Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
POLYNUCLEOTIDE VACCINES EXPRESSING CODON OPTIMIZED HIV-1
NEF AND MODIFIED HIV-1 NEF
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit, under 35 U.S.C. ~119(e), of U.S.
provisional application 60/172,442, filed December 17, 1999.
STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not Applicable
REFERENCE TO MICROFICHE APPENDIX
Not Applicable
FIELD OF THE INVENTION
The present invention relates to HIV Nef polynucleotide pharmaceutical
products, as well as the production and use thereof which, when directly
introduced
into living vertebrate tissue, preferably a mammalian host such as a human or
a
non-human mammal of commercial or domestic veterinary importance, express the
HIV Nef protein or biologically relevant portions thereof within the animal,
inducing
a cellular immune response which specifically recognizes human
immunodeficiency
virus-1 (HIV-1). The polynucleotides of the present invention are synthetic
DNA
molecules encoding codon optimized HIV-1 Nef and derivatives of optimized HIV-
1
Nef, including nef mutants which effect wild type characteristics of Nef, such
as
myristylation and down regulation of host CD4. The polynucleotide vaccines of
the
present invention should offer a prophylactic advantage to previously
uninfected
individuals and/or provide a therapeutic effect by reducing viral load levels
within an
infected individual, thus prolonging the asymptomatic phase of HIV-1
infection.
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BACKGROUND OF THE INVENTION
Human Immunodeficiency Virus-1 (HIV-1) is the etiological agent of
acquired human immune deficiency syndrome (AIDS) and related disorders. HIV-1
is an RNA virus of the Retroviridae family and exhibits the 5' LTR-gag-pol-env-
LTR 3' organization of all retroviruses. The integrated form of HIV-1, known
as the
provirus, is approximately 9.8 Kb in length. Each end of the viral genome
contains
flanking sequences known as long terminal repeats (LTRs). The HIV genes encode
at
least nine proteins and are divided into three classes; the major structural
proteins
(Gag, Pol, and Envy, the regulatory proteins (Tat and Rev); and the accessory
proteins
(Vpu, Vpr, Vif and Nef).
The gag gene encodes a 55-kilodalton (kDa) precursor protein (p55) which is
expressed from the unspliced viral mRNA and is proteolytically processed by
the HIV
protease, a product of the pol gene. The mature p55 protein products are p17
(matrix), p24 (capsid), p9 (nucleocapsid) and p6.
The pol gene encodes proteins necessary for virus replication; a reverse
transcriptase, a protease, integrase and RNAse H. These viral proteins are
expressed
as a Gag-Pol fusion protein, a 160 kDa precursor protein which is generated
via a
ribosomal frame shifting. The viral encoded protease proteolytically cleaves
the Pol
polypeptide away from the Gag-Pol fusion and further cleaves the Pol
polypeptide to
the mature proteins which provide protease (Pro, P10), reverse transcriptase
(RT,
P50), integrase (IN, p31) and RNAse H (RNAse, p15) activities.
The nef gene encodes an early accessory HIV protein (Nef) which has been
shown to possess several activities such as down regulating CD4 expression,
disturbing T-cell activation and stimulating HIV infectivity.
The env gene encodes the viral envelope glycoprotein that is translated as a
160-kilodalton (kDa) precursor (gp160) and then cleaved by a cellular protease
to
yield the external 120-kDa envelope glycoprotein (gp120) and the transmembrane
41-
kDa envelope glycoprotein (gp41). Gp120 and gp41 remain associated and are
displayed on the viral particles and the surface of HIV-infected cells.
The tat gene encodes a long form and a short form of the Tat protein, a RNA
binding protein which is a transcriptional transactivator essential for HIV-1
replication.
The rev gene encodes the 13 kDa Rev protein, a RNA binding protein. The
Rev protein binds to a region of the viral RNA termed the Rev response element
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(RRE). The Rev protein is promotes transfer of unspliced viral RNA from the
nucleus to the cytoplasm. The Rev protein is required for HIV late gene
expression
and in turn, HIV replication.
Gp120 binds to the CD4/chemokine receptor present on the surface of helper
T-lymphocytes, macrophages and other target cells in addition to other co-
receptor
molecules. X4 (macrophage tropic) virus show tropism for CD4/CXCR4 complexes
while a RS (T-cell line tropic) virus interacts with a CD4/CCRS receptor
complex.
After gp120 binds to CD4, gp41 mediates the fusion event responsible for virus
entry.
The virus fuses with and enters the target cell, followed by reverse
transcription of its
single stranded RNA genome into the double-stranded DNA via a RNA dependent
DNA polymerase. The viral DNA, known as provirus, enters the cell nucleus,
where
the viral DNA directs the production of new viral RNA within the nucleus,
expression
of early and late HIV viral proteins, and subsequently the production and
cellular
release of new virus particles. Recent advances in the ability to detect viral
load
within the host shows that the primary infection results in an extremely high
generation and tissue distribution of the virus, followed by a steady state
level of virus
(albeit through a continual viral production and turnover during this phase),
leading
ultimately to another burst of virus load which leads to the onset of clinical
AIDS.
Productively infected cells have a half life of several days, whereas
chronically or
latently infected cells have a 3-week half life, followed by non-productively
infected
cells which have a long half life (over 100 days) but do not significantly
contribute to
day to day viral loads seen throughout the course of disease.
Destruction of CD4 helper T lymphocytes, which are critical to immune
defense, is a major cause of the progressive immune dysfunction that is the
hallmark
of HIV infection. The loss of CD4 T-cells seriously impairs the body's ability
to fight
most invaders, but it has a particularly severe impact on the defenses against
viruses,
fungi, parasites and certain bacteria, including mycobacteria.
Effective treatment regimens for HIV-1 infected individuals have become
available recently. However, these drugs will not have a significant impact on
the
disease in many parts of the world and they will have a minimal impact in
halting the
spread of infection within the human population. As is true of many other
infectious
diseases, a significant epidemiologic impact on the spread of HIV-1 infection
will
only occur subsequent to the development and introduction of an effective
vaccine.
There are a number of factors that have contributed to the lack of successful
vaccine
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development to date. As noted above, it is now apparent that in a chronically
infected
person there exists constant virus production in spite of the presence of anti-
HIV-1
humoral and cellular immune responses and destruction of virally infected
cells. As
in the case of other infectious diseases, the outcome of disease is the result
of a
balance between the kinetics and the magnitude of the immune response and the
pathogen replicative rate and accessibility to the immune response. Pre-
existing
immunity may be more successful with an acute infection than an evolving
immune
response can be with an established infection. A second factor is the
considerable
genetic variability of the virus. Although anti-HIV-1 antibodies exist that
can
neutralize HIV-1 infectivity in cell culture, these antibodies are generally
virus
isolate-specific in their activity. It has proven impossible to define
serological
groupings of HIV-1 using traditional methods. Rather, the virus seems to
define a
serological "continuum" so that individual neutralizing antibody responses, at
best,
are effective against only a handful of viral variants. Given this latter
observation, it
would be useful to identify immunogens and related delivery technologies that
are
likely to elicit anti-HIV-1 cellular immune responses. It is known that in
order to
generate CTL responses antigen must be synthesized within or introduced into
cells,
subsequently processed into small peptides by the proteasome complex, and
translocated into the endoplasmic reticulum/Golgi complex secretory pathway
for
eventual association with major histocompatibility complex (MHC) class I
proteins.
CD8+ T lymphocytes recognize antigen in association with class I MHC via the T
cell
receptor (TCR) and the CD8 cell surface protein. Activation of naive CD8+ T
cells
into activated effector or memory cells generally requires both TCR engagement
of
antigen as described above as well as engagement of costimulatory proteins.
Optimal
induction of CTL responses usually requires "help" in the form of cytokines
from
CD4+ T lymphocytes which recognize antigen associated with MHC class II
molecules via TCR and CD4 engagement.
As introduced above, the nef gene encodes an early accessory HIV protein
(Nef) which has been shown to possess several activities such as down
regulating
CD4 expression, disturbing T-cell activation and stimulating HIV infectivity.
Zazopoulos and Haseltine (1992, Proc. Natl. Acad. Sci. 89: 6634-6638) disclose
mutations to the HIV-1 nef gene which effect the rate of virus replication.
The
authors show that the nef open reading frame mutated to encode Ala-2 in place
of
Gly-2 inhibits myristolation of the protein and results in delayed viral
replication rates
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in Jurkat cells and PBMCs.
