mice with a composition comprising an antigen,
verifying the presence of antibody production by removing a serum
sample, removing the spleen to obtain B lymphocytes, fusing the B
lymphocytes with myeloma cells to produce hybridomas, cloning the
hybridomas, selecting positive clones that produce antibodies to
the antigen, and isolating the antibodies from the hybridoma
cultures. Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well-established techniques.
Such isolation techniques include affinity chromatography with
Protein-A Sepharose, size-exclusion chromatography, and
ion-exchange chromatography. See, for example, Coligan et al.,
sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes et al.,
"Purification of Immunoglobulin G (IgG)," in: Methods in Molecular
Biology, Vol. 10, pages 79-104, Humana Press, 1992.
[0240] Methods of in vitro and in vivo multiplication of monoclonal
antibodies are well known to those skilled in the art.
Multiplication in vitro may be carried out in suitable culture
media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium,
optionally supplemented by a mammalian serum such as fetal calf
serum or trace elements and growth-sustaining supplements such as
normal mouse peritoneal exudate cells, spleen cells, thymocytes or
bone marrow macrophages. Production in vitro provides relatively
pure antibody preparations and allows scale-up to yield large
amounts of the desired antibodies. Large-scale hybridoma
cultivation can be carried out by homogenous suspension culture in
an airlift reactor, in a continuous stirrer reactor, or in
immobilized or entrapped cell culture. Multiplication in vivo may
be carried out by injecting cell clones into mammals
histocompatible with the parent cells, for example, syngeneic mice,
to cause growth of antibody-producing tumors. Optionally, the
animals are primed with a hydrocarbon, especially oils such as
pristane (tetramethylpentadecane) prior to injection. After one to
three weeks, the desired monoclonal antibody is recovered from the
body fluid of the animal.
[0241] Antibodies can also be derived from subhuman primate
antibody. General techniques for raising therapeutically useful
antibodies in baboons can be found, for example, in WO 91/11465,
1991, and Losman et al., Int. J. Cancer 46:310, 1990.
[0242] Alternatively, an antibody that specifically binds a STLV-3
subtype D polypeptide can be derived from a humanized monoclonal
antibody. Humanized monoclonal antibodies are produced by
transferring mouse complementarity determining regions from heavy
and light variable chains of the mouse immunoglobulin into a human
variable domain, and then substituting human residues in the
framework regions of the murine counterparts. The use of antibody
components derived from humanized monoclonal antibodies obviates
potential problems associated with the immunogenicity of murine
constant regions. General techniques for cloning murine
immunoglobulin variable domains are described, for example, by
Orlandi et al., Proc. Nat'l Acad. Sci. U.S.A. 86:3833, 1989.
Techniques for producing humanized monoclonal antibodies are
described, for example, by Jones et al., Nature 321:522, 1986;
Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science
239:1534, 1988; Carter et al., Proc. Nat'l Acad. Sci. U.S.A.
89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singer
et al., J. Immunol. 150:2844, 1993.
[0243] Antibodies can be derived from human antibody fragments
isolated from a combinatorial immunoglobulin library. See, for
example, Barbas et al., in: Methods: a Companion to Methods in
Enzymology, Vol. 2, page 119, 1991; Winter et al., Ann. Rev.
Immunol. 12:433, 1994. Cloning and expression vectors that are
useful for producing a human immunoglobulin phage library can be
obtained, for example, from STRATAGENE.RTM. Cloning Systems (La
Jolla, Calif.).
[0244] In addition, antibodies can be derived from a human
monoclonal antibody. Such antibodies are obtained from transgenic
mice that have been "engineered" to produce specific human
antibodies in response to antigenic challenge. In this technique,
elements of the human heavy and light chain loci are introduced
into strains of mice derived from embryonic stem cell lines that
contain targeted disruptions of the endogenous heavy and light
chain loci. The transgenic mice can synthesize human antibodies
specific for human antigens, and the mice can be used to produce
human antibody-secreting hybridomas. Methods for obtaining human
antibodies from transgenic mice are described by Green et al.,
Nature Genet. 7:13, 1994; Lonberg et al., Nature 368:856, 1994; and
Taylor et al., Int. Immunol. 6:579, 1994.
[0245] Antibodies include intact molecules as well as fragments
thereof, such as Fab, F(ab').sub.2, and Fv which are capable of
binding the epitopic determinant. Methods of making these fragments
are known in the art. (See for example, Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New
York, 1988). An epitope is any antigenic determinant on an antigen
to which the paratope of an antibody binds. Epitopic determinants
usually consist of chemically active surface groupings of molecules
such as amino acids or sugar side chains and usually have specific
three dimensional structural characteristics, as well as specific
charge characteristics.
[0246] Antibody fragments can be prepared by proteolytic hydrolysis
of the antibody or by expression in E. coli of DNA encoding the
fragment. Antibody fragments can be obtained by pepsin or papain
digestion of whole antibodies by conventional methods. For example,
antibody fragments can be produced by enzymatic cleavage of
antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent, and optionally a blocking group for the sulfhydryl
groups resulting from cleavage of disulfide linkages, to produce
3.5S Fab' monovalent fragments. Alternatively, an enzymatic
cleavage using pepsin produces two monovalent Fab' fragments and an
Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat. No.
4,331,647, and references contained therein; Nisonhoff et al.,
Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119,
1959; Edelman et al., Methods in Enzymology, Vol. 1, page 422,
Academic Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10
and 2.10.1-2.10.4).
[0247] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical, or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0248] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association may be noncovalent (Inbar et
al., Proc. Nat'l Acad. Sci. U.S.A. 69:2659, 1972). Alternatively,
the variable chains can be linked by an intermolecular disulfide
bond or cross-linked by chemicals such as glutaraldehyde. See, for
example, Sandhu, supra. Preferably, the Fv fragments comprise
V.sub.H and V.sub.L chains connected by a peptide linker. These
single-chain antigen binding proteins (sFv) are prepared by
constructing a structural gene comprising DNA sequences encoding
the V.sub.H and V.sub.L domains connected by an oligonucleotide.
The structural gene is inserted into an expression vector, which is
subsequently introduced into a host cell such as E. coli. The
recombinant host cells synthesize a single polypeptide chain with a
linker peptide bridging the two V domains. Methods for producing
sFvs are known in the art (see Whitlow et al., Methods: a Companion
to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et al.,
Science 242:423, 1988; U.S. Pat. No. 4,946,778; Pack et al.,
Bio/Technology 11:1271, 1993; and Sandhu, supra).
[0249] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing cells
(Larrick et al., Methods: a Companion to Methods in Enzymology,
Vol. 2, page 106, 1991).
[0250] Antibodies can be prepared using an intact polypeptide or
fragments containing small peptides of interest as the immunizing
antigen. The polypeptide or a peptide used to immunize an animal
can be derived from substantially purified polypeptide produced in
host cells, in vitro translated cDNA, or chemical synthesis, which
can be conjugated to a carrier protein, if desired. Such commonly
used carriers which are chemically coupled to the peptide include
keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum
albumin (BSA), and tetanus toxoid. The coupled peptide is then used
to immunize the animal (for example, a mouse, a rat, or a
rabbit).
[0251] Polyclonal or monoclonal antibodies can be further purified,
for example, by binding to and elution from a matrix to which the
polypeptide or a peptide to which the antibodies were raised is
bound. Those of skill in the art will know of various techniques
common in the immunology arts for purification and/or concentration
of polyclonal antibodies, as well as monoclonal antibodies (See for
example, Coligan et al., Unit 9, Current Protocols in Immunology,
Wiley Interscience, 1991).
[0252] It is also possible to use the anti-idiotype technology to
produce monoclonal antibodies, which mimic an epitope. For example,
an anti-idiotypic monoclonal antibody made to a first monoclonal
antibody will have a binding domain in the hypervariable region
that is the "image" of the epitope bound by the first monoclonal
antibody.
[0253] Effector molecules, such as therapeutic, diagnostic, or
detection moieties (for example labels), can be linked to an
antibody that specifically binds a STLV-3 subtype D polypeptide,
using any number of means known to those of skill in the art. Both
covalent and noncovalent attachment means may be used. The
procedure for attaching an effector molecule to an antibody varies
according to the chemical structure of the effector. Polypeptides
typically contain a variety of functional groups; for example,
carboxylic acid (COOH), free amine (--NH.sub.2) or sulfhydryl
(--SH) groups, which are available for reaction with a suitable
functional group on an antibody to result in the binding of the
effector molecule. Alternatively, the antibody is derivatized to
expose or attach additional reactive functional groups. The
derivatization may involve attachment of any of a number of linker
molecules such as those available from Pierce Chemical Company,
Rockford, Ill. The linker can be any molecule used to join the
antibody to the effector molecule. The linker is capable of forming
covalent bonds to both the antibody and to the effector molecule.
Suitable linkers are well known to those of skill in the art and
include, but are not limited to, straight or branched-chain carbon
linkers, heterocyclic carbon linkers, or peptide linkers. Where the
antibody and the effector molecule are polypeptides, the linkers
may be joined to the constituent amino acids through their side
groups (such as through a disulfide linkage to cysteine) or to the
alpha carbon amino and carboxyl groups of the terminal amino
acids.
[0254] In some circumstances, it is desirable to free the effector
molecule from the antibody when the immunoconjugate has reached its
target site. Therefore, in these circumstances, immunoconjugates
will comprise linkages that are cleavable near the target site.
Cleavage of the linker to release the effector molecule from the
antibody may be prompted by enzymatic activity or conditions to
which the immunoconjugate is subjected either inside the target
cell or near the target site.
[0255] In view of the large number of methods that have been
reported for attaching a variety of radiodiagnostic compounds,
radiotherapeutic compounds, label (for example enzymes or
fluorescent molecules) drugs, toxins, and other agents to
antibodies, one skilled in the art will be able to determine a
suitable method for attaching a given effector molecule to an
antibody or other polypeptide.
[0256] The immunoconjugates can be prepared by cloning techniques.
Examples of appropriate cloning and sequencing techniques, and
instructions sufficient to direct persons of skill through many
cloning exercises are found in Sambrook et al., Molecular Cloning:
A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor
Laboratory (1989), Berger and Kimmel (eds.), Guide to Molecular
Cloning Techniques, Academic Press, Inc., San Diego Calif. (1987),
or Ausubel et al. (eds.), Current Protocols in Molecular Biology,
Greene Publishing and Wiley-Interscience, NY (1987). Product
information from manufacturers of biological reagents and
experimental equipment also provide useful information. Such
manufacturers include the SIGMA chemical company (Saint Louis,
Mo.), R&D systems (Minneapolis, Minn.), Pharmacia LKB
Biotechnology (Piscataway, N.J.), CLONTECH.RTM. oratories, Inc.
(Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company
(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life
Technologies, Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika
Analytika (Fluka Chemie AG, Buchs, Switzerland), INVITROGEN.TM.
(San Diego, Calif.), and Applied Biosystems (Foster City, Calif.),
as well as many other commercial sources known to one of skill
[0257] Nucleic acids encoding native effector molecules or
anti-STLV-3 subtype D antibodies can be modified to form the
effector molecule, antibodies, or immunoconjugates. Modification by
site-directed mutagenesis is well known in the art. Nucleic acids
encoding effector molecule or anti-STLV-3 subtype D antibodies can
be amplified by in vitro methods. Amplification methods include the
polymerase chain reaction (PCR), the ligase chain reaction (LCR),
the transcription-based amplification system (TAS), the
self-sustained sequence replication system (3SR). A wide variety of
cloning methods, host cells, and in vitro amplification
methodologies are well known in the art.
[0258] In one embodiment, immunoconjugates are prepared by
inserting a cDNA which encodes an anti-STLV-3 subtype D polypeptide
scFv antibody into a vector which comprises the cDNA encoding the
effector molecule. The insertion is made so that the scFv and the
EM are read in frame that is in one continuous polypeptide, which
contains a functional Fv region and a functional EM region.
[0259] In addition to recombinant methods, the immunoconjugates,
effector molecules, and antibodies can also be constructed in whole
or in part using standard peptide synthesis. Solid phase synthesis
of the polypeptides of less than about 50 amino acids in length may
be accomplished by attaching the C-terminal amino acid of the
sequence to an insoluble support followed by sequential addition of
the remaining amino acids in the sequence. Techniques for solid
phase synthesis are described by Barany & Merrifield, "The
Peptides: Analysis, Synthesis, Biology," Vol. 2, Special Methods in
Peptide Synthesis, Part A. pp. 3-284; Merrifield et al. J. Am.
Chem. Soc. 85:2149-2156, 1963, and Stewart et al., Solid Phase
Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill., 1984.
Proteins of greater length may be synthesized by condensation of
the amino and carboxyl termini of shorter fragments. Methods of
forming peptide bonds by activation of a carboxyl terminal end (for
example, by the use of the coupling reagent
N,N'-dicycylohexylcarbodiimide) are known to those of skill
[0260] Once the nucleic acids encoding an EM, anti-STLV-3 subtype D
antibody, or an immunoconjugate, are isolated and cloned, one may
express the desired protein in a recombinantly engineered cell such
as bacteria, plant, yeast, insect and mammalian cells. It is
expected that those of skill in the art are knowledgeable in the
numerous expression systems available for expression of proteins
including E. coli, other bacterial hosts, yeast, and various higher
eukaryotic cells such as the COS, CHO, HeLa and myeloma cell
lines.
[0261] Antibodies can be covalently or non-covalently linked to a
detectable label. Detectable labels suitable for such use include
any composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical means.
Useful labels in the present disclosure include magnetic beads (for
example DYNABEADS.RTM.), fluorescent dyes (for example, fluorescein
isothiocyanate, Texas red, rhodamine, green fluorescent protein,
and the like), radiolabels (for example, .sup.3H, .sup.125I,
.sup.35S, .sup.14C, or .sup.32P), enzymes (for example, horseradish
peroxidase, alkaline phosphatase and others commonly used in an
ELISA), and colorimetric labels such as colloidal gold or colored
glass or plastic (for example, polystyrene, polypropylene, latex,
etc.) beads.
[0262] The detecting such labels are well known to those of skill
in the art. Thus, for example, radiolabels may be detected using
photographic film or scintillation counters, fluorescent markers
may be detected using a photodetector to detect emitted
illumination. Enzymatic labels are typically detected by providing
the enzyme with a substrate and detecting the reaction product
produced by the action of the enzyme on the substrate, and
colorimetric labels are detected by simply visualizing the colored
label.
Immunogenic Compositions and Therapeutic Methods
[0263] Any of the STLV-3 subtype D polypeptides and nucleic acid
molecules encoding the STLV-3 subtype D polypeptides disclosed
herein can be used as immunogens, or to produce immunogens to
elicit an immune response (for example as an immunogenic
composition) to a STLV-3 subtype D polypeptide or a to a STLV-3
subtype D polypeptide expressing virus. These compositions are of
use, for example, to reduce STLV-3 subtype D infection or a symptom
of STLV-3 subtype D infection. Following administration of a
therapeutically effective amount of the disclosed immunogenic
composition, the subject can be monitored for STLV-3 subtype D
infection, symptoms associated with STLV-3 subtype D infection, or
both. Disclosed herein are methods of administering the therapeutic
molecules disclosed herein (such as STLV-3 subtype D polypeptides
and nucleic acids encoding STLV-3 subtype D polypeptides) to reduce
STLV-3 subtype D infection. In several non-limiting examples, a
therapeutically effective amount of a STLV-3 subtype D polypeptide
encoded by nucleotides 747-2009 of SEQ ID NO: 1, nucleotides
1961-2494 of SEQ ID NO: 1, nucleotides 2416-5061 of SEQ ID NO: 1,
SEQ ID NO: 25, SEQ ID NO: 26, or a immunogenic fragment thereof is
administered to a subject.