Kaminchik et al. (1991, J. Virology 65(2): 583-588) disclose an amino-
terminal nef open reading frame mutated to encode Met-Ala-Ala in place of Met-
Gly-
Gly. The authors show that this mutant is deficient in myristolation.
Saksela et al. (1995, EMBO J. 14(3): 484-491) and Lee et al. (1995, EMBO J.
14(20): 5006-5015) show the importance of a proline rich motif in HIV-1 Nef
which
mediates binding to a SH3 domain of the Hck protein. The authors conclude that
this
motif is important in the enhancement of viral replication but not down-
regulation of
CD4 expression.
Calarota et al. (1998, The Lancet 351: 1320-1325) present human clinical data
concerning immunization of three HIV infected individuals with a DNA plasmid
expressing wild type Nef. The authors conclude that immunization with a Nef
encoding DNA plasmid induced a cellular immune response in the three
individuals.
However, two of the three patients were on alternative therapies during the
study, and
the authors conclude that the CTL response was most likely a boost to a pre-
existing
CTL response. In addition, the viral load increased substantially in two of
the three
patients during the course of the study.
Tobery et al. (1997, J. Exp. Med. 185(5): 909-920) constructed two ubiquitin
nef (Ub-nef) fusion constructs, one which encoded the Nef initiating
methionine and
the other with an Arg residue at the amino terminus of the Nef open reading
frame.
The authors state that vaccinia- or plasmid-based immunization of mice with a
Ub-nef
construct containing an Arg residue at the amino terminus induces a Nef-
specific CTL
response. The authors suggest the expressed fusion protein is more efficiently
presented to the MHC class I antigen presentation pathway, resulting in an
improved
cellular immune response.
Kim et al. (1997, J. Immunol. 158(2): 816-826) disclose that co-administration
of a plasmid DNA construct expressing IL-12 with a plasmid construct
expressing
Nef results in an improved cellular immune response in mice when compared to
inoculation with the Nef construct alone. The authors reported a reduction in
the
humoral response from the Nef / IL-12 co-administration as compared to
administration of the plasmid construct expressing Nef alone.
Moynier et al. (1998, Vaccine 16(16): 1523-1530) show varying humoral
responses in mice immunized with a DNA plasmid encoding Nef, depending upon
the
presence of absence of Freund's adjuvant. No data is disclosed regarding a
cellular
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immune response in mice vaccinated with the aforementioned DNA construct
alone.
Hanna et al. (1998, Cell 95:163-175) suggest that wild type Nef may play a
critical role in AIDS pathogenicity.
It would be of great import in the battle against A>DS to produce a
prophylactic- and/or therapeutic-based HIV vaccine which generates a strong
cellular
immune response against an HIV infection. The present invention addresses and
meets this needs by disclosing a class of DNA vaccines based on host delivery
and
expression of the early HIV gene, nef.
SUMMARY OF THE INVENTION
The present invention relates to synthetic DNA molecules (also referred to
herein as "polynucleotides") and associated DNA vaccines (also referred to
herein as
"polynucleotide vaccines") which elicit CTL responses upon administration to
the
host, such as a mammalian host and including primates and especially humans,
as
well as non-human mammals of commercial or domestic veterinary importance.
The CTL-directed vaccines of the present invention should lower transmission
rate to
previously uninfected individuals and/or reduce levels of the viral loads
within an
infected individual, so as to prolong the asymptomatic phase of HIV-1
infection. In
particular, the present invention relates to DNA vaccines which encode various
forms
of HIV-1 Nef, wherein administration, intracellular delivery and expression of
the
HIV-1 nef gene of interest elicits a host CTL and Th response. The preferred
synthetic DNA molecules of the present invention encode codon optimized
versions
of wild type HIV-1 Nef, codon optimized versions of HIV-1 Nef fusion proteins,
and
codon optimized versions of HIV-1 Nef derivatives, including but not limited
to nef
modifications involving introduction of an amino-terminal leader sequence,
removal
of an amino-terminal myristylation site and/or introduction of dileucine motif
mutations. The Nef-based fusion and modified proteins disclosed within this
specification may possess altered trafficking and/or host cell function while
retaining
the ability to be properly presented to the host MHC I complex and in turn
elicit a
host CTL and Th response.
A particular embodiment of the present invention relates to a DNA molecule
encoding HIV-1 Nef from the HIV-1 jfrl isolate wherein the codons are
optimized for
expression in a mammalian system such as a human. The DNA molecule which
encodes this protein is disclosed herein as SEQ ID NO:1, while the expressed
open
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reading frame is disclosed herein as SEQ ID N0:2.
In another embodiment of the present invention, a codon optimized DNA
molecule encoding a protein containing the human plasminogen activator (tpa)
leader
peptide fused with the NHz-terminus of the HIV-1 Nef polypeptide. The DNA
molecule which encodes this protein is disclosed herein as SEQ ID N0:3, while
the
expressed open reading frame is disclosed herein as SEQ 117 N0:4.
In an additional embodiment, the present invention relates to a DNA molecule
encoding optimized HIV-1 Nef wherein the open reading frame codes for
modifications at the amino terminal myristylation site (Gly-2 to Ala-2) and
substitution of the Leu-174-Leu-175 dileucine motif to Ala-174-Ala-175, herein
described as opt nef (G2A,LLAA). The DNA molecule which encodes this protein
is
disclosed herein as SEQ ID NO:S, while the expressed open reading frame is
disclosed herein as SEQ B7 N0:6.
Another additional embodiment of the present invention relates to a DNA
molecule encoding optimized HIV-1 Nef wherein the amino terminal myristylation
site and dileucine motif have been deleted, as well as comprising a tPA leader
peptide.
This DNA molecule, opt tpanef (LLAA), comprises an open reading frame which
encodes a Nef protein containing a tPA leader sequence fused to amino acid
residue
6-216 of HIV-1 Nef (jfrl), wherein Leu-174 and Leu-175 are substituted with
Ala-174
and Ala-175, herein referred to as opt tpanef (LLAA) is disclosed herein as
SEQ >D
N0:7, while the expressed open reading frame is disclosed herein as SEQ ID
N0:8.
The present invention also relates to non-codon optimized versions of DNA
molecules and associated DNA vaccines which encode the various wild type and
modified forms of the HIV Nef protein disclosed herein. Partial or fully codon
optimized DNA vaccine expression vector constructs are preferred, but it is
within the
scope of the present invention to utilize "non-codon optimized" versions of
the
constructs disclosed herein, especially modified versions of HIV Nef which are
shown
to promote a substantial cellular immune response subsequent to host
administration.
The DNA backbone of the DNA vaccines of the present invention are
preferably DNA plasmid expression vectors. DNA plasmid expression vectors
utilized in the present invention include but are not limited to constructs
which
comprise the cytomegalovirus promoter with the intron A sequence (CMV-intA)
and
a bovine growth hormone transcription termination sequence. In addition, the
DNA
plasmid vectors of the present invention preferably comprise an antibiotic
resistance
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marker, including but not limited to an ampicillin resistance gene, a neomycin
resistance gene or any other pharmaceutically acceptable antibiotic resistance
marker.
In addition, an appropriate polylinker cloning site and a prokaryotic origin
of
replication sequence are also preferred. Specific DNA vectors of the present
invention include but are not limited to V1, V1J (SEQ 1D N0:14), VlJneo (SEQ
>D
NO:15), VlJns (Figure 1A, SEQ 117 N0:16), V1R (SEQ ID N0:26), and any of the
aforementioned vectors wherein a nucleotide sequence encoding a leader
peptide,
preferably the human tPA leader, is fused directly downstream of the CMV-intA
promoter, including but not limited to VlJns-tpa, as shown in Figure 1B and
SEQ ID
N0:19.