[0264] In certain embodiments, the immunogenic composition includes
an adjuvant. An adjuvant can be a suspension of minerals, such as
alum, aluminum hydroxide, aluminum phosphate, on which antigen is
adsorbed; or water-in-oil emulsion in which antigen solution is
emulsified in oil (MF-59, Freund's incomplete adjuvant), sometimes
with the inclusion of killed mycobacteria (Freund's complete
adjuvant) to further enhance antigenicity (inhibits degradation of
antigen and/or causes influx of macrophages). In one embodiment,
the adjuvant is a mixture of stabilizing detergents,
micelle-forming agent, and oil available under the name PROVAX.RTM.
(IDEC Pharmaceuticals, San Diego, Calif.). An adjuvant can also be
an immunostimulatory nucleic acid, such as a nucleic acid including
a CpG motif.
[0265] In one example, the immunogenic composition is mixed with an
adjuvant containing two or more of a stabilizing detergent, a
micelle-forming agent, and an oil. Suitable stabilizing detergents,
micelle-forming agents, and oils are detailed in U.S. Pat. No.
5,585,103; U.S. Pat. No. 5,709,860; U.S. Pat. No. 5,270,202; and
U.S. Pat. No. 5,695,770, all of which are incorporated by reference
herein in their entirety. A stabilizing detergent is any detergent
that allows the components of the emulsion to remain as a stable
emulsion. Such detergents include polysorbate 80 (TWEEN)
(Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl;
manufactured by ICI Americas, Wilmington, Del.), TWEEN 40.TM.,
TWEEN 20.TM., TWEEN 60.TM., ZWITTERGENT.TM. 3-12, TEEPOL HB7.TM.,
and SPAN 85.TM.. These detergents are usually provided in an amount
of approximately 0.05 to 0.5%, such as at about 0.2%. A micelle
forming agent is an agent which is able to stabilize the emulsion
formed with the other components such that a micelle-like structure
is formed. Such agents generally cause some irritation at the site
of injection in order to recruit macrophages to enhance the
cellular response. Examples of such agents include polymer
surfactants described by BASF Wyandotte publications, for example,
Schmolka, J. Am. Oil. Chem. Soc. 54:110, 1977, and Hunter et al.,
J. Immunol. 129:1244, 1981, PLURONIC.TM. L62LF, L101, and L64,
PEG1000, and TETRONIC.TM. 1501, 150R1, 701, 901, 1301, and 130R1.
The chemical structures of such agents are well known in the art.
In one embodiment, the agent is chosen to have a
hydrophile-lipophile balance (HLB) of between 0 and 2, as defined
by Hunter and Bennett, J. Immun. 133:3167, 1984. The agent can be
provided in an effective amount, for example between 0.5 and 10%,
or in an amount between 1.25 and 5%.
[0266] The oil included in the composition is chosen to promote the
retention of the antigen in oil-in-water emulsion, to provide a
vehicle for the desired antigen, and preferably has a melting
temperature of less than 65.degree. C. such that emulsion is formed
either at room temperature (about 20.degree. C. to 25.degree. C.),
or once the temperature of the emulsion is brought down to room
temperature. Examples of such oils include squalene, Squalane,
EICOSANE.TM., tetratetracontane, glycerol, and peanut oil or other
vegetable oils. In one specific, non-limiting example, the oil is
provided in an amount between 1 and 10%, or between 2.5 and 5%. The
oil should be both biodegradable and biocompatible so that the body
can break down the oil over time, and so that no adverse affects,
such as granulomas, are evident upon use of the oil.
[0267] Immunogenic compositions can be formulated with an
appropriate solid or liquid carrier, depending upon the particular
mode of administration chosen. If desired, the disclosed
pharmaceutical compositions can also contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate. Excipients that can
be included in the disclosed compositions include flow conditioners
and lubricants, for example silicic acid, talc, stearic acid or
salts thereof, such as magnesium or calcium stearate, and/or
polyethylene glycol, or derivatives thereof.
[0268] Immunogenic compositions can be provided as parenteral
compositions, such as for injection or infusion. Such compositions
are formulated generally by mixing a disclosed therapeutic agent at
the desired degree of purity, in a unit dosage injectable form
(solution, suspension, or emulsion), with a pharmaceutically
acceptable carrier, for example one that is non-toxic to recipients
at the dosages and concentrations employed and is compatible with
other ingredients of the formulation. In addition, a disclosed
therapeutic agent can be suspended in an aqueous carrier, for
example, in an isotonic buffer solution at a pH of about 3.0 to
about 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to
6.0, or 3.5 to about 5.0. Useful buffers include sodium
citrate-citric acid and sodium phosphate-phosphoric acid, and
sodium acetate/acetic acid buffers. The active ingredient,
optionally together with excipients, can also be in the form of a
lyophilisate and can be made into a solution prior to parenteral
administration by the addition of suitable solvents. Solutions such
as those that are used, for example, for parenteral administration
can also be used as infusion solutions.
[0269] A form of repository or "depot" slow release preparation can
be used so that therapeutically effective amounts of the
preparation are delivered into the bloodstream over many hours or
days following transdermal injection or delivery. Such long acting
formulations can be administered by implantation (for example
subcutaneously or intramuscularly) or by intramuscular injection.
The compounds can be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, or as sparingly soluble derivatives,
for example, as a sparingly soluble salt.
[0270] Immunogenic compositions that include a disclosed
therapeutic agent can be delivered by way of a pump (see Langer,
supra; Sefton, CRC Crit. Ref Biomed. Eng. 14:201, 1987; Buchwald et
al., Surgery 88:507, 1980; Saudek et al., N. Engl. J. Med. 321:574,
1989) or by continuous subcutaneous infusions, for example, using a
mini-pump. An intravenous bag solution can also be employed. One
factor in selecting an appropriate dose is the result obtained, as
measured by the methods disclosed here, as are deemed appropriate
by the practitioner. Other controlled release systems are discussed
in Langer (Science 249:1527-33, 1990).
[0271] In one example, a pump is implanted (for example see U.S.
Pat. Nos. 6,436,091; 5,939,380; and 5,993,414). Implantable drug
infusion devices are used to provide subjects with a constant and
long-term dosage or infusion of a therapeutic agent. Such device
can be categorized as either active or passive.
[0272] Active drug or programmable infusion devices feature a pump
or a metering system to deliver the agent into the subject's
system. An example of such an active infusion device currently
available is the Medtronic SYNCHROMED.TM. programmable pump.
Passive infusion devices, in contrast, do not feature a pump, but
rather rely upon a pressurized drug reservoir to deliver the agent
of interest. An example of such a device includes the Medtronic
ISOMED.TM..
[0273] In particular examples, immunogenic compositions including a
disclosed therapeutic agent are administered by sustained-release
systems. Suitable examples of sustained-release systems include
suitable polymeric materials (such as, semi-permeable polymer
matrices in the form of shaped articles, for example films, or
mirocapsules), suitable hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, and
sparingly soluble derivatives (such as, for example, a sparingly
soluble salt). Sustained-release compositions can be administered
orally, parenterally, intracistemally, intraperitoneally, topically
(as by powders, ointments, gels, drops or transdermal patch), or as
an oral or nasal spray. Sustained-release matrices include
polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of
L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al.,
Biopolymers 22:547-556, 1983, poly(2-hydroxyethyl methacrylate));
(Langer et al., J. Biomed. Mater. Res. 15:167-277, 1981; Langer,
Chem. Tech. 12:98-105, 1982, ethylene vinyl acetate (Langer et al.,
Id.) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
[0274] Polymers can be used for ion-controlled release. Various
degradable and nondegradable polymeric matrices for use in
controlled drug delivery are known in the art (Langer, Accounts
Chem. Res. 26:537, 1993). For example, the block copolymer,
polaxamer 407 exists as a viscous yet mobile liquid at low
temperatures but forms a semisolid gel at body temperature. It has
shown to be an effective vehicle for formulation and sustained
delivery of recombinant interleukin-2 and urease (Johnston et al.,
Pharm. Res. 9:425, 1992; and Pec, J. Parent. Sci. Tech. 44(2):58,
1990). Alternatively, hydroxyapatite has been used as a
microcarrier for controlled release of proteins (Ijntema et al.,
Int. J. Pharm. 112:215, 1994). In yet another aspect, liposomes are
used for controlled release as well as drug targeting of the
lipid-capsulated drug (Betageri et al., Liposome Drug Delivery
Systems, Technomic Publishing Co., Inc., Lancaster, Pa., 1993).
Numerous additional systems for controlled delivery of therapeutic
proteins are known (for example, U.S. Pat. No. 5,055,303; U.S. Pat.
No. 5,188,837; U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728;
U.S. Pat. No. 4,837,028; U.S. Pat. No. 4,957,735; and U.S. Pat. No.
5,019,369; U.S. Pat. No. 5,055,303; U.S. Pat. No. 5,514,670; U.S.
Pat. No. 5,413,797; U.S. Pat. No. 5,268,164; U.S. Pat. No.
5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No. 5,506,206; U.S.
Pat. No. 5,271,961; U.S. Pat. No. 5,254,342; and U.S. Pat. No.
5,534,496).
[0275] Immunogenic compositions can be administered for therapeutic
treatments. In therapeutic applications, a therapeutically
effective amount of the immunogenic composition is administered to
a subject suffering from a disease, such as STLV-3 subtype D
infection. The immunogenic composition can be administered by any
means known to one of skill in the art (see Banga, "Parenteral
Controlled Delivery of Therapeutic Peptides and Proteins," in
Therapeutic Peptides and Proteins, Technomic Publishing Co., Inc.,
Lancaster, Pa., 1995) such as by intramuscular, subcutaneous, or
intravenous injection, but even oral, nasal, or anal administration
is contemplated. To extend the time during which the peptide or
protein is available to stimulate a response, the peptide or
protein can be provided as an implant, an oily injection, or as a
particulate system. The particulate system can be a microparticle,
a microcapsule, a microsphere, a nanocapsule, or similar particle
(see, for example, Banga, supra). A particulate carrier based on a
synthetic polymer has been shown to act as an adjuvant to enhance
the immune response, in addition to providing a controlled release.
Aluminum salts can also be used as adjuvants to produce an immune
response.
[0276] Immunogenic compositions can be formulated in unit dosage
form, suitable for individual administration of precise dosages. In
pulse doses, a bolus administration of an immunogenic composition
that includes a disclosed immunogen is provided, followed by a
time-period wherein no disclosed immunogen is administered to the
subject, followed by a second bolus administration. A
therapeutically effective amount of an immunogenic composition can
be administered in a single dose, or in multiple doses, for example
daily, during a course of treatment. In specific, non-limiting
examples, pulse doses of an immunogenic composition that include a
disclosed immunogen are administered during the course of a day,
during the course of a week, or during the course of a month.
[0277] Immunogenic compositions can be administered whenever the
effect (such as decreased signs, symptom, or laboratory results of
STLV-3 subtype D infection) is desired. Generally, the dose is
sufficient to treat or ameliorate symptoms or signs of disease
without producing unacceptable toxicity to the subject. Systemic or
local administration can be utilized.
[0278] Amounts effective for therapeutic use can depend on the
severity of the disease and the age, weight, general state of the
subject, and other clinical factors. Thus, the final determination
of the appropriate treatment regimen will be made by the attending
clinician. Typically, dosages used in vitro can provide useful
guidance in the amounts useful for in situ administration of the
pharmaceutical composition, and animal models may be used to
determine effective dosages for treatment of particular disorders.
Various considerations are described, for example in Gilman et al.,
eds., Goodman and Gilman: The Pharmacological Bases of
Therapeutics, 8th ed., Pergamon Press, 1990; and Remington's
Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton,
Pa., 1990. Typically, the dose range for a STLV-3 subtype D
polypeptide is from about 0.1 .mu.g/kg body weight to about 100
mg/kg body weight. Other suitable ranges include doses of from
about 1 .mu.g/kg to 10 mg/kg body weight. In one example, the dose
is about 1.0 .mu.g to about 50 mg, for example, 1 .mu.g to 1 mg,
such as 1 mg peptide per subject. The dosing schedule can vary from
daily to as seldom as once a year, depending on clinical factors,
such as the subject's sensitivity to the peptide and tempo of their
disease. Therefore, a subject can receive a first dose of a
disclosed therapeutic molecule, and then receive a second dose (or
even more doses) at some later time(s), such as at least one day
later, such as at least one week later.
[0279] The pharmaceutical compositions disclosed herein can be
prepared and administered in dose units. Solid dose units include
tablets, capsules, transdermal delivery systems, and suppositories.
The administration of a therapeutic amount can be carried out both
by single administration in the form of an individual dose unit or
else several smaller dose units and also by multiple
administrations of subdivided doses at specific intervals. Suitable
single or divided doses include, but are not limited to about 0.01,
0.1, 0.5, 1, 3, 5, 10, 15, 30, or 50 .mu.g protein/kg/day.
[0280] The nucleic acid constructs encoding antigenic STLV-3
subtype D polypeptides described herein are used, for example, in
combination, as pharmaceutical compositions (medicaments) for use
in therapeutic, for example, prophylactic regimens (such as
vaccines) and administered to subjects (for example, primate
subjects such as human subjects) to elicit an immune response
against STLV-3 subtype D. For example, the compositions described
herein can be administered to a human (or non-human) subject prior
to infection with STLV-3 subtype D to inhibit infection by or
replication of the virus. Thus, the pharmaceutical compositions
described above can be administered to a subject to elicit a
protective immune response against STLV-3 subtype D. To elicit an
immune response, a therapeutically effective (for example,
immunologically effective) amount of the nucleic acid constructs
are administered to a subject, such as a human (or non-human)
subject.
[0281] Immunization by nucleic acid constructs is well known in the
art and taught, for example, in U.S. Pat. No. 5,643,578 (which
describes methods of immunizing vertebrates by introducing DNA
encoding a desired antigen to elicit a cell-mediated or a humoral
response), and U.S. Pat. No. 5,593,972 and U.S. Pat. No. 5,817,637
(which describe operably linking a nucleic acid sequence encoding
an antigen to regulatory sequences enabling expression). U.S. Pat.
No. 5,880,103 describes several methods of delivery of nucleic
acids encoding immunogenic peptides or other antigens to an
organism. The methods include liposomal delivery of the nucleic
acids (or of the synthetic peptides themselves), and
immune-stimulating constructs, or ISCOMS.TM., negatively charged
cage-like structures of 30-40 nm in size formed spontaneously on
mixing cholesterol and QUIL A.TM. (saponin).
[0282] For administration of STLV-3 subtype D nucleic acid
molecules, the nucleic acid can be delivered intracellularly, for
example by expression from an appropriate nucleic acid expression
vector which is administered so that it becomes intracellular, such
as by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or
by direct injection, or by use of microparticle bombardment (such
as a gene gun; Biolistic, Dupont), or coating with lipids or
cell-surface receptors or transfecting agents, or by administering
it in linkage to a homeobox-like peptide which is known to enter
the nucleus (for example Joliot et al., Proc. Natl. Acad. Sci. USA
1991, 88:1864-8). The present disclosure includes all forms of
nucleic acid delivery, including synthetic oligos, naked DNA,
plasmid and viral, integrated into the genome or not.
[0283] In another approach to using nucleic acids for immunization,
an immunogenic STLV-3 subtype D polypeptide can also be expressed
by attenuated viral hosts or vectors or bacterial vectors.
Recombinant vaccinia virus, adeno-associated virus (AAV), herpes
virus, retrovirus, or other viral vectors can be used to express
the peptide or protein, thereby eliciting a CTL response. For
example, vaccinia vectors and methods useful in immunization
protocols are described in U.S. Pat. No. 4,722,848. BCG (Bacillus
Calmette Guerin) provides another vector for expression of the
peptides (see Stover, Nature 351:456-460, 1991).
[0284] In one example, a viral vector is utilized. These vectors
include, but are not limited to, adenovirus, herpes virus,
vaccinia, or an RNA virus such as a retrovirus. In one example, the
retroviral vector is a derivative of a murine or avian retrovirus.
Examples of retroviral vectors in which a single foreign gene can
be inserted include, but are not limited to: Moloney murine
leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),
murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
When the subject is a human, a vector such as the gibbon ape
leukemia virus (GaLV) can be utilized. A number of additional
retroviral vectors can incorporate multiple genes. All of these
vectors can transfer or incorporate a gene for a selectable marker
so that transduced cells can be identified and generated. By
inserting a nucleic acid sequence encoding a STLV-3 subtype D
polypeptide into the viral vector, along with another gene that
encodes the ligand for a receptor on a specific target cell, for
example, the vector is now target specific. Retroviral vectors can
be made target specific by attaching, for example, a sugar, a
glycolipid, or a protein. Preferred targeting is accomplished by
using an antibody to target the retroviral vector. Those of skill
in the art will know of, or can readily ascertain without undue
experimentation, specific polynucleotide sequences which can be
inserted into the retroviral genome or attached to a viral envelope
to allow target specific delivery of the retroviral vector
containing the polynucleotide encoding a STLV-3 subtype D
polypeptide.
[0285] Since recombinant retroviruses are defective, they need
assistance in order to produce infectious vector particles. This
assistance can be provided, for example, by using helper cell lines
that contain plasmids encoding all of the structural genes of the
retrovirus under the control of regulatory sequences within the
LTR. These plasmids are missing a nucleotide sequence that enables
the packaging mechanism to recognize an RNA transcript for
encapsidation. Helper cell lines that have deletions of the
packaging signal include, but are not limited to Q2, PA317, and
PA12, for example. These cell lines produce empty virions, since no
genome is packaged. If a retroviral vector is introduced into such
cells in which the packaging signal is intact, but the structural
genes are replaced by other genes of interest, the vector can be
packaged and vector virion produced.
[0286] Suitable formulations for the nucleic acid constructs,
include aqueous and non-aqueous solutions, isotonic sterile
solutions, which can contain anti-oxidants, buffers, and
bacteriostats, and aqueous and non-aqueous sterile suspensions that
can include suspending agents, solubilizers, thickening agents,
stabilizers, and preservatives. The formulations can be presented
in unit-dose or multi-dose sealed containers, such as ampules and
vials, and can be stored in a freeze-dried (lyophilized) condition
requiring only the addition of the sterile liquid carrier, for
example, water, immediately prior to use. Extemporaneous solutions
and suspensions can be prepared from sterile powders, granules, and
tablets. Preferably, the carrier is a buffered saline solution.
More preferably, the composition for use in the inventive method is
formulated to protect the nucleic acid constructs from damage prior
to administration. For example, the composition can be formulated
to reduce loss of the adenoviral vectors on devices used to
prepare, store, or administer the expression vector, such as
glassware, syringes, or needles. The compositions can be formulated
to decrease the light sensitivity and/or temperature sensitivity of
the components. To this end, the composition preferably comprises a
pharmaceutically acceptable liquid carrier, such as, for example,
those described above, and a stabilizing agent selected from the
group consisting of polysorbate 80, L-arginine,
polyvinylpyrrolidone, trehalose, and combinations thereof.
[0287] In therapeutic applications, a therapeutically effective
amount of the composition is administered to a subject prior to or
following exposure to or infection by STLV-3 subtype D. When
administered prior to exposure, the therapeutic application can be
referred to as a prophylactic administration (such as in the form
of a vaccine). Single or multiple administrations of the
compositions are administered depending on the dosage and frequency
as required and tolerated by the subject. In one embodiment, the
dosage is administered once as a bolus, but in another embodiment
can be applied periodically until a therapeutic result, such as a
protective immune response, is achieved. Generally, the dose is
sufficient to treat or ameliorate symptoms or signs of disease
without producing unacceptable toxicity to the subject. Systemic or
local administration can be utilized.
[0288] In the context of nucleic acid vaccines, naturally occurring
or synthetic immunostimulatory compositions that bind to and
stimulate receptors involved in innate immunity can be administered
along with nucleic acid constructs encoding the STLV-3 subtype D
polypeptides. For example, agents that stimulate certain Toll-like
receptors (such as TLR7, TLR8 and TLR9) can be administered in
combination with the nucleic acid constructs encoding STLV-3
subtype D polypeptides. In some embodiments, the nucleic acid
construct is administered in combination with immunostimulatory CpG
oligonucleotides.
[0289] Nucleic acid constructs encoding STLV-3 subtype D
polypeptides can be introduced in vivo as naked DNA plasmids. DNA
vectors can be introduced into the desired host cells by methods
known in the art, including but not limited to transfection,
electroporation (for example, transcutaneous electroporation),
microinjection, transduction, cell fusion, DEAE dextran, calcium
phosphate precipitation, use of a gene gun, or use of a DNA vector
transporter (See for example, Wu et al. J. Biol. Chem.,
267:963-967, 1992; Wu and Wu J. Biol. Chem., 263:14621-14624, 1988;
and Williams et al. Proc. Natl. Acad. Sci. USA 88:2726-2730, 1991).
As described in detail in the Examples, a needleless delivery
device, such as a BIOJECTOR.RTM. needleless injection device can be
utilized to introduce the therapeutic nucleic acid constructs in
vivo. Receptor-mediated DNA delivery approaches can also be used
(Curiel et al. Hum. Gene Ther., 3:147-154, 1992; and Wu and Wu, J.
Biol. Chem., 262:4429-4432, 1987). Methods for formulating and
administering naked DNA to mammalian muscle tissue are disclosed in
U.S. Pat. Nos. 5,580,859 and 5,589,466, both of which are herein
incorporated by reference. Other molecules are also useful for
facilitating transfection of a nucleic acid in vivo, such as a
cationic oligopeptide (for example, WO95/21931), peptides derived
from DNA binding proteins (for example, WO96/25508), or a cationic
polymer (for example, WO95/21931).
[0290] Another well known method that can be used to introduce
nucleic acid constructs encoding STLV-3 subtype D immunogens into
host cells is particle bombardment (also know as biolistic
transformation). Biolistic transformation is commonly accomplished
in one of several ways. One common method involves propelling inert
or biologically active particles at cells. This technique is
disclosed in, for example, U.S. Pat. Nos. 4,945,050, 5,036,006; and
5,100,792, all to Sanford et al., which are hereby incorporated by
reference. Generally, this procedure involves propelling inert or
biologically active particles at the cells under conditions
effective to penetrate the outer surface of the cell and to be
incorporated within the interior thereof. When inert particles are
utilized, the plasmid can be introduced into the cell by coating
the particles with the plasmid containing the exogenous DNA.
Alternatively, the target cell can be surrounded by the plasmid so
that the plasmid is carried into the cell by the wake of the
particle.
[0291] Alternatively, the vector can be introduced in vivo by
lipofection. For the past decade, there has been increasing use of
liposomes for encapsulation and transfection of nucleic acids in
vitro. Synthetic cationic lipids designed to limit the difficulties
and dangers encountered with liposome mediated transfection can be
used to prepare liposomes for in vivo transfection of a gene
encoding a marker (Felgner et. al. Proc. Natl. Acad. Sci. USA
84:7413-7417, 1987; Mackey, et al. Proc. Natl. Acad. Sci. USA
85:8027-8031, 1988; Ulmer et al. Science 259:1745-1748, 1993). The
use of cationic lipids can promote encapsulation of negatively
charged nucleic acids, and also promote fusion with negatively
charged cell membranes (Felgner and Ringold Science 337:387-388,
1989). Particularly useful lipid compounds and compositions for
transfer of nucleic acids are described in WO95/18863 and
WO96/17823, and in U.S. Pat. No. 5,459,127, herein incorporated by
reference.
[0292] As with the immunogenic polypeptide, the nucleic acid
compositions may be administered in a single dose, or multiple
doses separated by a time interval can be administered to elicit an
immune response against STLV-3 subtype D. For example, two doses,
or three doses, or four doses, or five doses, or six doses or more
can be administered to a subject over a period of several weeks,
several months or even several years, to optimize the immune
response.
EXAMPLES
Example 1
[0293] This example describes the material and methods used to
obtain STLV-3 subtype D sequences.
Sample Collection and Preparation
[0294] Self-identified hunters were recruited from 17 villages in
southern Cameroon and were trained to collect dried blood spots
(DBS) from freshly collected monkey bushmeat. Preliminary species
identification of hunted non-human primates (NHPs) was determined
using pictographs of NHPs common in the region. Hunters were not
given incentives for collection of the bushmeat samples but were
educated about the risks associated with direct contact with
primate samples and were instructed on appropriate prevention
measures. A total of 362 DBS from hunted NHPs was collected on
Whatman filter paper, air-dried at room temperature, and
temporarily stored in envelopes with silica gels. Specimens were
then stored at -20.degree. C. until processed. Nucleic acids were
extracted from DBS using the NUCLISENS.RTM. nucleic acid isolation
kits (Biomerieux, Durham, N.C.). Briefly, DBS were incubated in
lysis buffer for 2 hours at room temperature, nucleic acids were
eluted from a silica suspension with wash buffer containing
guanidine thiocyanate. Ethanol-precipitated nucleic acids were
resuspended in 50 .mu.l of water and stored at 4.degree. C. until
tested. DNA quality and yield were determined by semi-quantitative
PCR amplification of the .beta.-actin gene according to standard
procedures (Switzer et al. Transplantation 71:959-96, 2001).
Primate T-Cell Lymphoma/Leukaemia Viruses (PTLV) Sequence
Amplification and NHP Species Identification
[0295] NHP DNAs were tested for tax sequences using generic, nested
PCR assays capable of detecting viruses from all four major primate
T-cell lymphoma/leukaemia viruses (PTLV) groups as described by
Busch et al. (Transfusion 40:443-449, 2000, incorporated herein by
reference) and Van Dooren et al. (J. Gen. Virol. 85:507-519, 2004,
incorporated herein by reference). Phylogenetic resolution within
the identified PTLV groups was achieved by analysis of long
terminal repeat (LTR) sequences obtained with PCR primers specific
for each PTLV group. PCR amplification of overlapping regions of
the 5' and 3' STLV-1 LTR was performed using primers and conditions
described by Meertens et al. (Virology 287:275-285, 2001,
incorporated herein by reference). STLV-3 LTR sequences were
obtained using PCR primers and conditions reported by Wolfe et al.
(Proc. Natl. Acad. Sci. U.S.A. 102:7994-7999, 2005).
STLV-3 Subtype D Specific PCR Assay
[0296] Confirmation of primate species was done by analysis of
mitochondrial cytochrome oxidase subunit II (COXII) and
glucose-6-phosphate dehydrogenase (G6PD) sequences PCR-amplified
from DBS DNA using primers PCO2F2 and PCO2R1, or GPDF1 and GPDR1,
respectively (Switzer et al. Nature 434:376-380, 2005, incorporated
herein by reference).
[0297] PCR products were visualized on 1.8% agarose gels stained
with ethidium bromide and were purified with QIAQUICK.RTM. PCR or
gel purification kits (QIAGEN.RTM., Valencia, Calif.). Using an ABI
3130x1 sequencer, purified amplicons were either directly sequenced
on both strands using ABI PRISM.RTM. Big Dye terminator kits
(Foster City, Calif.), or after cloning into a TOPO.RTM. vector
(INVITROGEN.TM.).
Identification of a Novel PTLV Group
[0298] Using a PCR-based genome walking approach (Switzer et al. J.
Virol. 80:7427-7438, 2006, incorporated herein by reference), new
primer sets were designed to amplify partial fragments of the viral
genome (see Table 1). Larger tax sequences (658-bp and 656-bp) were
amplified from animals Cmo8699AB and Cni7867AB, respectively with
external primers 8699TF1 and PGTAXR1 and internal primers 8699TF2
and PGTAXR2, with 40 cycles of standard PCR conditions and
annealing temperatures of 45.degree. C. and 50.degree. C.,
respectively. Overlapping sequences from animal Cmo8699AB from the
3' end of the tax gene to LTR were obtained by semi-nested PCR
using the external and internal forward primers 8699TF6 and 8699TF8
with reverse primer PGTATA1+2R1. For animal Cni7867AB, the internal
primers 8699TF7 and PGTATA1+2R1 were used to amplify the tax-LTR
fragment. The remainder of the LTR sequences from both animals were
amplified by semi-nested PCR with external primers 8699LF3 and
PGPBSR1n and internal primers 8699LF4 and PGPBSR1n. The PCR
reactions included 40 cycles of standard PCR conditions, an
annealing temperature of 45.degree. C. and primer extension time of
2.5 minutes.
Nucleotide Sequence Accession Numbers
[0299] GenBank accession numbers for the STLV-1 LTR, STLV-3 LTR,
STLV-3 (Cmo8699AB) tax-LTR, and small tax sequences are
EU152271-EU152276, EU152277-EU152279, EU152280-EU152281, and
EU152282-EU152293, respectively.
TABLE-US-00010 TABLE 1 PCR Primers for Amplification of the Viral
Genome Primer Name Sequence SEQ ID NO: 8699TF1
GTACCCTGTCTACGTTTTCGGCGAT 4 PGTAXR1
GAIGA(T/C)TGIA(C/G)TAC(T/C)AAAGATGGCTG 5 8699TF2
TTACTGGCCACCTGTCCTGAACAC 6 PGTAXR2 TTIGGG(T/C)AIGGICCGGAAATCAT 7
8699TF6 CATCCGGACCAACTAGGGGCCTTC 8 8699TF8 CAGCCCACCCGCGCACCAGTAATT
9 PGTATA1 + 2R1 TCCTGAAC(T/C)GTC(T/C)(T/C)(T/C)(A/G)CGCTTTTATAG 10
8699TF7 AACAAAAATCCCTACCAAACGCTT 11 8699LF3
CTCTGACGTCTCTCCCTGCCTTGT 12 PGPBSR1n ATCCCGGACGAGCCCCCA 13 8699LF4
CCGGAAAAAACCTTAAACCACCCA 14
[0300] An 325-bp env gene region of STLV-3 subtype D was amplified
using generic and nested forward primers, PGENVF1 and PGENVF2, and
reverse primers, PGENVF2 and PGENVR2, respectively, in standard PCR
conditions as described by Switzer et al. (J. Virol. 80:7427-7438,
2006, incorporated herein by reference).
Sequence Analysis
[0301] Percent nucleotide divergence was calculated using the GAP
program in the Genetics Computer Group (GCG) Wisconsin package
(Womble, Methods Mol. Biol. 132:3-22, 2000). Sequences were aligned
using the Clustal W program followed by manual editing. Gaps were
removed and distance-based phylogenetic trees were generated using
the Kimura two-parameter model together with the neighbor-joining
method in the MEGA program (version 3.1) and maximum-likelihood
(ML) analysis in the PAUP* program (Switzer et al. Nature
434:376-380, 2005).
[0302] The reliability of the final topology of the trees was
tested with 1,000 bootstrap replicates. PTLV diversity was analyzed
using the phylogeny inferred from the larger tax sequences using
the TreePAT package of the TreeDyn software build 198.3. TreePAT
generates a visual representation of a phylogenetic tree in a
pairwise distance matrix of the branch lengths of the tree between
each pair of taxa, with a distance of zero for a taxa to itself.
Ranges of genetic distances, or classes, were empirically
investigated based on accepted PTLV taxonomic groups using
available full-length genomes. Colors are assigned to each class
with the distance matrix being colored so that taxa within a given
distance class appear with their respective colors as squares along
the diagonal of the matrix allowing for a visual comparison of
divergence levels between taxa and/or viral groups.