The present invention especially relates to a DNA vaccine and a
pharmaceutically active vaccine composition which contains this DNA vaccine,
and
the use as a prophylactic and/or therapeutic vaccine for host immunization,
preferably
human host immunization, against an HIV infection or to combat an existing HIV
condition. These DNA vaccines are represented by codon optimized DNA molecules
encoding HIV-1 Nef of biologically active Nef modifications or Nef-containing
fusion proteins which are ligated within an appropriate DNA plasmid vector,
with or
without a nucleotide sequence encoding a functional leader peptide. DNA
vaccines of
the present invention relate in part to codon optimized DNA molecules encoding
HIV-1 Nef of biologically active Nef modifications or Nef-containing fusion
proteins
ligated in DNA vectors V 1, V 1J (SEQ ID N0:14), V lJneo (SEQ ID NO:15), V
lJns
(Figure 1A, SEQ ID N0:16), V1R (SEQ ID N0:26), or any of the aforementioned
vectors wherein a nucleotide sequence encoding a leader peptide, preferably
the
human tPA leader, is fused directly downstream of the CMV-intA promoter,
including but not limited to VlJns-tpa, as shown in Figure 1B and SEQ >D
N0:19.
Especially preferred DNA vaccines of the present invention include codon
optimized
DNA vaccine constructs VlJns/nef, VlJns/tpanef, VlJns/tpanef(LLAA) and
VlJns/(G2A,LLAA), as exemplified in Example Section 2.
The present invention also relates to HIV Nef polynucleotide
pharmaceutical products, as well as the production and use thereof, wherein
the
DNA vaccines are formulated with an adjuvant or adjuvants which may increase
immunogenicity of the DNA polynucleotide vaccines of the present invention,
namely by increasing a humoral response to inoculation. A preferred adjuvant
is
an aluminum phosphate-based adjuvant or a calcium phosphate based adjuvant,
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with an aluminum phosphate adjuvant being especially preferred. Another
preferred adjuvant is a non-ionic block copolymer, preferably comprising the
blocks of polyoxyethylene (POE) and polyoxypropylene (POP) such as a POE-
POP-POE block copolymer. These adjuvanted forms comprising the DNA
vaccines disclosed herein are useful in increasing humoral responses to DNA
vaccination without imparting a negative effect on an appropriate cellular
immune
response.
As used herein, a DNA vaccine or DNA polynucleotide vaccine or
polynucleotide vaccine is a DNA molecule (i.e., "nucleic acid",
"polynucleotide")
which contains essential regulatory elements such that upon introduction into
a living,
vertebrate cell, it is able to direct the cellular machinery to produce
translation
products encoded by the respective nef genes of the present invention.
BRIEF DESCRIPTION OF THE FIGURES
Figure lA-B show a schematic representation of DNA vaccine expression
vectors VlJns (A) and VlJns/tpa utilized for HIV-1 nef and HIV-1 modified nef
constructs.
Figure 2A-B show a nucleotide sequence comparison between wild type
nef(jrfl) and codon optimized nef. The wild type nef gene from the jrfl
isolate
consists of 648 nucleotides capable of encoding a 216 amino acid polypeptide.
WT,
wild type sequence (SEQ >D N0:9); opt, codon-optimized sequence (contained
within
SEQ m NO:1). The Nef amino acid sequence is shown in one-letter code (SEQ m
N0:2).
Figure 3A-C show nucleotide sequences at junctions between nef coding
sequence and plasmid backbone of nef expression vectors V lJns/nef (Figure
3A),
VlJns/nef(G2A,LLAA) (Figure 3B), VlJns/tpanef (Figure 3C) and
V lJns/tpanef(LLAA) (Figure 3C, also). 5' and 3' flanking sequences of codon
optimized nef or codon optimized nef mutant genes are indicated by bold/italic
letters;
nef and nef mutant coding sequences are indicated by plain letters. Also
indicated (as
underlined) are the restriction endonuclease sites involved in construction of
respective nef expression vectors. VlJns/tpanef and VlJns/tpanef(LLAA) have
identical sequences at the junctions.
Figure 4 shows a schematic presentation of nef and nef derivatives. Amino
acid residues involved in Nef derivatives are presented. Glycine 2 and
Leucine174
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and 175 are the sites involved in myristylation and dileucine motif,
respectively. For
both versions of the tpanef fusion genes, the putative leader peptide cleavage
sites are
indicated with "*", and a exogenous serine residue introduced during the
construction
of the mutants is underlined.
Figure 5 shows Western blot analysis of nef and modified nef proteins
expressed in transfected 293 cells. 293 cells grown in 100 mm culture dish
were
transfected with respective codon optimized nef constructs. Sixty hours post
transfection, supernatant and cells were collected separately and separated on
10%
SDS-PAGE under reducing conditions. The proteins were transferred into a PVDF
membrane and probed with a mixture of Gag mAb and Nef mAbs, both at 1:2000
dilution. The protein signals were detected with ECL. (A) cells transfected
with
VlJns/gag only; (B) cells transfected with VlJns/gag and VlJns/nef; (C) cells
transfected with VlJns/gag and VlJns/nef(G2A, LLAA); (D) cells transfected
with
V lJns/gag and V lJns/tpanef; (E) cells transfected with V lJns/gag and
VlJns/tpanef(LLAA). The low case letter c and m represent medium and cellular
fractions, respectively. M.W. = molecular weight marker.
Figure 6 shows an Elispot assay of cell-mediated responses to Nef peptides.
Three strains of mice, Balb/c, C57BIJ6 and C3H, were immunized with 50 mcg of
V lJns/nef (codon optimized) and boosted twice with a two-week interval. Two
weeks following the final immunization, splenocytes were isolated and tested
in an
Elispot assay against respective Nef peptide pools. As a control, splenocytes
were
from non-immunized naive mice were tested in parallel. Nef peptide pool A
consists
of all 21 Nef peptides; Nef peptide pool B consists of 11 non-overlapping
peptide
started from residue 1; Nef peptide pool C consists of 10 non-overlapping
peptides
started from residue 11. SFC, INF-gamma secreting spot-forming cells.
Figure 7A-C show Nef-specific CD8 and CD4 epitope mapping. The
immunization regime is as per Figure 6. Mouse splenocytes were isolated and
fractionated into CD8+ and CD8- cells using Miltenyi's magnetic cell
separator. The
resultant CD8+ and CD8- cells were then tested in an Elispot assay against
individual
Nef peptides. SFC, INF-gamma secreting spot-forming cells. The mice strains
tested
are Balb/c mice (Figure 7A), C57BL/6 mice (Figure 7B), and C3H mice (Figure
7C).
Figure 8A-C show identification of a Nef CTL epitope. Splenocytes from nef
immunized C57BL/6 mice were stimulated in vitro with peptide-pulsed,
irradiated
naiva splenocytes for 7 days. Following the in vitro stimulation, cells were
harvested
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and tested in a standard S~Cr-releasing assay using peptide pulsed EL-4 cells
as
targets. Open symbol, specific killings of EL-4 cells without peptide; solid
symbol,
specific killing of EL-4 cells with peptide. Panel A - peptide Nef 51-70;
Panel B -
peptide Nef 60-68, Panel C - peptide Nef 58-70.
Figure 9A-B shows a comparison of the immunogenicity of codon optimized
DNA vaccine vectors expressing Nef and modified forms of Nef C57BL/6 mice,
five
per group, were immunized with 100 mcg of the indicated nef constructs.
Fourteen
days following immunization, splenocytes were collected and tested against the
Nef
CD8 (aa58-66) and CD4 (aa81-100) peptides. Identical immunization regimens
were
used for both experiments. In experiment 1 (Panel A), three codon optimized
nef
constructs were tested, namely, VlJns/nef, VlJns/tpanef(LLAA) and
VlJns/nef(G2A,LLAA), whereas in experiment 2 (Panel B) all four codon
optimized
nef constructs were tested. The data represent means plus standard deviation
of 5
mice per group.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to synthetic DNA molecules (also referred to
herein as "nucleic acid" molecules or "polynucleotides") and associated DNA
vector
vaccines (also referred to herein as "polynucleotide vaccines") which elicit
CTL and
humoral responses upon administration to the host, including primates and
especially
humans. In particular, the present invention relates to DNA vector vaccines
which
encode various forms of HIV-1 Nef, wherein administration, intracellular
delivery
and expression of the HIV-1 nef gene of interest elicits a host CTL and Th
response.
The synthetic DNA molecules of the present invention encode codon optimized
versions of wild type HIV-1 Nef, codon optimized versions of HIV-1 Nef fusion
proteins, and codon optimized versions of HIV-1 Nef derivatives, including but
not
limited to nef modifications involving introduction of an amino-terminal
leader
sequence, removal of an amino-terminal myristylation site and/or introduction
of
dileucine motif mutations. In some instances the Nef-based fusion and modified
proteins disclosed within this specification possess altered trafficking
and/or host cell
function while retaining the ability to be properly presented to the host MHC
I
complex. Those skilled in the art will recognize that the use of nef genes
from HIV-2
strains which express Nef proteins having analogous function to HIV-1 Nef
would be
expected to generate immune responses analogous to those described herein for
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HIV-1 constructs.
In order to generate a CTL response, the immunogen must be synthesized
within (MHCI presentation) or introduced into cells (MHCII presentation). For
intracellular synthesized immunogens, the protein is expressed and then
processed
into small peptides by the proteasome complex, and translocated into the
endoplasmic
reticulum/Golgi complex secretory pathway for eventual association with major
histocompatibility complex (MHC) class I proteins. CD8+ T lymphocytes
recognize
antigen in association with class I MHC via the T cell receptor (TCR).
Activation of
naive CD8+ T cells into activated effector or memory cells generally requires
both
TCR engagement of antigen as described above as well as engagement of
co-stimulatory proteins. Optimal induction of CTL responses usually requires
"help"
in the form of cytokines from CD4+ T lymphocytes which recognize antigen
associated with MHC class II molecules via TCR.
The HIV-1 genome employs predominantly uncommon codons compared to
highly expressed human genes. Therefore, the nef open reading frame has been
synthetically manipulated using optimal codons for human expression. As noted
above, a preferred embodiment of the present invention relates to DNA
molecules
which comprise a HIV-1 nef open reading frame, whether encoding full length
nef or
a modification or fusion as described herein, wherein the codon usage has been
optimized for expression in a mammal, especially a human.
In a particular embodiment of the present invention, a DNA molecule
encoding HIV-1 Nef from the HIV-1 jfrl isolate wherein the codons are
optimized for
expression in a mammalian system such as a human. The nucleotide sequence of
the
codon optimized version of HIV-1 jrfl nef gene is disclosed herein as SEQ ID
NO:1,
as shown herein:
GATCTGCCAC CATGGGCGGC AAGTGGTCCA AGAGGTCCGT GCCCGGCTGG TCCACCGTGA
GGGAGAGGAT GAGGAGGGCC GAGCCCGCCG CCGACAGGGT GAGGAGGACC GAGCCCGCCG
CCGTGGGCGT GGGCGCCGTG TCCAGGGACC TGGAGAAGCA CGGCGCCATC ACCTCCTCCA
ACACCGCCGC CACCAACGCC GACTGCGCCT GGCTGGAGGC CCAGGAGGAC GAGGAGGTGG
3O GCTTCCCCGT GAGGCCCCAG GTGCCCCTGA GGCCCATGAC CTACAAGGGC GCCGTGGACC
TGTCCCACTT CCTGAAGGAG AAGGGCGGCC TGGAGGGCCT GATCCACTCC CAGAAGAGGC
AGGACATCCT GGACCTGTGG GTGTACCACA CCCAGGGCTA CTTCCCCGAC TGGCAGAACT
ACACCCCCGG CCCCGGCATC AGGTTCCCCC TGACCTTCGG CTGGTGCTTC AAGCTGGTGC
CCGTGGAGCC CGAGAAGGTG GAGGAGGCCA ACGAGGGCGA GAACAACTGC CTGCTGCACC
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CCATGTCCCA GCACGGCATC GAGGACCCCG AGAAGGAGGT GCTGGAGTGG AGGTTCGACT
CCAAGCTGGC CTTCCACCAC GTGGCCAGGG AGCTGCACCC CGAGTACTAC AAGGACTGCT
AAAGCCCGGG C (SEQ ID N0:1).
As can be discerned from comparing native to optimized codon usage in
Figure 2A-B, the following codon usage for mammalian optimization is
preferred:
Met (ATG), Gly (GGC), Lys (AAG), Trp (TGG), Ser (TCC), Arg (AGG), Val (GTG),
Pro (CCC), Thr (ACC), Glu (GAG); Leu (CTG), His (CAC), Ile (ATC), Asn (AAC),
Cys (TGC), Ala (GCC), Gln (CAG), Phe (TTC) and Tyr (TAC). For an additional
discussion relating to mammalian (human) codon optimization, see WO 97/31115
(PCT/US97/02294), which is hereby incorporated by reference.
The open reading frame for SEQ ID NO:l above comprises an initiating
methionine residue at nucleotides 12-14 and a "TAA" stop codon from
nucleotides
660-662. The open reading frame of SEQ ID NO:1 provides for a 216 amino acid
HIV-1 Nef protein expressed through utilization of a codon optimized DNA
vaccine
vector. The 216 amino acid HIV-1 Nef (jfrl) protein is disclosed herein as SEQ
ID
N0:2, and as follows:
Met Gly Gly Lys Trp Ser Lys Arg Ser Val Pro Gly Trp Ser Thr Val
Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Arg Val Arg Arg
Thr Glu Pro Ala Ala Val Gly Val Gly Ala Val Ser Arg Asp Leu Glu
Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Ala Thr Asn Ala Asp
Cys Ala Trp Leu Glu Ala Gln Glu Asp Glu Glu Val Gly Phe Pro Val
Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Gly Ala Val Asp
Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile His
Ser Gln Lys Arg Gln Asp Ile Leu Asp Leu Trp Val Tyr His Thr Gln
Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Ile Arg
Phe Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu Val Pro Val Glu Pro
Glu Lys Val Glu Glu Ala Asn Glu Gly Glu Asn Asn Cys Leu Leu His
Pro Met Ser Gln His Gly Ile Glu Asp Pro Glu Lys Glu Val Leu Glu
Trp Arg Phe Asp Ser Lys Leu Ala Phe His His Val Ala Arg Glu Leu
His Pro Glu Tyr Tyr Lys Asp Cys (SEQ ID N0:2).
HIV-1 Nef is a 206 amino acid cytosolic protein which associates with the
inner surface of the host cell plasma membrane through myristylation of Gly-2
(Franchini et al., 1986, Virology 155: 593-599). While not all possible Nef
functions
have been elucidated, it has become clear that correct trafficking of Nef to
the inner
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plasma membrane promotes viral replication by altering the host intracellular
environment to facilitate the early phase of the HIV-1 life cycle and by
increasing the
infectivity of progeny viral particles. In one aspect of the invention
regarding
codon-optimized, protein-modified polypeptides, either the DNA vaccine vector
molecule or the HIV-1 nef construct is modified to contain a nucleotide
sequence
which encodes a heterologous leader peptide such that the amino terminal
region of
the expressed protein will contain the leader peptide. The diversity of
function that
typifies eukaryotic cells depends upon the structural differentiation of their
membrane
boundaries. To generate and maintain these structures, proteins must be
transported
from their site of synthesis in the endoplasmic reticulum to predetermined
destinations throughout the cell. This requires that the trafficking proteins
display
sorting signals that are recognized by the molecular machinery responsible for
route
selection located at the access points to the main trafficking pathways.
Sorting
decisions for most proteins need to be made only once as they traverse their
biosynthetic pathways since their final destination, the cellular location at
which they
perform their function, becomes their permanent residence. Maintenance of
intracellular integrity depends in part on the selective sorting and accurate
transport of
proteins to their correct destinations. Defined sequence motifs exist in
proteins which
can act as 'address labels'. A number of sorting signals have been found
associated
with the cytoplasmic domains of membrane proteins. An effective induction of
CTL
responses often required sustained, high level endogenous expression of an
antigen. In
light of its diverse biological activities, vaccines composed of wild-type Nef
could
potentially have adverse effects on the host cells. As membrane-association
via
myristylation is an essential requirement for most of Nef's function, mutants
lacking
myristylation, by glycine-to-alanine change, change of the dileucine motif
andJor by
substitution with a tpa leader sequence as described herein, will be
functionally
defective, and therefore will have improved safety profile compared to wild-
type Nef
for use as an HIV-1 vaccine component.
In a preferred and exemplified embodiment of this portion of the invention,
either the DNA vector or the HIV-1 nef nucleotide sequence is modified to
include
the human tissue-specific plasminogen activator (tPA) leader. As shown in
Figure lA-B for the DNA vector VlJns, a DNA vector which may be utilized to
practice the present invention may be modified by known recombinant DNA
methodology to contain a leader signal peptide of interest, such that
downstream
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cloning of the modified HIV-1 protein of interest results in a nucleotide
sequence
which encodes a modified HIV-1 tPA/Nef protein. In the alternative, as noted
above,
insertion of a nucleotide sequence which encodes a leader peptide may be
inserted
into a DNA vector housing the open reading frame for the Nef protein of
interest.