Dating the Origin of STLV-3
[0303] Additional molecular analyses were performed to estimate the
divergence times of the most recent common ancestor (MRCA) of
STLV-3 (from animal Cmo8699AB). The molecular clock hypothesis was
not rejected for the 881-bp alignment of PTLV and BLV tax sequences
in both the PAUP* and Tree-Puzzle analyses (P=0.012 and 0.858,
respectively). For this analyses, the molecular clock was enforced
and calibrated the tree using a value of 40,000-60,000 years ago
(ya) estimated for the origin of the Melanesian HTLV-1. By using
these dates and methods, the evolutionary rate for PTLV was
estimated to be 9.17.times.10.sup.-7 to 1.38.times.10.sup.-6
substitutions/site/year which is consistent with rates determined
previously both with and without enforcing a molecular clock. The
evolutionary rate for STLV-3 (from animal Cmo8699AB) is estimated
to be 2.11.times.10.sup.-6 to 3.16.times.10.sup.-6 and the MRCA is
inferred to have occurred about 92,072-138,560 ya suggesting an
ancient origin and perhaps the identification of one of the oldest
viruses in the PTLV-3 group.
[0304] A total of 362 DBS representing 12 primate and prosimian
species were collected from 4 sites in southern Cameroon. From
these, 215 DBS (60%) had adequate blood spot quality and quantity
for nucleic acid extraction. Of the 215 samples tested, 170 (79%)
yielded adequate DNA integrity through the amplification of the
.beta.-actin gene (Table 2). The presence of blood clots and
limited volumes of blood on some DBS may account for the poor DNA
yield of some samples.
TABLE-US-00011 TABLE 2 Distribution of PTLV in wild-caught simian
and pro-simian species # .beta.-actin # tax # STLV-1 # STLV- Common
# DBS positive positive.sup.2 LTR 3 LTR Species name
extracted.sup.1 (%) (%) positive positive Old World Monkeys
Cercocebus agilis agile 6 3 (50) 3 (100) 2 1 mangabey Cercopithecus
moustached 41 32 (78) 0 0 0 cephus monkey Cercopithecus mona mona
40 36 (90) 1 (2.7) 0 1 monkey Cercopithecus De Brazza's 1 1 (100) 0
0 0 neglectus monkey Cercopithecus spot-nosed 98 73 (74.5) 7 (9.6)
4 2 nictitans monkey Cercopithecus crowned 9 8 (88.8) 0 0 0
pogonias monkey Colobus guereza guereza 3 2 (66.7) 0 0 0 colobus
Lophocebus grey- 10 9 (90) 1 (11.1) 0 1 albigena cheeked monkey
Prosimian Arctocebus aureus golden 2 1 (50) 0 0 0 angwantibo
Arctocebus calabar calabarensis angwantibo 2 2 (100) 0 0 0 Galago
alleni Allen's galago 1 1 (100) 0 0 0 Perodicticus potto potto 2 2
(100) 0 0 0 Total 215 170 (79.1) 12 (7.1) 6 (3.5) 5 (2.9)
.sup.1DBS, dried blood spots .sup.2samples testing negative for
.beta.-actin sequences were not tested for PTLV sequences
[0305] High PTLV diversity and geographic distribution were
observed among wild monkeys hunted for bush meat in southern
Cameroon. Of the 170 samples screened, 12 (7%) from four NHP
species were positive for PTLV tax sequences using a generic PCR
assay (Table 3). Phylogenetic analysis of the short tax sequences
from these 12 samples showed that 7 NHPs (two Cercocebus agilis and
five Cercopithecus nictitans) were infected with STLV-1, while 3
NHPs (C. agilis, C. nictitans, and Lophocebus albigena) were
infected with STLV-3 (FIG. 2 and Table 3). No evidence was observed
of infection of C. agili with STLV-2, HTLV-4-like STLV, or dual
STLV-1 and STLV-3s.
[0306] The samples obtained from animals Cmo8699AB and Cni7867AB,
both collected near the same village but from two different primate
species, were found to contain nearly identical STLV sequences with
highest nucleotide identity to viruses in the PTLV-3 group, but
exhibited high divergence in this small region of tax (FIG. 2,
Table 4). BLAST analysis of these divergent tax sequences
identified sequence similarity (.about.92-93%) to very short
STLV-3-like tax sequences (.about.219-bp) from four C. nictitans
from southern Cameroon (Cni217, Cni227, Cni3034, and Cni3038;
GenBank accession numbers AY039033, AF412120, AM746663, and,
AM746660, respectively) (Table 4). However, further phylogenetic
analysis of STLV-3 (from animal Cmo8699AB) and STLV-3 (from animal
Cni7867AB) including the small tax sequences from 3 of the 4 C.
nictitans (Cni3034 was omitted because it had a shorter but
identical tax sequence to Cni3038) and other STLV-3-infected
species (L. albigena, C. agilis, and C. cephus) from the same
region showed that the new STLV-3 (viral subtype D) viruses cluster
tightly with high bootstrap support (99) as a distinct monophyletic
subtype of STLV-3 (FIG. 3). Since there is generally less than 3%
nucleotide divergence within viral subtypes and up to 15%
nucleotide divergence between viral subtypes in the tax region, the
7% divergence seen in the tax sequences of STLV-3 (from animal
Cmo8699AB) and STLV-3 (from animal Cni7867AB), and the clustering
of these viruses outside the diversity of other STLV-3-like viruses
demonstrate that this virus (denoted STLV-3 subtype D) is a new and
highly divergent PTLV-3 subtype (FIG. 3, Table 4). Complete LTR
sequences were obtained for 11 of 12 PTLV-positive samples using
overlapping primer pairs.
TABLE-US-00012 TABLE 3 PTLV infection of wild-caught nonhuman
primates from Cameroon PTLV No. Code Species Common name Site
Province (subtype) 1 Cag9812NL Cercocebus agilis agile mangabey
Ngoila East STLV-1 (f) 2 Cag9813NL Cercocebus agilis agile mangabey
Ngoila East STLV-1 (f) 3 Cag9748NL Cercocebus agilis agile mangabey
Ngoila East STLV-3 (b) 4 Cmo8699AB Cercopithecus mona mona monkey
Abat Southwest STLV-3 (d) 5 Cni10026NL Cercopithecus nictitans
spot-nosed monkey Ngoila East STLV-1.sup.1 6 Cni10225NL
Cercopithecus nictitans spot-nosed monkey Ngoila East STLV-1 (d) 7
Cni8284NY Cercopithecus nictitans spot-nosed monkey Nyabissan South
STLV-1 (d) 8 Cni8286NY Cercopithecus nictitans spot-nosed monkey
Nyabissan South STLV-1 (d) 9 Cni8348NY Cercopithecus nictitans
spot-nosed monkey Nyabissan South STLV-1 (d) 10 Cni7882AB
Cercopithecus nictitans spot-nosed monkey Abat Southwest STLV-3 (b)
11 Cni7867AB Cercopithecus nictitans spot-nosed monkey Abat
Southwest STLV-3 (d) 12 Lal9589NL Lophocebus albigena grey-cheeked
monkey Ngoila East STLV-3 (b) .sup.1subtype not determined.
Phylogenetic Resolution of a New STLV-3 Subtype
[0307] The identification of a new STLV lineage in DBS from animals
Cmo8699AB and Cni7867AB was investigated further by additional
analyses of a larger tax sequence (1015-bp) obtained from the DBS
DNA of these two monkeys. The tax sequences from both monkeys were
nearly identical (99.9%) despite nucleic acid extraction, PCR
amplification, and sequencing for both animals all being done on
different days. Analysis of mitochondrial DNA (mtDNA) sequences
also confirmed the different Cercopithecus species of each monkey
and the absence of admixtures of specimens from different NHP
species. Nearly identical STLVs have also been previously reported
in monkeys and apes living in close geographic proximity indicating
the relative ease with which these viruses jump species boundaries.
The STLV-3(Cmo8699AB) tax sequences were nearly equidistant from
all other PTLV groups sharing approximately 72-74% nucleotide
identity with PTLV-1, PTLV-2, and PTLV-4 sequences but having
closer genetic identity to the PTLV-3 group (82-84%) in this highly
conserved region where intragroup sequence identity is typically
>90% (Table in FIG. 7). The nucleotide identities of tax
sequences from Cmo8699AB and Cni7867AB are more consistent with the
observed intergroup sequence identity that ranges from 71 to 83%
(Table in FIG. 7). Indeed phylogenetic analysis of 881-bp tax
sequences (FIG. 4) from these two monkeys (Cmo8699AB and Cni7867AB)
with other PTLVs, using BLV as an outgroup, inferred a new lineage
with very high bootstrap support (99) from the diversity of other
PTLV-3 subtypes (larger tax sequences representing PTLV-3 subtype C
were not available for inclusion in this analysis), suggesting a
long independent evolution and the possibility of a yet to be
identified human counterpart for these viruses.
[0308] Similar results were obtained by analysis of 275-bp LTR
sequences (FIG. 5), where STLV-3 (from animal Cmo8699AB) and STLV-3
(from animal Cni7867AB) had only 70-74% identity to LTRs from
members of the PTLV-3 group which share greater than 84% nucleotide
identity between subtypes A and B. LTR sequences from other
STLV-3-infected C. agilis and C. nictitans from Cameroon reported
elsewhere were not available at GENBANK.RTM. and thus were not
included in the current phylogenetic analysis. Combined, the
phylogenetic analyses of the tax sequences (FIGS. 3 and 4) and LTR
(FIG. 5) show that STLV-3 (from animal Cmo8699AB) and STLV-3 (from
animal Cni7867AB) both form a distinct cluster with high bootstrap
support from the other known PTLV-3 subtypes. Based on nomenclature
proposed by others, our results demonstrate that these viruses are
members of a novel PTLV-3 subtype tentatively named as STLV-3 West
African subtype D.
[0309] Partial fragments of the env, tax, and LTR regions for the
two novel STLV-3-like samples were sequenced (see Table 4). The
174-bp tax fragments for CMO8699AB and CNI7867AB are identical,
both showing 91% sequence homology to STLV-3 (CTO-604) as well as
the two recently described HTLV-3 viruses (2026ND and Pyl43) from
separate individuals in southern Cameroon with reported primate
contact. Using specifically designed primers, sequence analysis of
the entire tax fragment (1018-bp) for both specimens yielded 85%
sequence homology to STLV-3 (TG-2117 and PH969) found in baboons.
Analysis of the env gene region revealed that CMO8699AB and
CNI7867AB shared 95% genetic identity to STLV-1 (Tan90) and 80% to
STLV-3 (CTO-NG409), respectively. Cloning of a portion of the tax
gene fragment for the former specimen resulted in identical clones,
indicating that the recombinant sequences are not due to mixed
infections of STLV-1 and STLV-3.
TABLE-US-00013 TABLE 4 STLV-3 subtype D Genome Sequences Nucleotide
Virus Gene/Region SEQ ID NO: positions STLV-3 subtype D LTR 1 7-706
STLV-3 subtype D Gag 1 747-2009 STLV-3 subtype D Protease 1
1961-2494 STLV-3 subtype D Pol 1 2416-5061 STLV-3 subtype D Env 1
5054-6535 STLV-3 subtype D tax 1 (SEQ ID NO: 25) 5054-5057 and
7232-8280 STLV-3 subtype D rex 1 (SEQ ID NO: 25) 4995-5057 and
7232-7717
[0310] Phylogenetic analysis of the tax region for these novel
sequences clearly supports a significant divergence from the PTLV-3
cluster, indicating that these are the first sequences of a new
PTLV group found in the same region of Cameroon.
[0311] Primate COII genes were amplified and sequenced to confirm
species identification. Eleven of the 12 PTLV-positive specimens
were correctly identified using the pictographs and confirmed
through mitochondrial DNA analysis. One sample, identified as C.
agilis, had high sequence homology to C. torquatus.
[0312] The tax region showed 98% homology to STLV-3 (PPAF2), STLV-3
(CT604), as well as HTLV-3 (2026ND), but diverged in a rooted
phylogenetic tree analysis of this region. Sequencing and
phylogenetic analysis of the LTR region revealed 94% identity and
clustered with the LTR of STLV-3 CT604 (C. torquatus).
[0313] The use of field-collected DBS in collaboration with hunters
provides a good surveillance tool for emerging infections at the
primate-hunter interface. Samples collected on DBS yielded
sufficient viral DNA for PCR analysis and sequencing.
[0314] Based on the samples collected, the prevalence of PTLV among
wild monkeys hunted for bush meat in southern Cameroon was found to
be 7%, which is comparable to previously published reports. Four of
the 8 primate species collected and tested were shown to harbor
PTLVs.
[0315] Sequence analyses of the env, tax, and LTR gene regions of
CMO8699AB and CNI7867AB indicate that this novel group is highly
divergent from all known PTLV-3 subtypes. The discovery of a novel
PTLV subtype (identified herein as STLV-3 subtype D) in the same
region where two novel HTLV groups were identified, contributes to
the growing evidence that PTLVs have greater diversity and
geographic distribution than previously acknowledged. More
surveillance of wild primates in contact with human populations
particularly via bush meat hunting is needed.
[0316] Bush meat hunting, a common practice in many parts of
Africa, has been suggested to be an ideal interface for
cross-species transmission of retroviruses between primates and
humans. Contact with bodily fluids and blood during hunting and
butchering of bush meat exposes humans to a plethora of
retroviruses, and increases the likelihood of emerging diseases in
humans.
Broad STLV-3 Diversity in Wild NHPs from Cameroon
[0317] Sequence analysis of the STLV-3 LTR sequences from animals
Cni7882AB, Cag9748NL, and Lal9589NL showed that all were infected
with distinct STLV-3s. LTR sequences (283-bp) from animal Cag9748NL
shared the greatest identity (.gtoreq.97%) with those from HTLV-3
(Pyl43) and STLV-3 (Cto604) from a red-capped mangabey from
Cameroon. The 282-bp LTR sequence from Cni7882AB shared the highest
nucleotide identity (99%) to STLV-3 (CtoNG409), a red-capped
mangabey from neighboring Nigeria. The phylogeographic clustering
of these sequences supports further the proposed subtype
classification of STLV-3 by geographic origin rather than by host
species. In contrast, the 432-bp LTR sequence from L. albigena
(Lal9589NL) was more divergent sharing only 10-16% nucleotide
identity with all PTLV-3 LTR sequences. Similar to the phylogenetic
relationships inferred with the small tax sequences, the LTR
sequence from L. albigena (Lal9589NL) formed a new lineage within
the diversity of other PTLV-3 sequences from west-central Africa
(FIG. 5). Although these results will need to be confirmed with
additional LTR sequences from this virus and from other
STLV-3-infected L. albigena, these findings demonstrate a host
range and geographical distribution of STLV-3 that is more
widespread than previously considered.
Phylogenetic Analysis of STLV-1 Diversity
[0318] To investigate further the genetic relationships inferred
with the small PTLV-1-like tax sequences, we obtained LTR sequences
for 6 of 7 PTLV-1-positive samples using established primer pair
combinations (see Meertens et al., Virology. 287(2):275-85, 2001;
Slattery et al., Genome Res. 9(6):525-40, 1999; Wolfe et al., Proc
Natl Acad Sci USA.; 102(22):7994-9, 2005. Phylogenetic analysis of
these sequences, including those identified from a study of
infected primate hunters in Cameroon (Wolfe et al., Proc Natl Acad
Sci USA.; 102(22):7994-9, 2005), revealed that four C. nictitans
sequences all clustered in the central African HTLV-1 subtype D
clade consisting of STLV-1 from Mandrillus sphinx and Cercopithecus
pogonias and HTLV-1 sequences from Cameroon (FIG. 6).
Interestingly, the STLV-1(Cni10225NL) LTR sequence was closest
phylogenetically to the HTLV-1(1842LE) strain from a primate hunter
from Cameroon (Wolfe et al., Proc Natl Acad Sci USA.;
102(22):7994-9, 2005) (FIG. 6). Similarly, LTR sequences from two
C. agilis (Cag9812NL and Cag9813NL) clustered within the HTLV-1F
clade (FIG. 6). Combined, these results support further the primate
origin of the HTLV-1D and -1F subtypes. STLV-1 LTR sequences could
not be amplified from DBS samples from a C. nictitans (Cni10026NL)
that was positive for STLV-1 tax sequences possibly due to either
low viral load in this animal, a lower sensitivity of the LTR
primers, or genetic variances at the LTR primer binding sites.
Screening for Novel STLV-3 Subtype Sequences in Primate Hunters
[0319] Given the prevalence of the STLV-3 subtype D virus in at
least two monkey species in Cameroon it was determined whether this
new subtype was also present among primate hunters in Cameroon.
PBMC DNA samples were available from a previous study from 63
primate hunters with a wide range of WB seroreactivity to HTLV.
HTLV sequences were not previously detected in the PBMC DNA of
these persons using either generic or group-specific primers (Wolfe
et al., Proc Natl Acad Sci USA.; 102(22):7994-9, 2005). All 63
primate hunters also tested negative for STLV-3 (Cmo8699AB)
tax-specific sequences suggesting the absence of this virus in this
subset of persons with broad WB seroreactivity to HTLV.
Example 2
PCR Assay for STLV-3 Subtype D Tax Sequences
[0320] This example describes an exemplary PCR assay for STLV-3
subtype D infection using primers to the STLV-3 subtype D tax
Sequences
[0321] To screen humans for evidence of STLV-3 subtype D-like
infection a nested PCR assay was developed to detect STLV-3 subtype
D tax sequences. Similar strategies have been used to detect the
novel HTLV-3 and HTLV-4 viruses in primate hunters from Cameroon.
Peripheral blood mononuclear cell (PBMC) DNA was available for
testing from 63 primate hunters in Cameroon with seroreactivity to
HTLV antigens in the Genelabs Diagnostics HTLV Western blot 2.4
kit. The WB profiles for these samples included HTLV-1-like (n=2),
HTLV-2-like (n=4), HTLV-positive but untypeable (n=8), and
HTLV-indeterminate (n=49). PBMC DNA from all 63 hunters tested
negative for sequences using primers that can detect PTLV-1,
PTLV-2, PTLV-3, and PTLV-4. The external P5TAXF3 (5' CCC TCA AGG
TCC TCA CCC CGC CGC 3', SEQ ID NO: 21) and P5TAXR3 (5' TAA CGG CCA
GGT CAT TGG AGG TGT 3', SEQ ID NO: 22) and internal PCR primers
P5TAXF2 (5' AAG TTC CTC CCT CCT TCT TCC ATG 3', SEQ ID NO: 23) and
P5TAXR1 (TGG TAG AGG TAT AAG CAC ACG ATG GTG 3', SEQ ID NO: 24)
were used to amplify 244-bp and 174-bp STLV-3 subtype D sequences,
respectively, using standard PCR conditions. The assay could
reliably detect 10 copies of STLV-3 subtype D (Cmo8699AB) tax
plasmid sequences in a background of human DNA. STLV-3 subtype D
tax sequences were not amplified from PTLV-1, PTLV-2, PTLV-3, and
HTLV-4 cell line or tax-containing plasmid DNA, or from HTLV
nonreactive blood donor DNAs showing the high sensitivity and
specificity of the assay.
[0322] PCR products were visualized on 1.8% agarose gels stained
with ethidium bromide and were purified with Qiaquick.RTM. PCR or
gel purification kits (QIAGEN.RTM., Valencia, Calif.). Using an ABI
3130x1 sequencer, purified amplicons were then either directly
sequenced on both strands using ABI PRISM Big Dye terminator kits
(Foster City, Calif.) or after cloning into a TOPO vector
(INVITROGEN.TM., Carlsbad, Calif.).
Example 3
Production of Antibodies to STLV-3 Subtype D Polypeptides
[0323] Polyclonal or monoclonal antibodies (including humanized
monoclonal antibodies) and fragments of monoclonal antibodies such
as Fab, F(ab')2 and Fv fragments, as well as any other agent
capable of specifically binding to an STLV-3 subtype D polypeptide,
may be produced to the STLV-3 subtype D virion, or any of the
STLV-3 subtype D polypeptides (for example STLV-3 subtype D
envelope, protease, polymerase, tax, rex, or capsid polypeptides).
Optimally, antibodies raised against an STLV-3 subtype D
polypeptide would specifically bind the STLV-3 subtype D
polypeptide of interest (or a virion containing the STLV-3 subtype
D polypeptide of interest). That is, such antibodies would
recognize and bind the protein and would not substantially
recognize or bind to other proteins found in human or other cells.
The determination that an antibody specifically detects the STLV-3
subtype D polypeptide is made by any one of a number of standard
immunoassay methods; for instance, the Western blotting technique
(Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
[0324] To determine that a given antibody preparation (such as one
produced in a mouse) specifically detects the STLV-3 subtype D
polypeptide by Western blotting, total cellular protein is
extracted from murine myeloma cells and electrophoresed on a
SDS-polyacrylamide gel. The proteins are then transferred to a
membrane (for example, nitrocellulose) by Western blotting, and the
antibody preparation is incubated with the membrane. After washing
the membrane to remove non-specifically bound antibodies, the
presence of specifically bound antibodies is detected by the use of
an anti-mouse antibody conjugated to an enzyme such as alkaline
phosphatase; application of the substrate
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium results
in the production of a dense blue compound by immuno-localized
alkaline phosphatase. Antibodies which specifically detect an
STLV-3 subtype D polypeptide will, by this technique, be shown to
bind to the STLV-3 subtype D polypeptide band (which will be
localized at a given position on the gel determined by its
molecular weight). Non-specific binding of the antibody to other
proteins (such as serum albumin) may occur and may be detectable as
a weak signal on the Western blot. The non-specific nature of this
binding will be recognized by one skilled in the art by the weak
signal obtained on the Western blot relative to the strong primary
signal arising from the specific antibody-STLV-3 subtype D
polypeptide binding.
[0325] A substantially pure virion can be obtained, or
substantially pure STLV-3 subtype D polypeptide suitable for use as
an immunogen is isolated by purification or recombinant expression.
Concentration of protein in the final preparation is adjusted, for
example, by concentration on an Amicon filter device, to the level
of a few micrograms per milliliter. Monoclonal or polyclonal
antibody to the protein can then be prepared as described by Harlow
and Lane (Antibodies, A Laboratory Manual, Cold Spring Harbor
Press. 1988).
[0326] Alternatively, antibodies may be raised against synthetic
STLV-3 subtype D polypeptide synthesized on a commercially
available peptide synthesizer based upon the predicted amino acid
sequence of the STLV-3 subtype D polypeptide (Harlow and Lane,
Antibodies, A Laboratory Manual, Cold Spring Harbor Press.
1988).
[0327] Another method of raising antibodies against a STLV-3
subtype D polypeptide is by subcutaneous injection of a DNA vector
which expresses the STLV-3 subtype D polypeptide into laboratory
animals, such as mice. Delivery of the recombinant vector into the
animals may be achieved using a hand-held form of the Biolistic
system (Sanford et al., 1987, Particulate Sci. Technol. 5:27-37) as
described by Tang et al. (Nature 356:152-4, 1992). Expression
vectors suitable for this purpose may include those which express
the STLV-3 subtype D polypeptide under the transcriptional control
of either the human .beta.-actin promoter or the cytomegalovirus
(CMV) promoter.
Monoclonal Antibody Production by Hybridoma Fusion
[0328] Monoclonal antibody to epitopes of the STLV-3 subtype D
polypeptides identified and isolated as described can be prepared
from murine hybridomas according to the classical method of Kohler
and Milstein (Nature 256:495, 1975) or derivative methods thereof.
Briefly, a mouse is repetitively inoculated with a few micrograms
of the selected protein over a period of a few weeks. The mouse is
then sacrificed, and the antibody-producing cells of the spleen
isolated. The spleen cells are fused by means of polyethylene
glycol with mouse myeloma cells, and the excess unfused cells
destroyed by growth of the system on selective media comprising
aminopterin (HAT media). The successfully fused cells are diluted
and aliquots of the dilution placed in wells of a microtiter plate
where growth of the culture is continued. Antibody-producing clones
are identified by detection of antibody in the supernatant fluid of
the wells by immunoassay procedures, such as ELISA, as originally
described by Engvall (Enzymol. 70:419, 1980), and derivative
methods thereof. Selected positive clones can be expanded and their
monoclonal antibody product harvested for use. Detailed procedures
for monoclonal antibody production are described in Harlow and Lane
(Antibodies: A Laboratory Manual. 1988, Cold Spring Harbor
Laboratory, New York).
Polyclonal Antibody Production by Immunization
[0329] Polyclonal antiserum containing antibodies to heterogeneous
epitopes of a single protein can be prepared by immunizing suitable
animals with the expressed protein, which can be unmodified or
modified to enhance immunogenicity. Effective polyclonal antibody
production is affected by many factors related both to the antigen
and the host species. For example, small molecules tend to be less
immunogenic than others and may require the use of carriers and
adjuvant. Also, host animals vary in response to site of
inoculations and dose, with both inadequate or excessive doses of
antigen resulting in low titer antisera. Small doses (ng level) of
antigen administered at multiple intradermal sites appears to be
most reliable. An effective immunization protocol for rabbits can
be found in Vaitukaitis et al. (J. Clin. Endocrinol. Metab.
33:988-91, 1971).
[0330] Booster injections can be given at regular intervals, and
antiserum harvested when antibody titer thereof, as determined
semi-quantitatively, for example, by double immunodiffusion in agar
against known concentrations of the antigen, begins to fall. See,
for example, Ouchterlony et al. (In: Handbook of Experimental
Immunology, Wier, D. (ed.). Chapter 19. Blackwell. 1973). Plateau
concentration of antibody is usually in the range of 0.1 to 0.2
mg/ml of serum (about 12 .mu.M). Affinity of the antisera for the
antigen is determined by preparing competitive binding curves, as
described, for example, by Fisher (Manual of Clinical Immunology,
Chapter 42. 1980).
Labeled Antibodies
[0331] Antibodies of the present invention can be conjugated with
various labels for their direct detection (see Chapter 9, Harlow
and Lane, Antibodies: A Laboratory Manual. 1988). The label, which
may include, but is not limited to, a radiolabel, enzyme,
fluorescent probe, or biotin, is chosen based on the method of
detection available to the user.
Example 4
Vaccines
[0332] This disclosure provides substances suitable for use as
vaccines for the prevention of STLV-3 subtype D infection and
methods for administering them. Particular vaccines are directed
against STLV-3 subtype D, and may include antigens obtained from
STLV-3 subtype D. In one embodiment, the vaccine contains a nucleic
acid vector encoding a surface protein of STLV-3 subtype D, such as
a capsid protein or a envelope protein or other gene products found
to elicit appropriate humoral and/or cell mediated immune
responses.
[0333] This disclosure also provides a method of vaccinating a
subject against STLV-3 subtype D infection, comprising
administering to a susceptible subject an effective amount of the
peptide or polypeptide encoded by an isolated DNA molecule encoding
a polypeptide or combination of polypeptides expressed by the DNA
molecule, and a suitable acceptable carrier. In one embodiment,
naked DNA is administered to the subject in an effective amount to
vaccinate the subject against STLV-3 subtype D infection.
[0334] The vaccine can be made using synthetic peptide or
recombinantly-produced polypeptide described above as antigen.
Typically, a vaccine will include from about 1 to 50 micrograms of
antigen, for example from about 15 to about 45 micrograms.
Typically, the vaccine is formulated so that a dose includes about
0.5 milliliters. The vaccine may be administered by any route known
in the art, for example parenteral, subcutaneous or
intramuscular.
[0335] There are a number of strategies for amplifying an antigen's
effectiveness, particularly as related to the art of vaccines. For
example, cyclization of a peptide can increase the peptide's
antigenic and immunogenic potency. See U.S. Pat. No. 5,001,049.
More conventionally, an antigen can be conjugated to a suitable
carrier, usually a protein molecule. This procedure can allow
multiple copies of an antigen, such as a peptide, to be conjugated
to a single larger carrier molecule. Additionally, the carrier may
possess properties which facilitate transport, binding, absorption
or transfer of the antigen.
[0336] For parenteral administration, such as subcutaneous
injection, examples of suitable carriers are the tetanus toxoid,
the diphtheria toxoid, serum albumin and lamprey, or keyhole
limpet, hemocyanin because they provide the resultant conjugate
with minimum genetic restriction. Conjugates including these
universal carriers can function as T cell clone activators in
individuals having very different gene sets. The conjugation
between a peptide and a carrier can be accomplished using one of
the methods known in the art. Specifically, the conjugation can use
bifunctional cross-linkers as binding agents as detailed, for
example, by Means and Feeney, "A recent review of protein
modification techniques," Bioconjugate Chem. 1:2-12 (1990).
[0337] Vaccines against STLV-3 subtype D infection can be made from
the STLV-3 subtype D envelope glycoproteins and others. These
proteins can be purified and used for vaccination (Lasky, J. Med.
Virol. 31:59, 1990). MHC-binding peptides from cells infected with
the human herpesvirus can be identified for vaccine candidates per
the methodology of Marloes, et al. Eur. J. Immunol. 21:2963-2970,
1991. The STLV-3 subtype D antigen may be combined or mixed with
various solutions and other compounds as is known in the art. For
example, it may be administered in water, saline or buffered
vehicles with or without various adjuvants or immunodiluting
agents. Examples of such adjuvants or agents include aluminum
hydroxide, aluminum phosphate, aluminum potassium sulfate (alum),
beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions,
oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin,
lipid X, Corynebacterium parvum (Propionibacterium acnes),
Bordetella pertussis, polyribonucleotides, sodium alginate,
lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole,
DEAE-dextran, blocked copolymers or other synthetic adjuvants. Such
adjuvants are available commercially from various sources, for
example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.)
or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco
Laboratories, Detroit, Mich.). Other suitable adjuvants are
Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide), or a
mixture of Amphigen and Alhydrogel. Only aluminum is approved for
human use.
[0338] The proportion of antigen and adjuvant can be varied over a
broad range so long as both are present in effective amounts. For
example, aluminum hydroxide can be present in an amount of about
0.5% of the vaccine mixture (Al.sub.2O.sub.3 basis). On a per-dose
basis, the amount of the antigen can range from about 0.1 .mu.g to
about 100 .mu.g protein per subject, for example about to about 50
.mu.g per dose, or about 15 .mu.g to about 45 .mu.g. A suitable
dose size is about 0.5 ml. Accordingly, a dose for intramuscular
injection, for example, would comprise 0.5 ml containing 45 .mu.g
of antigen in admixture with 0.5% aluminum hydroxide. After
formulation, the vaccine may be incorporated into a sterile
container which is then sealed and stored at a low temperature, for
example 4.degree. C., or it may be freeze-dried. Lyophilization
permits long-term storage in a stabilized form.
[0339] The vaccines may be administered by any conventional method
for the administration of vaccines including oral and parenteral
(e.g., subcutaneous or intramuscular) injection. Intramuscular
administration is preferred. The treatment may consist of a single
dose of vaccine or a plurality of doses over a period of time.
Also, the antigen could be a component of a recombinant vaccine
which is adaptable for oral administration. Vaccines of this
disclosure may be combined with other vaccines for other diseases
to produce multivalent vaccines. A pharmaceutically effective
amount of the antigen can be employed with a pharmaceutically
acceptable carrier such as a protein or diluent useful for the
vaccination of mammals, particularly humans. Other vaccines may be
prepared according to methods well-known to those skilled in the
art.
[0340] Those of skill will readily recognize that it is only
necessary to expose a mammal to appropriate epitopes in order to
elicit effective immunoprotection. The epitopes are typically
segments of amino acids which are a small portion of the whole
protein. Using recombinant genetics, it is routine to alter a
natural protein's primary structure to create derivatives embracing
epitopes that are identical to or substantially the same as
(immunologically equivalent to) the naturally occurring epitopes.