Regardless of the cloning strategy, the end result is a polynucleotide vaccine
which
comprises vector components for effective gene expression in conjunction with
nucleotide sequences which encode a modified HIV-1 Nef protein of interest,
including but not limited to a HIV-1 Nef protein which contains a leader
peptide. The
amino acid sequence of the human tPA leader utilized herein is as follows:
MDAMKRGLCCVLLLCGAVFVSPSEISS (SEQ m N0:19).
It has been shown that myristylation of Gly-2 in conjunction with a dileucine
motif in the carboxy region of the protein is essential for Nef-induced down
regulation of CD4 (Aiken et al., 1994, Cell 76: 853-864) via endocytosis. It
has also
been shown that Nef expression promotes down regulation of MHCI (Schwartz et
al.,
1996, Nature Medicine 2(3): 338-342) via endocytosis. The present invention
relates
in part to DNA vaccines which encode modified Nef proteins altered in
trafficking
and/or functional properties. The modifications introduced into the DNA
vaccines of
the present invention include but are not limited to additions, deletions or
substitutions to the nef open reading frame which results in the expression of
a
modified Nef protein which includes an amino terminal leader peptide,
modification
or deletion of the amino terminal myristylation site, and modification or
deletion of
the dileucine motif within the Nef protein and which alter function within the
infected
host cell. Therefore, a central theme of the DNA molecules and DNA vaccines of
the
present invention is (1) host administration and intracellular delivery of a
codon
optimized nef-based DNA vector vaccine; (2) expression of a modified Nef
protein
which is immunogenic in terms of eliciting both CTL and Th responses; and,
(3) inhibiting or at least altering known early viral functions of Nef which
have been
shown to promote HIV-1 replication and load within an infected host.
In another preferred and exemplified embodiment of the present invention, the
nef coding region is altered, resulting in a DNA vaccine which expresses a
modified
Nef protein wherein the amino terminal Gly-2 myristylation residue is either
deleted
or modified to express alternate amino acid residues.
In another preferred and exemplified embodiment of the present invention, the
nef coding region is altered, resulting in a DNA vaccine which expresses a
modified
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Nef protein wherein the dileucine motif is either deleted or modified to
express
alternate amino acid residues.
Therefore, the present invention relates to an isolated DNA molecule,
regardless of codon usage, which expresses a wild type or modified Nef protein
as
described herein, including but not limited to modified Nef proteins which
comprise a
deletion or substitution of Gly 2, a deletion or substitution of Leu 174 and
Leu 17S
and/or inclusion of a leader sequence.
The present invention also relates to a substantially purified protein
expressed
from the DNA polynucleotide vaccines of the present invention, especially the
purified proteins set forth below as SEQ ID NOs: 2, 4, 6, and 8. These
purified
proteins may be useful as protein-based HIV vaccines.
In a specific embodiment of the invention as it relates DNA vaccines encoding
modified forms of HIV-1, an open reading frame which encodes a Nef protein
which
comprises a tPA leader sequence fused to amino acid residue 6-216 of HIV-1 Nef
1S (jfrl) is referred to herein as opt tpanef. The nucleotide sequence
comprising the open
reading frame of opt tpanef is disclosed herein as SEQ ID N0:3, as shown
below:
CATGGATGCA ATGAAGAGAG GGCTCTGCTG TGTGCTGCTG CTGTGTGGAG CAGTCTTCGT
TTCGCCCAGC GAGATCTCCT CCAAGAGGTC CGTGCCCGGC TGGTCCACCG TGAGGGAGAG
GATGAGGAGG GCCGAGCCCG CCGCCGACAG GGTGAGGAGG ACCGAGCCCG CCGCCGTGGG
2O CGTGGGCGCC GTGTCCAGGG ACCTGGAGAA GCACGGCGCC ATCACCTCCT CCAACACCGC
CGCCACCAAC GCCGACTGCG CCTGGCTGGA GGCCCAGGAG GACGAGGAGG TGGGCTTCCC
CGTGAGGCCC CAGGTGCCCC TGAGGCCCAT GACCTACAAG GGCGCCGTGG ACCTGTCCCA
CTTCCTGAAG GAGAAGGGCG GCCTGGAGGG CCTGATCCAC TCCCAGAAGA GGCAGGACAT
CCTGGACCTG TGGGTGTACC ACACCCAGGG CTACTTCCCC GACTGGCAGA ACTACACCCC
ZS CGGCCCCGGC ATCAGGTTCC CCCTGACCTT CGGCTGGTGC TTCAAGCTGG TGCCCGTGGA
GCCCGAGAAG GTGGAGGAGG CCAACGAGGG CGAGAACAAC TGCCTGCTGC ACCCCATGTC
CCAGCACGGC ATCGAGGACC CCGAGAAGGA GGTGCTGGAG TGGAGGTTCG ACTCCAAGCT
GGCCTTCCAC CACGTGGCCA GGGAGCTGCA CCCCGAGTAC TACAAGGACT GCTAAAGCC
(SEQ ID N0:3).
30 The open reading frame for SEQ ID N0:3 comprises an initiating methionine
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residue at nucleotides4 and a stop
2- "TAA" codon
from
nucleotides
713-715.
The
open reading frameEQ ID N0:3 Nef
of S provides
for a
237 amino
acid HIV-1
protein which comprises nce fused 6-216
a tPA leader seque to amino of
acids HIV-1
Nef, including acid residues
the dileucine 174 and
motif at amino 175. This
237
amino acid tPA/Nef as SEQ :4,
(jfrl) fusion ID and
protein is disclosed N0 is
herein
shown as follows:
Met Asp Ala Met Arg Gly CysCys Val Leu Cys Gly
Lys Leu Leu Leu
Ala Val Phe Val Pro Ser IleSer Ser Arg Val Pro
Ser Glu Lys Ser
Gly Trp Ser Thr Arg Glu MetArg Arg Glu Ala Ala
Val Arg Ala Pro
Asp Arg Val Arg Thr Glu AlaAla Val Val Ala Val
Arg Pro Gly Gly
Ser Arg Asp Leu Lys His AlaIle Thr Ser Thr Ala
Glu Gly Ser Asn
Ala Thr Asn Ala Cys Ala LeuGlu Ala Glu Glu Glu
Asp Trp Gln Asp
Val Gly Phe Pro Arg Pro ValPro Leu Pro Thr Tyr
Val Gln Arg Met
Lys Gly Ala Val Leu Ser PheLeu Lys Lys Gly Leu
Asp His Glu Gly
Glu Gly Leu Ile Ser Gln ArgGln Asp Leu Leu Trp
His Lys Ile Asp
Val Tyr His Thr Gly Tyr ProAsp Trp Asn Thr Pro
Gln Phe Gln Tyr
Gly Pro Gly Ile Phe Pro ThrPhe Gly Cys Lys Leu
Arg Leu Trp Phe
Val Pro Val Glu Glu Lys GluGlu Ala Glu Glu Asn
Pro Val Asn Gly
Asn Cys Leu Leu Pro Met GlnHis Gly Glu Pro Glu
His Ser Ile Asp
Lys Glu Val Leu Trp Arg AspSer Lys Ala His His
Glu Phe Leu Phe
Val Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys (SEQ ID N0:4).
Therefore, this exemplified Nef protein, Opt tPA-Nef, contains both a tPA
leader sequence as well as deleting the myristylation site of Gly-2A DNA
molecule
encoding HIV-1 Nef from the HIV-1 jfrl isolate wherein the codons are
optimized for
expression in a mammalian system such as a human.
In another specific embodiment of the present invention, a DNA molecule is
disclosed which encodes optimized HIV-1 Nef wherein the open reading frame
codes
for modifications at the amino terminal myristylation site (Gly-2 to Ala-2)
and
substitution of the Leu-174-Leu-175 dileucine motif to Ala-174-Ala-175. This
open
reading frame is herein described as opt nef (G2A,LLAA) and is disclosed as
SEQ ID
NO:S, which comprises an initiating methionine residue at nucleotides 12-14
and a
"TAA" stop codon from nucleotides 660-662. The nucleotide sequence of this
codon
optimized version of HIV-1 jrfl nef gene with the above mentioned
modifications is
disclosed herein as SEQ ID NO:S, as follows:
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GATCTGCCAC CATGGCCGGC AAGTGGTCCA AGAGGTCCGTGCCCGGCTGGTCCACCGTGA
GGGAGAGGAT GAGGAGGGCC GAGCCCGCCG CCGACAGGGTGAGGAGGACCGAGCCCGCCG
CCGTGGGCGT GGGCGCCGTG TCCAGGGACC TGGAGAAGCACGGCGCCATCACCTCCTCCA
ACACCGCCGC CACCAACGCC GACTGCGCCT GGCTGGAGGCCCAGGAGGACGAGGAGGTGG
S GCTTCCCCGT GAGGCCCCAG GTGCCCCTGA GGCCCATGACCTACAAGGGCGCCGTGGACC
TGTCCCACTT CCTGAAGGAG AAGGGCGGCC TGGAGGGCCTGATCCACTCCCAGAAGAGGC
AGGACATCCT GGACCTGTGG GTGTACCACA CCCAGGGCTACTTCCCCGACTGGCAGAACT
ACACCCCCGG CCCCGGCATC AGGTTCCCCC TGACCTTCGGCTGGTGCTTCAAGCTGGTGC
CCGTGGAGCC CGAGAAGGTG GAGGAGGCCA ACGAGGGCGAGAACAACTGCGCCGCCCACC
CCATGTCCCA GCACGGCATC GAGGACCCCG AGAAGGAGGTGCTGGAGTGGAGGTTCGACT
CCAAGCTGGC CTTCCACCAC GTGGCCAGGG AGCTGCACCCCGAGTACTACAAGGACTGCT
AAAGCCCGGG C (SEQ ID N0:5).
The open reading frame of SEQ m NO:S ,LLAA),
encodes Nef (G2A
disclosed herein as SEQ ID N0:6, as
follows:
1S Met Ala Gly Lys Trp Ser Lys Arg Ser Gly Trp Thr Val
Val Pro Ser
Arg Glu Arg Met Arg Arg Ala Glu Pro Asp Arg Arg Arg
Ala Ala Val
Thr Glu Pro Ala Ala Val Gly Val Gly Ser Arg Leu Glu
Ala Val Asp
Lys His Gly Ala Ile Thr Ser Ser Asn Ala Thr Ala Asp
Thr Ala Asn
Cys Ala Trp Leu Glu Ala Gln Glu Asp Val Gly Pro Val
Glu Glu Phe
Arg Pro Gln Val Pro Leu Arg Pro Met Lys Gly Val Asp
Thr Tyr Ala
Leu Ser His Phe Leu Lys Glu Lys Gly Glu Gly Ile His
Gly Leu Leu
Ser Gln Lys Arg Gln Asp Ile Leu Asp Val Tyr Thr Gln
Leu Trp His
Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Gly Pro Ile Arg
Thr Pro Gly
Phe Pro Leu Thr Phe Gly Trp Cys Phe Val Pro Glu Pro
Lys Leu Val
2S Glu Lys Val Glu Glu Ala Asn Glu Gly Asn Cys Ala His
Glu Asn Ala
Pro Met Ser Gln His Gly Ile Glu Asp Lys Glu Leu Glu
Pro Glu Val
Trp Arg Phe Asp Ser Lys Leu Ala Phe Val Ala Glu Leu
His His Arg
His Pro Glu Tyr Tyr Lys Asp Cys Ser (SEQ ID N0:6).
An additional embodiment of the present invention relates to another DNA
molecule encoding optimized HIV-1 Nef wherein the amino terminal myristylation
site and dileucine motif have been deleted, as well as comprising a tPA leader
peptide.
This DNA molecule, opt tpanef (LLAA) comprises an open reading frame which
encodes a Nef protein containing a tPA leader sequence fused to amino acid
residue
6-216 of HIV-1 Nef (jfrl), wherein Leu-174 and Leu-17S are substituted with
Ala-174
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and Ala-17S (Ala-19S and Ala-196 in
this tPA-based fusion protein). The
nucleotide
sequence comprising the open reading
frame of opt tpanef (LLAA) is disclosed
herein
as SEQ >D N0:7, as shown below:
CATGGATGCA ATGAAGAGAG GGCTCTGCTG TGTGCTGCTGCTGTGTGGAG CAGTCTTCGT
S TTCGCCCAGC GAGATCTCCT CCAAGAGGTC CGTGCCCGGCTGGTCCACCG TGAGGGAGAG
GATGAGGAGG GCCGAGCCCG CCGCCGACAG GGTGAGGAGGACCGAGCCCG CCGCCGTGGG
CGTGGGCGCC GTGTCCAGGG ACCTGGAGAA GCACGGCGCCATCACCTCCT CCAACACCGC
CGCCACCAAC GCCGACTGCG CCTGGCTGGA GGCCCAGGAGGACGAGGAGG TGGGCTTCCC
CGTGAGGCCC CAGGTGCCCC TGAGGCCCAT GACCTACAAGGGCGCCGTGG ACCTGTCCCA
IO CTTCCTGAAG GAGAAGGGCG GCCTGGAGGG CCTGATCCACTCCCAGAAGA GGCAGGACAT
CCTGGACCTG TGGGTGTACC ACACCCAGGG CTACTTCCCCGACTGGCAGA ACTACACCCC
CGGCCCCGGC ATCAGGTTCC CCCTGACCTT CGGCTGGTGCTTCAAGCTGG TGCCCGTGGA
GCCCGAGAAG GTGGAGGAGG CCAACGAGGG CGAGAACAACTGCGCCGCCC ACCCCATGTC
CCAGCACGGC ATCGAGGACC CCGAGAAGGA GGTGCTGGAGTGGAGGTTCG ACTCCAAGCT
IS GGCCTTCCAC CACGTGGCCA GGGAGCTGCA CCCCGAGTACTACAAGGACT GCTAAAGCCC
(SEQ ID N0:7).
The open reading frame of SEQ >D N0:7
encoding tPA-Nef (LLAA),
disclosed herein as SEQ ID N0:8, is
as follows:
Met Asp Ala Met Lys Arg Gly Leu Cys Leu Leu Leu Cys Gly
Cys Val
ZO Ala Val Phe Val Ser Pro Ser Glu Ile Lys Arg Ser Val Pro
Ser Ser
Gly Trp Ser Thr Val Arg Glu Arg Met Ala Glu Pro Ala Ala
Arg Arg
Asp Arg Val Arg Arg Thr Glu Pro Ala Gly Val Gly Ala Val
Ala Val
Ser Arg Asp Leu Glu Lys His Gly Ala Ser Ser Asn Thr Ala
Ile Thr
Ala Thr Asn Ala Asp Cys Ala Trp Leu Gln Glu Asp Glu Glu
Glu Ala
ZS Val Gly Phe Pro Val Arg Pro Gln Val Arg Pro Met Thr Tyr
Pro Leu
Lys Gly Ala Val Asp Leu Ser His Phe Glu Lys Gly Gly Leu
Leu Lys
Glu Gly Leu Ile His Ser Gln Lys Arg Ile Leu Asp Leu Trp
Gln Asp
Val Tyr His Thr Gln Gly Tyr Phe Pro Gln Asn Tyr Thr Pro
Asp Trp
Gly Pro Gly Ile Arg Phe Pro Leu Thr Trp Cys Phe Lys Leu
Phe Gly
30 Val Pro Val Glu Pro Glu Lys Val Glu Asn Glu Gly Glu Asn
Glu Ala
Asn Cys Ala Ala His Pro Met Ser Gln Ile Glu Asp Pro Glu
His Gly
Lys Glu Val Leu Glu Trp Arg Phe Asp Leu Ala Phe His His
Ser Lys
Val Ala Arg Glu Leu His Pro Glu Tyr Asp Cys (SEQ ID N0:8).
Tyr Lys
The present invention also relates DNA molecule, regardless
in part to any of
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codon usage, which expresses a wild type or modified Nef protein as described
herein, including but not limited to modified Nef proteins which comprise a
deletion
or substitution of Gly 2, a deletion of substitution of Leu 174 and Leu 175
and/or
inclusion of a leader sequence. Therefore, partial or fully codon optimized
DNA
vaccine expression vector constructs are preferred since such constructs
should result
in increased host expression. However, it is within the scope of the present
invention
to utilize "non-codon optimized" versions of the constructs disclosed herein,
especially modified versions of HIV Nef which are shown to promote a
substantial
cellular immune response subsequent to host administration.