Such derivatives may include peptide fragments, amino acid
substitutions, amino acid deletions and amino acid additions of the
amino acid sequence for the viral polypeptides from the human
herpesvirus. For example, it is known in the protein art that
certain amino acid residues can be substituted with amino acids of
similar size and polarity without an undue effect upon the
biological activity of the protein. The human herpesvirus proteins
have significant tertiary structure and the epitopes are usually
conformational. Thus, modifications should generally preserve
conformation to produce a protective immune response.
Example 5
Peptide Synthesis and Purification
[0341] The peptides provided by the present disclosure, such as
STLV-3 subtype D polypeptides, can be chemically synthesized by any
of a number of manual or automated methods of synthesis known in
the art. For example, solid phase peptide synthesis (SPPS) is
carried out on a 0.25 millimole (mmole) scale using an Applied
Biosystems Model 431A Peptide Synthesizer and using
9-fluorenylmethyloxycarbonyl (Fmoc) amino-terminus protection,
coupling with dicyclohexylcarbodiimide/hydroxybenzotriazole or
2-(1H-benzo-triazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate/hydroxybenzotriazole (HBTU/HOBT), and using
p-hydroxymethylphenoxymethylpolystyrene (HMP) or Sasrin resin for
carboxyl-terminus acids or Rink amide resin for carboxyl-terminus
amides.
[0342] Fmoc-derivatized amino acids are prepared from the
appropriate precursor amino acids by tritylation and
triphenylmethanol in trifluoroacetic acid, followed by Fmoc
derivitization as described by Atherton et al. Solid Phase Peptide
Synthesis, IRL Press: Oxford, 1989.
[0343] Sasrin resin-bound peptides are cleaved using a solution of
1% TFA in dichloromethane to yield the protected peptide. Where
appropriate, protected peptide precursors are cyclized between the
amino- and carboxyl-termini by reaction of the amino-terminal free
amine and carboxyl-terminal free acid using diphenylphosphorylazide
in nascent peptides wherein the amino acid sidechains are
protected.
[0344] HMP or Rink amide resin-bound products are routinely cleaved
and protected sidechain-containing cyclized peptides deprotected
using a solution comprised of trifluoroacetic acid (TFA),
optionally also comprising water, thioanisole, and ethanedithiol,
in ratios of 100:5:5:2.5, for 0.5-3 hours at room temperature.
[0345] Crude peptides are purified by preparative high pressure
liquid chromatography (HPLC), for example using a Waters Delta-Pak
C18 column and gradient elution with 0.1% TFA in water modified
with acetonitrile. After column elution, acetonitrile is evaporated
from the eluted fractions, which are then lyophilized. The identity
of each product so produced and purified may be confirmed by fast
atom bombardment mass spectroscopy (FABMS) or electrospray mass
spectroscopy (ESMS).
Example 6
Assembly of the STLV-3 Subtype D Genome
[0346] This example describes exemplary procedures for assembling
the viral genome of STLV-3 subtype D with the sequences provided in
Example 8.
[0347] As shown in FIG. 1, the sequences provided herein span the
entire genome of STLV-3 subtype D. FIG. 1 is a schematic
representation of the genome of STLV-3 subtype D. At the top of
FIG. 1 is a block representation of the genes making up the STLV-3
subtype D genome. Positions of the primers used in sequencing the
genome are shown relative to the genome (shown in kilobases kB).
Shown below the position of the primers in block diagrams are the
position of sequenced portions of the STLV-3 subtype D genomes
obtained from each animal. Using the sequences obtained, the entive
genome of STLV-3 subtype D was assembled. An exemplary sequence of
the genome of the STLV-3 subtype D virus is set forth as SEQ ID NO:
1.
Example 7
Isolation of STLV-3 Subtype D
[0348] This example describes how STLV-3 subtype D is isolated
using the STLV-3 subtype D nucleic acid sequences disclosed herein.
Using primers designed from the STLV-3 subtype D nucleic acid
sequences disclosed herein, the entire genome of STLV-3 subtype D
was sequenced and cloned. Nucleic acid vectors including the entire
STLV-3 subtype D genome are introduced into cell cultures of
primate cells, for example primate leukocytes, thereby producing
STLV-3 subtype D virus. STLV-3 subtype D virus can subsequently be
isolated from supernatants, for example by centrifugation to remove
cellular material. Viral particles can be further purified, for
example with gradient centrifugation or immunoaffinity
chromatography, for example using antibodies raised against the
STLV-3 subtype D polypeptides disclosed herein.
[0349] The presence of isolated STLV-3 subtype D viruses is
confirmed by PCR with STLV-3 subtype D specific primers.
Alternatively, the presence of isolated STLV-3 subtype D viruses is
confirmed with antibodies that specifically recognize STLV-3
subtype D polypeptides.
[0350] While this disclosure has been described with an emphasis
upon particular embodiments, it will be obvious to those of
ordinary skill in the art that variations of the particular
embodiments may be used, and it is intended that the disclosure may
be practiced otherwise than as specifically described herein.
Features, characteristics, compounds, chemical moieties, or
examples described in conjunction with a particular aspect,
embodiment, or example of the invention are to be understood to be
applicable to any other aspect, embodiment, or example of the
invention. Accordingly, this disclosure includes all modifications
encompassed within the spirit and scope of the disclosure as
defined by the following claims.
Sequence CWU 1
1
2618913DNASTLV-3 subtype D 1tgacagtgac agcaagcccc aaggcgagcc
acaactacta gccaaagggc atacagttga 60atcatctgtc taggggacgt ctcgcaccca
gagtatgtcc aaagaacacc agggctctga 120cgtctctccc tgccttgtct
cccggaaaaa accttaaacc acccatttcc tcatgtttgc 180ccaaggctct
gacgataacc ctgaaaaatt tgactaacaa ataaaggaac ctggacccta
240taaaagggga gagcgaccta aaaatgggat caaccttttc tccccaacgc
cctttcgcgc 300cccgcggaca gccactgtcc gggctactcc tggcctacct
agatcattgc tccgcgcccg 360agccattctt ctgcagccaa gcggcacctt
gcaccttcgc ttctcctgtc ctggtaagat 420cccactgggt agagctaggc
cgttactccc tggccgctcc cctggagctc ctttgcttag 480ctcttaaggt
cgctctctcc ttctcgttag ggtccaagga ctaactttac ttccgtgtct
540cggtctcctt tctttggcgg tctcgtctaa agtcgaaagt aacacctcaa
actgtcagca 600gcgaggcctg gcccggggcc agcgcctgtg agctttactc
ggctcggagc caggggctca 660gaaagtaaag gctgtagctg ccagcctttg
aggggaacca aaaacaggtg ggggctcgtc 720cgggattgat caccctccta
ttaaacatgg gaaattcata cagccgtgcc gccaacccca 780tccccaaggc
cccaaaaggg ctagcaattc accactggtt aaactttcta caagctgcct
840atcggctgca accggggccc tcagagtttg atttccatca gttacgaaat
tttcttaaat 900tagctataaa aacccctgtt tggctaaacc ccatcaatta
ttccgtccta gctgaactcg 960ttcctaaaaa ttatccaggc agaatccaag
aaattatagc catcctaatc caagaaacct 1020ctacgcagga ggttccccca
tccgccccac cggccagcga accccaaaat cccccgcctt 1080atccagaacc
agggcaagcc ataccccagt gcctacctgt tctgcacccc catggtgccc
1140ctgccgccca tcgcccttgg cagatgaaag atctccaagc tataaaacag
gaagttacct 1200cttccgcacc agggagccct cagttcatgc aaaccgtgcg
cctggcagtc caacaatttg 1260acccgactgc caaagacctc catgacctct
tacaatacct gtgctcctca ctagttgcct 1320ccctgcacca ccagcagctc
gagaccctca tcgctcaggc tgaaacccaa gggataaccg 1380gatataatcc
cctggccggc cccctgcgag tacaggccaa caacccaact cagcaagggc
1440tccggcgaga ataccaaaac ttatggctgt cggccttttc tgccctccca
ggaaatacta 1500aagaccccac ctgggcggca atcctccagg gccccgagga
accgttttgc acattcgtag 1560aaagacttaa tgtggcccta gacaacggcc
tccctgaagg aacccccaaa gagcctattc 1620ttcggtcctt agcatattct
aatgccaaca aagaatgcca gaaactccta caagcccgag 1680ggcagacaaa
cggtccctta ggggacatgc tcagagcttg ccaggcgtgg acgccccggg
1740acaaaaacaa agtactaatg gtccaaccta aaaagacacc tcccccaaat
caaccatgct 1800tccggtgcgg gcaggcgggc cactggagca gagactgtaa
acaacctcgt ccccccccag 1860gcccatgtcc gctctgtcaa gaccccaccc
actggaagcg agattgcccg cagctaaaac 1920cagatcctga agaaggcatg
ttgttagatc tgccttgtga agacccagcg gccagagacc 1980aaaaaaactt
catagggggg gaggactagc ctccccccaa acagtgctgc cttttatacc
2040attatcccag caaaaacaac cagtcctaca cgtccgagta tccttcccag
gtaccccccc 2100agtaagcatc caggcgcttt tagacacagg ggcagatgta
accgtcctcc cagcccgtct 2160atgcccccct gacctaaaat tacaagacac
cactgtcctt ggagccagcg ggccaagcac 2220cgacaagttt aaagttctac
cctgttttac gtatgtccat ctgcccttcc gaggacgacc 2280agtaacctta
ccatcatgct taattgatat taataatcaa tgggccattc taggccgaga
2340tgtcctccag caatgccaaa gttcccttta ccttgcagac caaccctctc
gcgttctacc 2400aatccagaca cctagtgtca ttgggctgga acatctcccc
ccgcccccag aagttccaca 2460atttccgtta aaccagagcg cctccaggcc
ttgactgacc tggtatccaa ggcgctggag 2520gccaaataca tagaacctta
tcaaggacca ggcaataatc caattttccc ggtcaaaaaa 2580ccgaatggaa
aatggcgctt catccatgat ctccgggcca ccaactgcct cactaaaacc
2640ctaacttccc cgtctcccgg cccccccgac cttaccagtc tgccccaagg
cctcccacat 2700cttcgaacca ttgacctgac tgacgccttt tttcaaatcc
cactgcctgt tgccttccag 2760ccctattttg catttaccct ccctcagccc
aacaaccatg gccccggggc tcggtattcc 2820tggaaagtac taccccaagg
gtttaaaaat agcccaactc tatttgaaca acaactctct 2880catatactca
cacctgtaag acaggccttt ccaaaatcta tagtcattca gtacatggat
2940gacatactct tggccagccc tacccttgaa gagtccatcg ttctcgccca
ggaaataacc 3000aatgctctag cccaggaggg cttgcccatg tccacagaaa
aaacccaatc cactcctggt 3060cccatacact ttctcggaca aaccatatcc
aaaaaataca taacttatga aaccctccct 3120accatacatg tcaagcctaa
ttggacctta acagaattac agtccacctt aggggaattg 3180caatgggtat
ccaaagggac tcctacactc cgctcatccc tccatcaatt atatacggcc
3240ctccgaggtc atcatgaccc ccgcgatacc atacaactta ccccaccaca
actacaagcg 3300ctcaacacgc ttcaaaaggc tctgacccac aattgcagaa
gcagaatagt cagtaatctg 3360cctatcctgg ccctcataat gctccgcccc
acaggcacta cagcagttct ttttcaaaca 3420aaacaaaagt ggccacttgt
ctggctgcac accccccacc cggccactag tctgcgcctt 3480tggggacaat
tattggccaa tgccatcatt actctagata agtactcact acaacactat
3540ggccaggtat gcaaatcctt tcatcataac atatctaatc aggcccttac
ccactaccta 3600cacacgtcag accagtcaag tgttgccatt ctcctacagc
actcgcatag gttccataat 3660ctcggggccc aaccatcggg accatggaaa
ggcctcctac aagtacccca aatcttccaa 3720aatgttgcca cacttagccc
tccattcact atttcacctg tggttatcaa ccacgcccct 3780tgcctctttt
ccgatggatc caactctcag gctgccttca ctatctggga taaaaaaata
3840attcaccaac aagtccttcc tcttcctacc gccagctcgg ctcaagcagg
ggaacttttt 3900gccctattag cggccctacg agaatgcaaa ccctggtcat
cactaaacat attcttagac 3960tcaaagtttc ttgttggcca gctccggcgc
ctggcccttg gggctttcat aggtccatcc 4020acccaatgtg acttacactc
gcaactcctg ccgctcttgt ataacaaaac catttatgtt 4080catcatgtaa
gaagccacac cttattacag gaccctatat cccgcctcaa tgaggctacc
4140gatgccctca tgctcgcacc ccttctgccc ctcagtccag cgacccttca
tgaaatcacc 4200cactgcaacc cccctgcact gtgcaaccat ggggctacag
caactgagac taaggctatt 4260gtccgggcat gtcacacctg taagataacc
aatccccaag ggagactgcc ccagggtcac 4320attcgcagag ggcacgcccc
aaacactatc tggcaaggag atgtcactca cctacaatac 4380aaaaaatata
aatactgcct tttagtctgg gtcgatactt actcaggagc agtagctgtg
4440tcgtgccggc gtaaagaaac cagctcagaa tgtgtggcct cgctgctagc
agccatttcc 4500atcctaggaa aaccacacac cattaataca gacaatgggg
cagcatattt gtcccaggaa 4560ttccaacaat tttgtacctc actctccata
aaacacacca ctcatgtccc ctacaatccc 4620accagttccg gattagtgga
aagaactaat ggaatcctaa aaaccttaat ctccaaatac 4680ctcctagatg
accaccactt gcccctggac acagccattt ccaaaacttt gtggaccata
4740aaccatctca atgtcctctc ttcctgccaa aagacacgat ggcagttaca
tcaagctcaa 4800cccctgcccc ccgttcctga gaatttgccc cttcctgaac
cagtgccaaa atggtattat 4860tataaaatcc caggtcttac cagttcaagg
tggagtgggc ctgtacaatc tgttaaagaa 4920gcagccggag cggccctcat
cccggtaggt actaggcaca tctggattcc gtggcgtctc 4980ctgaaacgag
gtgcatgccc aagacccgga gacagcgtaa ccaccgaatc aaaacacaaa
5040gaccttcaac tccatgggta agtctagtct ctttatttgc ctcttttgct
catacatggc 5100tagtctcttt gtccctggcg accccagtcg gtgcacactt
tttataggag cctcctccta 5160ccactccagt ccctgcgggt ctaactaccc
tcaatgtact tggacactcg acctagtgtc 5220acttaccagg gatcaaagtc
taaaccctcc atgcccagat ctagtcacct actcccagta 5280tcacagacct
tattccttgt atctttttcc ccattggatt actaaaccga atcgtcaagg
5340ccttggttat tactctgcct cctactcaga tccctgtgct atcaagtgcc
cctacctagg 5400atgtcaatct tggacatgtc cctatacagg acctatgtcc
agcccatact ggaagtacac 5460ctcagaccta aatttcaccc aaaaggtgtc
ctctgtcacc ctccatctac atttctcaaa 5520atgcggatcc tccttctctc
ttttactcga cgcacccggt tatgaccccg tatggttcct 5580ttcctcccaa
actacacagg ccccacctac acccgcccct ctgacacaag actccgactt
5640ccaacatatc ttggagccct ctgtgccctg gagctccaaa atcctcaacc
ttatcctctt 5700aactcttaaa agcactaact actcctgcat ggtttgcgtt
gaccgctcca gcctctcctc 5760atggcatgtc ttgtatgacc cactaaaagt
tcccaagcaa cacgaacccc gtgcccgggc 5820cctcttgcgg ccctctctgg
ccattccaat aactaatacc acacccccct ttccttggtc 5880ccattgctac
tgcccccttc tacaggctgt catctccaat aactgcaaca actcagttat
5940actgcccccc ttctctctgt cccctgtcct cgatctctcc aagcctcgtc
agcgccgagc 6000cgtccccatc gccgtttggc tggtgtccgc cctagcggtc
ggtacaggta tagccggcgg 6060caccaccggg tccctatcct tggcatccag
caggagcctg ctacatgaag tagaccaaga 6120tataagccat ctcactcaag
ccatagttaa gaaccataac aatatccttc gggttgctca 6180atacgctgca
caaaaccgac gaggcctaga tttactcttc tgggaacaag gaggtctatg
6240caaggctatc agggaacaat gttgttttct caatatcagc aatacccacg
tgtctgtgct 6300ccaagagaga ccccccttag aaaaaagggt gattaccggt
tggggactca attgggacct 6360cggcctatcc caatgggccc gtgaagccct
ccagaccggt attaccctgt tagccctctt 6420cctcctactt atcatggtag
gcccttgtgt cctgcgccag ctacaggccc tcctgttccg 6480cctacagcac
cgtagccacc catactccct cctcaatcgc gaaaccaacc tataacacct
6540ctgcaacctc ctgtagcaat gagccatagt cctcgcccct accagaaacc
cacatacagc 6600ataggcccga agaatctccc caaatatcca tgccttgact
ccagtaatcc atgtacccaa 6660agtattcccc taatgcctcc tcacaatcca
cgcgaagttg gaaattctct cgttccaaaa 6720agtctatata acccgtcaac
aaattgcaaa acccctcaaa ccccagtaag tctatacaat 6780ccaactgctg
ccgccgctcc ttttttctcc tctttctctc ctctttttcc tcgtgacacc
6840tcctccggcg ctcttctctt cttttccgac cccgccagta gcttagcaat
tgcttctgct 6900cctgagcaag gtcttctaag cgacccttcc aatatcctga
atcctttgta ctagatccca 6960gaggacgccc tcggggtcgc ctaccacccc
cctgcagcat gtccacttga tcttttcccg 7020attgatcaca caactccaat
aaagcttcca ccggtgtgag aggatcttcg gccgccagta 7080tcggtggtcc
cacactccta gaccgagagg tcaagctgcc cccggaagta gagacgcagg
7140aatacaccac aggcatagtc cccgcagttg tggtctctgg agtcagtaaa
ggcatcttcc 7200taaaataccc tgtaaaataa tctcctgtca gcccactttc
caggtttcgg gcagagcctg 7260ctctacgggt accctgtcta cgttttcggc
gattgtgtgc aggccgattg gtgccccatt 7320tccggggggc tttgttccgc
ccggctacat cggcacgcct tactggccac ctgtcctgaa 7380caccagatca
cctgggaccc catcgatgga cgcgttgtca gctcgcctct acaatacctt
7440atccctcgcc tcccctcctt ccccacccaa agaacttccc gcaccctcaa
ggtcctcacc 7500ccgccgccca ctgctacaac ccccaaagtt cctccctcct
tcttccatgc agtcaggaaa 7560cacacccctt tccgaaacaa ctgcctcgag
ctcaccttgg gagagcaact acccgccatg 7620tctttccccg accccggcct
ccgaccccaa aatgtctata ccatgtgggg aagcaccatc 7680gtgtgcttat
acctctacca actcacacct ccaatgacct ggccgttaat cccacatgtc
7740attttttgcc atccggacca actaggggcc ttcctaacaa aaatccctac
caaacgcttg 7800gaagaactct tatacaaact attcttaagt acaggggcca
tacttatcct acctgaaaat 7860tgcttcccaa ctaccctgtt tcagcccacc
cgcgcaccag taattcaagc cccctggcac 7920tcaggcctac tcccatacct
aaaggaaatt gtcacccccg ggctgatttg ggtgtttact 7980gacggtagtt
ctatgatttc cggaccctgc cccaaggaag ggcagccatc tttggtggtc
8040caatcatcta cattcatttt ccaaaaattt caaaccaaag cctatcaccc
agccttcctc 8100ctgtcccata aattaatcca atactcctcg ttccattccc
tccatctact ttttgaagaa 8160tacaccactg tccccttttc tttattgttt
aacgaaaaag aggcaaatga cagtgacagc 8220aagccccaag gcgagccaca
actactagcc aaagggcata cagttgaatc atctgtctag 8280gggacgtctc
gcacccagag tatgtccaaa gaacaccagg gctctgacgt ctctccctgc
8340cttgtctccc ggaaaaaacc ttaaaccacc catttcctca tgtttgccca
aggctctgac 8400gataaccctg aaaaatttga ctaacaaata aaggaacctg
gaccctataa aaggggagag 8460cgacctaaaa atgggatcaa ccttttctcc
ccaacgccct ttcgcgcccc gcggacagcc 8520actgtccggg ctactcctgg
cctacctaga tcattgctcc gcgcccgagc cattcttctg 8580cagccaagcg
gcaccttgca ccttcgcttc tcctgtcctg gtaagatccc actgggtaga
8640gctaggccgt tactccctgg ccgctcccct ggagctcctt tgcttagctc
ttaaggtcgc 8700tctctccttc tcgttagggt ccaaggacta actttacttc
cgtgtctcgg tctcctttct 8760ttggcggtct cgtctaaagt cgaaagtaac
acctcaaact gtcagcagcg aggcctggcc 8820cggggccagc gcctgtgagc
tttactcggc tcggagccag gggctcagaa agtaaaggct 8880gtagctgcca
gcctttgagg ggaaccaaaa aca 8913220DNAArtificial sequenceTheoretical
nucleic acid molecule illustrating percent sequence identity.
2atggtggacc cggtgggctt 20320DNAArtificial sequenceTheoretical
nucleic acid molecule illustrating percent sequence identity.
3acgggggatc cggcgggcct 20425DNAArtificial sequenceSynthetic
oligonucleotide primer. 4gtaccctgtc tacgttttcg gcgat
25526DNAArtificial sequenceSynthetic oligonucleotide primer.
5gangantgna ntacnaaaga tggctg 26624DNAArtificial sequenceSynthetic
oligonucleotide primer. 6ttactggcca cctgtcctga acac
24723DNAArtificial sequenceSynthetic oligonucleotide primer.
7ttngggnang gnccggaaat cat 23824DNAArtificial sequenceSynthetic
oligonucleotide primer. 8catccggacc aactaggggc cttc
24924DNAArtificial sequenceSynthetic oligonucleotide primer.
9cagcccaccc gcgcaccagt aatt 241027DNAArtificial sequenceSynthetic
oligonucleotide primer. 10tcctgaacng tcnnnncgct tttatag
271124DNAArtificial sequenceSynthetic oligonucleotide primer.
11aacaaaaatc cctaccaaac gctt 241224DNAArtificial sequenceSynthetic
oligonucleotide primer. 12ctctgacgtc tctccctgcc ttgt
241318DNAArtificial sequenceSynthetic oligonucleotide primer.
13atcccggacg agccccca 181424DNAArtificial sequenceSynthetic
oligonucleotide primer. 14ccggaaaaaa ccttaaacca ccca
2415493PRTSTLV-3 subtype D 15Met Gly Lys Ser Ser Leu Phe Ile Cys
Leu Phe Cys Ser Tyr Met Ala 1 5 10 15 Ser Leu Phe Val Pro Gly Asp
Pro Ser Arg Cys Thr Leu Phe Ile Gly 20 25 30 Ala Ser Ser Tyr His
Ser Ser Pro Cys Gly Ser Asn Tyr Pro Gln Cys 35 40 45 Thr Trp Thr
Leu Asp Leu Val Ser Leu Thr Arg Asp Gln Ser Leu Asn 50 55 60 Pro
Pro Cys Pro Asp Leu Val Thr Tyr Ser Gln Tyr His Arg Pro Tyr 65 70
75 80 Ser Leu Tyr Leu Phe Pro His Trp Ile Thr Lys Pro Asn Arg Gln
Gly 85 90 95 Leu Gly Tyr Tyr Ser Ala Ser Tyr Ser Asp Pro Cys Ala
Ile Lys Cys 100 105 110 Pro Tyr Leu Gly Cys Gln Ser Trp Thr Cys Pro
Tyr Thr Gly Pro Met 115 120 125 Ser Ser Pro Tyr Trp Lys Tyr Thr Ser
Asp Leu Asn Phe Thr Gln Lys 130 135 140 Val Ser Ser Val Thr Leu His
Leu His Phe Ser Lys Cys Gly Ser Ser 145 150 155 160 Phe Ser Leu Leu
Leu Asp Ala Pro Gly Tyr Asp Pro Val Trp Phe Leu 165 170 175 Ser Ser
Gln Thr Thr Gln Ala Pro Pro Thr Pro Ala Pro Leu Thr Gln 180 185 190
Asp Ser Asp Phe Gln His Ile Leu Glu Pro Ser Val Pro Trp Ser Ser 195
200 205 Lys Ile Leu Asn Leu Ile Leu Leu Thr Leu Lys Ser Thr Asn Tyr
Ser 210 215 220 Cys Met Val Cys Val Asp Arg Ser Ser Leu Ser Ser Trp
His Val Leu 225 230 235 240 Tyr Asp Pro Leu Lys Val Pro Lys Gln His
Glu Pro Arg Ala Arg Ala 245 250 255 Leu Leu Arg Pro Ser Leu Ala Ile
Pro Ile Thr Asn Thr Thr Pro Pro 260 265 270 Phe Pro Trp Ser His Cys
Tyr Cys Pro Leu Leu Gln Ala Val Ile Ser 275 280 285 Asn Asn Cys Asn
Asn Ser Val Ile Leu Pro Pro Phe Ser Leu Ser Pro 290 295 300 Val Leu
Asp Leu Ser Lys Pro Arg Gln Arg Arg Ala Val Pro Ile Ala 305 310 315
320 Val Trp Leu Val Ser Ala Leu Ala Val Gly Thr Gly Ile Ala Gly Gly
325 330 335 Thr Thr Gly Ser Leu Ser Leu Ala Ser Ser Arg Ser Leu Leu
His Glu 340 345 350 Val Asp Gln Asp Ile Ser His Leu Thr Gln Ala Ile
Val Lys Asn His 355 360 365 Asn Asn Ile Leu Arg Val Ala Gln Tyr Ala
Ala Gln Asn Arg Arg Gly 370 375 380 Leu Asp Leu Leu Phe Trp Glu Gln
Gly Gly Leu Cys Lys Ala Ile Arg 385 390 395 400 Glu Gln Cys Cys Phe
Leu Asn Ile Ser Asn Thr His Val Ser Val Leu 405 410 415 Gln Glu Arg
Pro Pro Leu Glu Lys Arg Val Ile Thr Gly Trp Gly Leu 420 425 430 Asn
Trp Asp Leu Gly Leu Ser Gln Trp Ala Arg Glu Ala Leu Gln Thr 435 440
445 Gly Ile Thr Leu Leu Ala Leu Phe Leu Leu Leu Ile Met Val Gly Pro
450 455 460 Cys Val Leu Arg Gln Leu Gln Ala Leu Leu Phe Arg Leu Gln
His Arg 465 470 475 480 Ser His Pro Tyr Ser Leu Leu Asn Arg Glu Thr
Asn Leu 485 490 16420PRTSTLV-3 subtype D 16Met Gly Asn Ser Tyr Ser
Arg Ala Ala Asn Pro Ile Pro Lys Ala Pro 1 5 10 15 Lys Gly Leu Ala
Ile His His Trp Leu Asn Phe Leu Gln Ala Ala Tyr 20 25 30 Arg Leu
Gln Pro Gly Pro Ser Glu Phe Asp Phe His Gln Leu Arg Asn 35 40 45
Phe Leu Lys Leu Ala Ile Lys Thr Pro Val Trp Leu Asn Pro Ile Asn 50
55 60 Tyr Ser Val Leu Ala Glu Leu Val Pro Lys Asn Tyr Pro Gly Arg
Ile 65 70 75 80 Gln Glu Ile Ile Ala Ile Leu Ile Gln Glu Thr Ser Thr
Gln Glu Val 85 90 95 Pro Pro Ser Ala Pro Pro Ala Ser Glu Pro Gln
Asn Pro Pro Pro Tyr 100 105 110 Pro Glu Pro Gly Gln Ala Ile Pro Gln
Cys Leu Pro Val Leu His Pro 115 120 125 His Gly Ala Pro Ala Ala His
Arg Pro Trp Gln Met Lys Asp Leu Gln 130 135 140 Ala Ile Lys Gln Glu
Val Thr Ser Ser Ala Pro Gly Ser Pro Gln Phe 145 150 155 160 Met Gln
Thr Val Arg Leu Ala Val Gln Gln Phe Asp Pro Thr Ala Lys 165 170 175
Asp Leu His Asp Leu Leu Gln Tyr Leu Cys Ser Ser Leu Val Ala Ser 180
185
190 Leu His His Gln Gln Leu Glu Thr Leu Ile Ala Gln Ala Glu Thr Gln
195 200 205 Gly Ile Thr Gly Tyr Asn Pro Leu Ala Gly Pro Leu Arg Val
Gln Ala 210 215 220 Asn Asn Pro Thr Gln Gln Gly Leu Arg Arg Glu Tyr
Gln Asn Leu Trp 225 230 235 240 Leu Ser Ala Phe Ser Ala Leu Pro Gly
Asn Thr Lys Asp Pro Thr Trp 245 250 255 Ala Ala Ile Leu Gln Gly Pro
Glu Glu Pro Phe Cys Thr Phe Val Glu 260 265 270 Arg Leu Asn Val Ala
Leu Asp Asn Gly Leu Pro Glu Gly Thr Pro Lys 275 280 285 Glu Pro Ile