The DNA backbone of the DNA vaccines of the present invention are
preferably DNA plasmid expression vectors. DNA plasmid expression vectors are
well known in the art and the present DNA vector vaccines may be comprised of
any
such expression backbone which contains at least a promoter for RNA polymerase
transcription, and a transcriptional terminator 3' to the HIV nef coding
sequence. In
one preferred embodiment, the promoter is the Rous sarcoma virus (RSV) long
terminal repeat (LTR) which is a strong transcriptional promoter. A more
preferred
promoter is the cytomegalovirus promoter with the intron A sequence (CMV-
intA).
A preferred transcriptional terminator is the bovine growth hormone
terminator. In
addition, to assist in large scale preparation of an HIV nef DNA vector
vaccine, an
antibiotic resistance marker is also preferably included in the expression
vector.
Ampicillin resistance genes, neomycin resistance genes or any other
pharmaceutically
acceptable antibiotic resistance marker may be used. In a preferred embodiment
of
this invention, the antibiotic resistance gene encodes a gene product for
neomycin
resistance. Further, to aid in the high level production of the pharmaceutical
by
fermentation in prokaryotic organisms, it is advantageous for the vector to
contain an
origin of replication and be of high copy number. Any of a number of
commercially
available prokaryotic cloning vectors provide these benefits. In a preferred
embodiment of this invention, these functionalities are provided by the
commercially
available vectors known as pUC. It is desirable to remove non-essential DNA
sequences. Thus, the lacZ and lacI coding sequences of pUC are removed in one
embodiment of the invention.
DNA expression vectors exemplified herein are also disclosed in PCT
International Application No. PCT/US94/02751, International Publication
No. WO 94/21797, hereby incorporated by reference. A first DNA expression
vector
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is the expression vector pnRSV, wherein the rous sarcoma virus (RSV) long
terminal
repeat (LTR) is used as the promoter. A second embodiment relates to plasmid
V1, a
mutated pBR322 vector into which the CMV promoter and the BGH transcriptional
terminator is cloned. Another embodiment regarding DNA vector backbones
relates
to plasmid V1J. Plasmid V1J is derived from plasmid Vl and removes promoter
and
transcription termination elements in order to place them within a more
defined
context, create a more compact vector, and to improve plasmid purification
yields.
Therefore, V1J also contains the CMVintA promoter and (BGH) transcription
termination elements which control the expression of the HIV nef-based genes
disclosed herein. The backbone of V1J is provided by pUCl8. It is known to
produce high yields of plasmid, is well-characterized by sequence and
function, and is
of minimum size. The entire lac operon was removed and the remaining plasmid
was
purified from an agarose electrophoresis gel, blunt-ended with the T4 DNA
polymerise, treated with calf intestinal alkaline phosphatase, and ligated to
the
CMVintA/BGH element. In another DNA expression vector, the ampicillin
resistance
gene is removed from V1J and replaced with a neomycin resistance gene, to
generate
VlJneo. A DNA expression vector specifically exemplified herein is VlJns,
which is
the same as V 1J except that a unique Sfi 1 restriction site has been
engineered into the
single Kpn 1 site at position 2114 of V 1J-neo. The incidence of Sfi 1 sites
in human
genomic DNA is very low (approximately 1 site per 100,000 bases). Thus, this
vector
allows careful monitoring for expression vector integration into host DNA,
simply by
Sfi 1 digestion of extracted genomic DNA. Another DNA expression vector for
use as
the backbone to the HIV-1 nef-based DNA vaccines of the present invention is V
1R.
In this vector, as much non-essential DNA as possible is "trimmed" from the
vector to
produce a highly compact vector. This vector is a derivative of VlJns. This
vector
allows larger inserts to be used, with less concern that undesirable sequences
are
encoded and optimizes uptake by cells when the construct encoding specific
influenza
virus genes is introduced into surrounding tissue.
It will be evident upon review of the teaching within this specification that
numerous vector/Nef antigen constructs may be generated. While the exemplified
constructs (V lJns/nef, V lJns/tpanef, V lJns/tpanef(LLAA) and V
lJns/(G2A,LLAA)
are preferred, any number of vector/Nef antigen combinations are within the
scope of
the present invention, especially wild type or modified Nef proteins which
comprise a
deletion or substitution of Gly 2, a deletion of substitution of Leu 174 and
Leu 175
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and/or inclusion of a leader sequence. Therefore, the present invention
especially
relates to DNA vaccines and a pharmaceutically active vaccine composition
which
contains this DNA vector vaccine, and the use as prophylactic and/or
therapeutic
vaccine for host immunization, preferably human host immunization, against an
HIV
infection or to combat an existing HIV condition. These DNA vaccines are
represented by codon optimized DNA molecules encoding HIV-1 Nef of
biologically
active Nef modifications or Nef-containing fusion proteins which are ligated
within
an appropriate DNA plasmid vector, with or without a nucleotide sequence
encoding
a functional leader peptide. DNA vaccines of the present invention include but
in no
way are limited to codon optimized DNA molecules encoding HIV-1 Nef of
biologically active Nef modifications or Nef-containing fusion proteins
ligated in
DNA vectors Vl, V1J (SEQ >D N0:14), VlJneo (SEQ ID NO:15), VlJns (Figure 1A,
SEQ ID N0:16), V1R (SEQ ID N0:26), or any of the aforementioned vectors
wherein a nucleotide sequence encoding a leader peptide, preferably the human
tPA
leader, is fused directly downstream of the CMV-intA promoter, including but
not
limited to V lJns-tpa, as shown in Figure 1B and SEQ ID N0:19. Especially
preferred
DNA vaccines of the present invention include as VlJns/nef, VlJns/tpanef,
VlJns/tpanef(LLAA) and VlJns/(G2A,LLAA), as exemplified in Example Section 2.
The DNA vector vaccines of the present invention may be formulated in any
pharmaceutically effective formulation for host administration. Any such
formulation
may be, for example, a saline solution such as phosphate buffered saline
(PBS). It
will be useful to utilize pharmaceutically acceptable formulations which also
provide
long-term stability of the DNA vector vaccines of the present invention.
During
storage as a pharmaceutical entity, DNA plasmid vaccines undergo a
physiochemical
change in which the supercoiled plasmid converts to the open circular and
linear form.
A variety of storage conditions (low pH, high temperature, low ionic strength)
can
accelerate this process. Therefore, the removal and/or chelation of trace
metal ions
(with succinic or malic acid, or with chelators containing multiple phosphate
ligands)
from the DNA plasmid solution, from the formulation buffers or from the vials
and
closures, stabilizes the DNA plasmid from this degradation pathway during
storage.
In addition, inclusion of non-reducing free radical scavengers, such as
ethanol or
glycerol, are useful to prevent damage of the DNA plasmid from free radical
production that may still occur, even in apparently demetalated solutions.
Furthermore, the buffer type, pH, salt concentration, light exposure, as well
as the
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type of sterilization process used to prepare the vials, may be controlled in
the
formulation to optimize the stability of the DNA vaccine. Therefore,
formulations
that will provide the highest stability of the DNA vaccine will be one that
includes a
demetalated solution containing a buffer (phosphate or bicarbonate) with a pH
in the
range of 7-8, a salt (NaCI, KCl or LiCI) in the range of 100-200 mM, a metal
ion
chelator (e.g., EDTA, diethylenetriaminepenta-acetic acid (DTPA), malate,
inositol
hexaphosphate, tripolyphosphate or polyphosphoric acid), a non-reducing free
radical
scavenger (e.g. ethanol, glycerol, methionine or dimethyl sulfoxide) and the
highest
appropriate DNA concentration in a sterile glass vial, packaged to protect the
highly
purified, nuclease free DNA from light. A particularly preferred formulation
which
will enhance long term stability of the DNA vector vaccines of the present
invention
would comprise a Tris-HCl buffer at a pH from about 8.0 to about 9.0; ethanol
or
glycerol at about 3°lo w/v; EDTA or DTPA in a concentration range up to
about
5 mM; and NaCI at a concentration from about 50 mM to about 500 mM. The use of
such stabilized DNA vector vaccines and various alternatives to this preferred
formulation range is described in detail in PCT International Application No.
PCT/US97/06655, PCT International Publication No. WO 97/40839, which is hereby
incorporated by reference.