Leu Arg Ser Leu Ala Tyr Ser Asn Ala Asn Lys Glu Cys 290 295 300 Gln
Lys Leu Leu Gln Ala Arg Gly Gln Thr Asn Gly Pro Leu Gly Asp 305 310
315 320 Met Leu Arg Ala Cys Gln Ala Trp Thr Pro Arg Asp Lys Asn Lys
Val 325 330 335 Leu Met Val Gln Pro Lys Lys Thr Pro Pro Pro Asn Gln
Pro Cys Phe 340 345 350 Arg Cys Gly Gln Ala Gly His Trp Ser Arg Asp
Cys Lys Gln Pro Arg 355 360 365 Pro Pro Pro Gly Pro Cys Pro Leu Cys
Gln Asp Pro Thr His Trp Lys 370 375 380 Arg Asp Cys Pro Gln Leu Lys
Pro Asp Pro Glu Glu Gly Met Leu Leu 385 390 395 400 Asp Leu Pro Cys
Glu Asp Pro Ala Ala Arg Asp Gln Lys Asn Phe Ile 405 410 415 Gly Gly
Glu Asp 420 17176PRTSTLV-3 subtype D 17Pro Ser Gly Gln Arg Pro Lys
Lys Leu His Arg Gly Gly Gly Leu Ala 1 5 10 15 Ser Pro Gln Thr Val
Leu Pro Phe Ile Pro Leu Ser Gln Gln Lys Gln 20 25 30 Pro Val Leu
His Val Arg Val Ser Phe Pro Gly Thr Pro Pro Val Ser 35 40 45 Ile
Gln Ala Leu Leu Asp Thr Gly Ala Asp Val Thr Val Leu Pro Ala 50 55
60 Arg Leu Cys Pro Pro Asp Leu Lys Leu Gln Asp Thr Thr Val Leu Gly
65 70 75 80 Ala Ser Gly Pro Ser Thr Asp Lys Phe Lys Val Leu Pro Cys
Phe Thr 85 90 95 Tyr Val His Leu Pro Phe Arg Gly Arg Pro Val Thr
Leu Pro Ser Cys 100 105 110 Leu Ile Asp Ile Asn Asn Gln Trp Ala Ile
Leu Gly Arg Asp Val Leu 115 120 125 Gln Gln Cys Gln Ser Ser Leu Tyr
Leu Ala Asp Gln Pro Ser Arg Val 130 135 140 Leu Pro Ile Gln Thr Pro
Ser Val Ile Gly Leu Glu His Leu Pro Pro 145 150 155 160 Pro Pro Glu
Val Pro Gln Phe Pro Leu Asn Gln Ser Ala Ser Arg Pro 165 170 175
18880PRTSTLV-3 subtype D 18His Trp Ala Gly Thr Ser Pro Pro Ala Pro
Arg Ser Ser Thr Ile Ser 1 5 10 15 Val Lys Pro Glu Arg Leu Gln Ala
Leu Thr Asp Leu Val Ser Lys Ala 20 25 30 Leu Glu Ala Lys Tyr Ile
Glu Pro Tyr Gln Gly Pro Gly Asn Asn Pro 35 40 45 Ile Phe Pro Val
Lys Lys Pro Asn Gly Lys Trp Arg Phe Ile His Asp 50 55 60 Leu Arg
Ala Thr Asn Cys Leu Thr Lys Thr Leu Thr Ser Pro Ser Pro 65 70 75 80
Gly Pro Pro Asp Leu Thr Ser Leu Pro Gln Gly Leu Pro His Leu Arg 85
90 95 Thr Ile Asp Leu Thr Asp Ala Phe Phe Gln Ile Pro Leu Pro Val
Ala 100 105 110 Phe Gln Pro Tyr Phe Ala Phe Thr Leu Pro Gln Pro Asn
Asn His Gly 115 120 125 Pro Gly Ala Arg Tyr Ser Trp Lys Val Leu Pro
Gln Gly Phe Lys Asn 130 135 140 Ser Pro Thr Leu Phe Glu Gln Gln Leu
Ser His Ile Leu Thr Pro Val 145 150 155 160 Arg Gln Ala Phe Pro Lys
Ser Ile Val Ile Gln Tyr Met Asp Asp Ile 165 170 175 Leu Leu Ala Ser
Pro Thr Leu Glu Glu Ser Ile Val Leu Ala Gln Glu 180 185 190 Ile Thr
Asn Ala Leu Ala Gln Glu Gly Leu Pro Met Ser Thr Glu Lys 195 200 205
Thr Gln Ser Thr Pro Gly Pro Ile His Phe Leu Gly Gln Thr Ile Ser 210
215 220 Lys Lys Tyr Ile Thr Tyr Glu Thr Leu Pro Thr Ile His Val Lys
Pro 225 230 235 240 Asn Trp Thr Leu Thr Glu Leu Gln Ser Thr Leu Gly
Glu Leu Gln Trp 245 250 255 Val Ser Lys Gly Thr Pro Thr Leu Arg Ser
Ser Leu His Gln Leu Tyr 260 265 270 Thr Ala Leu Arg Gly His His Asp
Pro Arg Asp Thr Ile Gln Leu Thr 275 280 285 Pro Pro Gln Leu Gln Ala
Leu Asn Thr Leu Gln Lys Ala Leu Thr His 290 295 300 Asn Cys Arg Ser
Arg Ile Val Ser Asn Leu Pro Ile Leu Ala Leu Ile 305 310 315 320 Met
Leu Arg Pro Thr Gly Thr Thr Ala Val Leu Phe Gln Thr Lys Gln 325 330
335 Lys Trp Pro Leu Val Trp Leu His Thr Pro His Pro Ala Thr Ser Leu
340 345 350 Arg Leu Trp Gly Gln Leu Leu Ala Asn Ala Ile Ile Thr Leu
Asp Lys 355 360 365 Tyr Ser Leu Gln His Tyr Gly Gln Val Cys Lys Ser
Phe His His Asn 370 375 380 Ile Ser Asn Gln Ala Leu Thr His Tyr Leu
His Thr Ser Asp Gln Ser 385 390 395 400 Ser Val Ala Ile Leu Leu Gln
His Ser His Arg Phe His Asn Leu Gly 405 410 415 Ala Gln Pro Ser Gly
Pro Trp Lys Gly Leu Leu Gln Val Pro Gln Ile 420 425 430 Phe Gln Asn
Val Ala Thr Leu Ser Pro Pro Phe Thr Ile Ser Pro Val 435 440 445 Val
Ile Asn His Ala Pro Cys Leu Phe Ser Asp Gly Ser Asn Ser Gln 450 455
460 Ala Ala Phe Thr Ile Trp Asp Lys Lys Ile Ile His Gln Gln Val Leu
465 470 475 480 Pro Leu Pro Thr Ala Ser Ser Ala Gln Ala Gly Glu Leu
Phe Ala Leu 485 490 495 Leu Ala Ala Leu Arg Glu Cys Lys Pro Trp Ser
Ser Leu Asn Ile Phe 500 505 510 Leu Asp Ser Lys Phe Leu Val Gly Gln
Leu Arg Arg Leu Ala Leu Gly 515 520 525 Ala Phe Ile Gly Pro Ser Thr
Gln Cys Asp Leu His Ser Gln Leu Leu 530 535 540 Pro Leu Leu Tyr Asn
Lys Thr Ile Tyr Val His His Val Arg Ser His 545 550 555 560 Thr Leu
Leu Gln Asp Pro Ile Ser Arg Leu Asn Glu Ala Thr Asp Ala 565 570 575
Leu Met Leu Ala Pro Leu Leu Pro Leu Ser Pro Ala Thr Leu His Glu 580
585 590 Ile Thr His Cys Asn Pro Pro Ala Leu Cys Asn His Gly Ala Thr
Ala 595 600 605 Thr Glu Thr Lys Ala Ile Val Arg Ala Cys His Thr Cys
Lys Ile Thr 610 615 620 Asn Pro Gln Gly Arg Leu Pro Gln Gly His Ile
Arg Arg Gly His Ala 625 630 635 640 Pro Asn Thr Ile Trp Gln Gly Asp
Val Thr His Leu Gln Tyr Lys Lys 645 650 655 Tyr Lys Tyr Cys Leu Leu
Val Trp Val Asp Thr Tyr Ser Gly Ala Val 660 665 670 Ala Val Ser Cys
Arg Arg Lys Glu Thr Ser Ser Glu Cys Val Ala Ser 675 680 685 Leu Leu
Ala Ala Ile Ser Ile Leu Gly Lys Pro His Thr Ile Asn Thr 690 695 700
Asp Asn Gly Ala Ala Tyr Leu Ser Gln Glu Phe Gln Gln Phe Cys Thr 705
710 715 720 Ser Leu Ser Ile Lys His Thr Thr His Val Pro Tyr Asn Pro
Thr Ser 725 730 735 Ser Gly Leu Val Glu Arg Thr Asn Gly Ile Leu Lys
Thr Leu Ile Ser 740 745 750 Lys Tyr Leu Leu Asp Asp His His Leu Pro
Leu Asp Thr Ala Ile Ser 755 760 765 Lys Thr Leu Trp Thr Ile Asn His
Leu Asn Val Leu Ser Ser Cys Gln 770 775 780 Lys Thr Arg Trp Gln Leu
His Gln Ala Gln Pro Leu Pro Pro Val Pro 785 790 795 800 Glu Asn Leu
Pro Leu Pro Glu Pro Val Pro Lys Trp Tyr Tyr Tyr Lys 805 810 815 Ile
Pro Gly Leu Thr Ser Ser Arg Trp Ser Gly Pro Val Gln Ser Val 820 825
830 Lys Glu Ala Ala Gly Ala Ala Leu Ile Pro Val Gly Thr Arg His Ile
835 840 845 Trp Ile Pro Trp Arg Leu Leu Lys Arg Gly Ala Cys Pro Arg
Pro Gly 850 855 860 Asp Ser Val Thr Thr Glu Ser Lys His Lys Asp Leu
Gln Leu His Gly 865 870 875 880 19182PRTSTLV-3 subtype D 19Met Pro
Lys Thr Arg Arg Gln Arg Asn His Arg Ile Lys Thr Gln Arg 1 5 10 15
Pro Ser Thr Pro Trp Pro Thr Phe Gln Val Ser Gly Arg Ala Cys Ser 20
25 30 Thr Gly Thr Leu Ser Thr Phe Ser Ala Ile Val Cys Arg Pro Ile
Gly 35 40 45 Ala Pro Phe Pro Gly Gly Phe Val Pro Pro Gly Tyr Ile
Gly Thr Pro 50 55 60 Tyr Trp Pro Pro Val Leu Asn Thr Arg Ser Pro
Gly Thr Pro Ser Met 65 70 75 80 Asp Ala Leu Ser Ala Arg Leu Tyr Asn
Thr Leu Ser Leu Ala Ser Pro 85 90 95 Pro Ser Pro Pro Lys Glu Leu
Pro Ala Pro Ser Arg Ser Ser Pro Arg 100 105 110 Arg Pro Leu Leu Gln
Pro Pro Lys Phe Leu Pro Pro Ser Ser Met Gln 115 120 125 Ser Gly Asn
Thr Pro Leu Ser Glu Thr Thr Ala Ser Ser Ser Pro Trp 130 135 140 Glu
Ser Asn Tyr Pro Pro Cys Leu Ser Pro Thr Pro Ala Ser Asp Pro 145 150
155 160 Lys Met Ser Ile Pro Cys Gly Glu Ala Pro Ser Cys Ala Tyr Thr
Ser 165 170 175 Thr Asn Ser His Leu Gln 180 20350PRTSTLV-3 subtype
D 20Met Ala His Phe Pro Gly Phe Gly Gln Ser Leu Leu Tyr Gly Tyr Pro
1 5 10 15 Val Tyr Val Phe Gly Asp Cys Val Gln Ala Asp Trp Cys Pro
Ile Ser 20 25 30 Gly Gly Leu Cys Ser Ala Arg Leu His Arg His Ala
Leu Leu Ala Thr 35 40 45 Cys Pro Glu His Gln Ile Thr Trp Asp Pro
Ile Asp Gly Arg Val Val 50 55 60 Ser Ser Pro Leu Gln Tyr Leu Ile
Pro Arg Leu Pro Ser Phe Pro Thr 65 70 75 80 Gln Arg Thr Ser Arg Thr
Leu Lys Val Leu Thr Pro Pro Pro Thr Ala 85 90 95 Thr Thr Pro Lys
Val Pro Pro Ser Phe Phe His Ala Val Arg Lys His 100 105 110 Thr Pro
Phe Arg Asn Asn Cys Leu Glu Leu Thr Leu Gly Glu Gln Leu 115 120 125
Pro Ala Met Ser Phe Pro Asp Pro Gly Leu Arg Pro Gln Asn Val Tyr 130
135 140 Thr Met Trp Gly Ser Thr Ile Val Cys Leu Tyr Leu Tyr Gln Leu
Thr 145 150 155 160 Pro Pro Met Thr Trp Pro Leu Ile Pro His Val Ile
Phe Cys His Pro 165 170 175 Asp Gln Leu Gly Ala Phe Leu Thr Lys Ile
Pro Thr Lys Arg Leu Glu 180 185 190 Glu Leu Leu Tyr Lys Leu Phe Leu
Ser Thr Gly Ala Ile Leu Ile Leu 195 200 205 Pro Glu Asn Cys Phe Pro
Thr Thr Leu Phe Gln Pro Thr Arg Ala Pro 210 215 220 Val Ile Gln Ala
Pro Trp His Ser Gly Leu Leu Pro Tyr Leu Lys Glu 225 230 235 240 Ile
Val Thr Pro Gly Leu Ile Trp Val Phe Thr Asp Gly Ser Ser Met 245 250
255 Ile Ser Gly Pro Cys Pro Lys Glu Gly Gln Pro Ser Leu Val Val Gln
260 265 270 Ser Ser Thr Phe Ile Phe Gln Lys Phe Gln Thr Lys Ala Tyr
His Pro 275 280 285 Ala Phe Leu Leu Ser His Lys Leu Ile Gln Tyr Ser
Ser Phe His Ser 290 295 300 Leu His Leu Leu Phe Glu Glu Tyr Thr Thr
Val Pro Phe Ser Leu Leu 305 310 315 320 Phe Asn Glu Lys Glu Ala Asn
Asp Ser Asp Ser Lys Pro Gln Gly Glu 325 330 335 Pro Gln Leu Leu Ala
Lys Gly His Thr Val Glu Ser Ser Val 340 345 350 2124DNAArtificial
sequenceSynthetic oligonucleotide primer. 21ccctcaaggt cctcaccccg
ccgc 242224DNAArtificial sequenceSynthetic oligonucleotide primer.
22taacggccag gtcattggag gtgt 242324DNAArtificial sequenceSynthetic
oligonucleotide primer. 23aagttcctcc ctccttcttc catg
242427DNAArtificial sequenceSynthetic oligonucleotide primer.
24tggtagaggt ataagcacac gatggtg 27251053DNASTLV-3 subtype D
25atggcccact ttccaggttt cgggcagagc ctgctctacg ggtaccctgt ctacgttttc
60ggcgattgtg tgcaggccga ttggtgcccc atttccgggg ggctttgttc cgcccggcta
120catcggcacg ccttactggc cacctgtcct gaacaccaga tcacctggga
ccccatcgat 180ggacgcgttg tcagctcgcc tctacaatac cttatccctc
gcctcccctc cttccccacc 240caaagaactt cccgcaccct caaggtcctc
accccgccgc ccactgctac aacccccaaa 300gttcctccct ccttcttcca
tgcagtcagg aaacacaccc ctttccgaaa caactgcctc 360gagctcacct
tgggagagca actacccgcc atgtctttcc ccgaccccgg cctccgaccc
420caaaatgtct ataccatgtg gggaagcacc atcgtgtgct tatacctcta
ccaactcaca 480cctccaatga cctggccgtt aatcccacat gtcatttttt
gccatccgga ccaactaggg 540gccttcctaa caaaaatccc taccaaacgc
ttggaagaac tcttatacaa actattctta 600agtacagggg ccatacttat
cctacctgaa aattgcttcc caactaccct gtttcagccc 660acccgcgcac
cagtaattca agccccctgg cactcaggcc tactcccata cctaaaggaa
720attgtcaccc ccgggctgat ttgggtgttt actgacggta gttctatgat
ttccggaccc 780tgccccaagg aagggcagcc atctttggtg gtccaatcat
ctacattcat tttccaaaaa 840tttcaaacca aagcctatca cccagccttc
ctcctgtccc ataaattaat ccaatactcc 900tcgttccatt ccctccatct
actttttgaa gaatacacca ctgtcccctt ttctttattg 960tttaacgaaa
aagaggcaaa tgacagtgac agcaagcccc aaggcgagcc acaactacta
1020gccaaagggc atacagttga atcatctgtc tag 105326549DNASTLV-3 subtype
D 26atgcccaaga cccggagaca gcgtaaccac cgaatcaaaa cacaaagacc
ttcaactcca 60tggcccactt tccaggtttc gggcagagcc tgctctacgg gtaccctgtc
tacgttttcg 120gcgattgtgt gcaggccgat tggtgcccca tttccggggg
gctttgttcc gcccggctac 180atcggcacgc cttactggcc acctgtcctg
aacaccagat cacctgggac cccatcgatg 240gacgcgttgt cagctcgcct
ctacaatacc ttatccctcg cctcccctcc ttccccaccc 300aaagaacttc
ccgcaccctc aaggtcctca ccccgccgcc cactgctaca acccccaaag
360ttcctccctc cttcttccat gcagtcagga aacacacccc tttccgaaac
aactgcctcg 420agctcacctt gggagagcaa ctacccgcca tgtctttccc
cgaccccggc ctccgacccc 480aaaatgtcta taccatgtgg ggaagcacca
tcgtgtgctt atacctctac caactcacac 540ctccaatga 549
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