The DNA vector vaccines of the present invention may, in addition to
generating a strong CTL-based immune response, provide for a measurable
humoral response subsequent immunization. This response may occur with or
without the addition of adjuvant to the respective vaccine formulation. To
this
end, the DNA vector vaccines of the present invention may also be formulated
with an adjuvant or adjuvants which may increase immunogenicity of the DNA
polynucleotide vaccines of the present invention. A number of these adjuvants
are
known in the art and are available for use in a DNA vaccine, including but not
limited to particle bombardment using DNA-coated gold beads, co-administration
of DNA vaccines with plasmid DNA expressing cytokines, chemokines, or
costimulatory molecules, formulation of DNA with cationic lipids or with
experimental adjuvants such as saponin, monophosphoryl lipid A or other
compounds which increase immunogenicity of the DNA vaccine. One preferred
adjuvant for use in the DNA vector vaccines of the present invention are one
or
more forms of an aluminum phosphate-based adjuvant. Aluminum phosphate is
known in the art for use with live, killed or subunit vaccines, but is only
recently
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disclosed as a useful adjuvant in DNA vaccine formulations. The artisan may
alter the ratio of DNA to aluminum phosphate to provide for an optimal immune
response. In addition, the aluminum phosphate-based adjuvant possesses a molar
POa/Al ratio of approximately 0.9, and may again be altered by the skilled
artisan
to provide for an optimal immune response: An additional mineral-based
adjuvant
may be generated from one or more forms of a calcium phosphate. These
mineral-based adjuvants are useful in increasing humoral responses to DNA
vaccination without imparting a negative effect on an appropriate cellular
immune
response. Complete guidance for use of these mineral-based compounds for use
as DNA vaccines adjuvants are disclosed in PCT International Application No.
PCT/US98/02414, PCT International Publication No. WO 98/35562, which are
hereby incorporated by reference in their entirety. Another preferred adjuvant
is a
non-ionic block copolymer which shows adjuvant activity with DNA vaccines.
The basic structure comprises blocks of polyoxyethylene (POE) and
polyoxypropylene (POP) such as a POE-POP-POE block copolymer. Newman et
al. (1998, Critical Reviews in Therapeutic Drug Carrier Systems 15(2): 89-142)
review a class of non-ionic block copolymers which show adjuvant activity. The
basic structure comprises blocks of polyoxyethylene (POE) and polyoxypropylene
(POP) such as a POE-POP-POE block copolymer. Newman et al. id., disclose
that certain POE-POP-POE block copolymers may be useful as adjuvants to an
influenza protein-based vaccine, namely higher molecular weight POE-POP-POE
block copolymers containing a central POP block having a molecular weight of
over about 9000 daltons to about 20,000 daltons and flanking POE blocks which
comprise up to about 20% of the total molecular weight of the copolymer (see
also
U.S. Reissue Patent No. 36,665, U.S. Patent No. 5,567,859, U.S. Patent No.
5,691,387, U.S. Patent No. 5,696,298 and U.S. Patent No. 5,990,241, all issued
to
Emanuele, et al., regarding these POE-POP-POE block copolymers).
WO 96/04932 further discloses higher molecular weight POE/POP block
copolymers which have surfactant characteristics and show biological efficacy
as
vaccine adjuvants. The above cited references within this paragraph are hereby
incorporated by reference in their entirety. It is therefore within the
purview of
the skilled artisan to utilize available adjuvants which may increase the
immune
response of the polynucleotide vaccines of the present ivention in comparison
to
administration of a non-adjuvanted polynucleotide vaccine.
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The DNA vector vaccines of the present invention are administered to the host
by any means known in the art, such as enteral and parenteral routes. These
routes of
delivery include but are not limited to intramusclar injection,
intraperitoneal injection,
intravenous injection, inhalation or intranasal delivery, oral delivery,
sublingual
administration, subcutaneous administration, transdermal administration,
transcutaneous administration, percutaneous administration or any form of
particle
bombardment, such as a biolostic device such as a "gene gun" or by any
available
needle-free injection device. The preferred methods of delivery of the HIV-1
Nef-
based DNA vaccines disclosed herein are intramuscular injection and needle-
free
injection. An especially preferred method is intramuscular delivery.
The amount of expressible DNA to be introduced to a vaccine recipient will
depend on the strength of the transcriptional and translational promoters used
in the
DNA construct, and on the immunogenicity of the expressed gene product. In
general, an immunologically or prophylactically effective dose of about 1 ~.g
to
greater than about 20 mg, and preferably in doses from about 1 mg to about 5
mg is
administered directly into muscle tissue. As noted above, subcutaneous
injection,
intradermal introduction, impression through the skin, and other modes of
administration such as intraperitoneal, intravenous, inhalation and oral
delivery are
also contemplated. It is also contemplated that booster vaccinations are to be
provided in a fashion which optimizes the overall immune response to the Nef-
based
DNA vector vaccines of the present invention.
The aforementioned polynucleotides, when directly introduced into a
vertebrate in vivo, express the respective HIV-1 Nef protein within the animal
and in
turn induce a cytotoxic T lymphocyte (CTL) response within the host to the
expressed
Nef antigen. To this end, the present invention also relates to methods of
using the
HIV-1 Nef-based polynucleotide vaccines of the present invention to provide
effective immunoprophylaxis, to prevent establishment of an HIV-1 infection
following exposure to this virus, or as a post-HIV infection therapeutic
vaccine to
mitigate the acute HIV-1 infection so as to result in the establishment of a
lower virus
load with beneficial long term consequences. As noted above, the present
invention
contemplates a method of administration or use of the DNA nef-based vaccines
of the
present invention using an any of the known routes of introducing
polynucleotides
into living tissue to induce expression of proteins.
Therefore, the present invention provides for methods of using a DNA nef-
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based vaccine utilizing the various parameters disclosed herein as well as any
additional parameters known in the art, which, upon introduction into
mammalian
tissue induces in vivo, intracellular expression of these DNA nef-based
vaccines. This
intracellular expression of the Nef-based immunogen induces a CTL and humoral
response which provides a substantial level of protection against an existing
HIV-1
infection or provides a substantial level of protection against a future
infection in a
presently uninfected host.
The following examples are provided to illustrate the present invention
without, however, limiting the same hereto.
EXAMPLE 1
Vaccine Vectors
Vl - Vaccine vector V 1 was constructed from pCMVIE-AKI-DHFR (Whang
et al., 1987, J. Virol. 61: 1796). The AKI and DHFR genes were removed by
cutting
the vector with EcoRI and self-ligating. This vector does not contain intron A
in the
CMV promoter, so it was added as a PCR fragment that had a deleted internal
SacI
site [at 1855 as numbered in Chapman, et al., (1991, Nuc. Acids Res. 19:
3979)]. The
template used for the PCR reactions was pCMVintA-Lux, made by ligating the
HindIII and NheI fragment from pCMV6a120 (see Chapman et al., ibid.), which
includes hCMV-IEl enhancer/promoter and intron A, into the HindIII and XbaI
sites
of pBL3 to generate pCMVIntBL. The 1881 base pair luciferase gene fragment
(HindIII-SmaI Klenow filled-in) from RSV-Lux (de Wet et al., 1987, Mol. Cell
Biol.
7: 725) was ligated into the SaII site of pCMVIntBL, which was Klenow filled-
in and
phosphatase treated. The primers that spanned intron A are: 5' primer:
5'-CTATATAAGCAGAGCTCGTTTAG-3' (SEQ m NO:10); 3' primer:
5'-GTAGCAAAGATCTAAGGACGGTGACTGCAG-3' (SEQ >D NO:11). The
primers used to remove the SacI site are: sense primer, 5'-GTATGTGTCTG
AAAATGAGC GTGGAGATTGGGCTCGCAC-3' (SEQ >D N0:12) and the
antisense primer, 5'-GTGCGAGCCCAATCTCCACGCTCATTTTCAGAC
ACATAC-3' (SEQ >D N0:13). The PCR fragment was cut with Sac I and Bgl II and
inserted into the vector which had been cut with the same enzymes.
V1J- Vaccine vector V1J was generated to remove the promoter and
transcription termination elements from vector Vl in order to place them
within a
more defined context, create a more compact vector, and to improve plasmid
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purification yields. V1J is derived from vectors V1 and pUCl8, a commercially
available plasmid. V1 was digested with SspI and EcoRI restriction enzymes
producing two fragments of DNA. The smaller of these fragments, containing the
CMVintA promoter and Bovine Growth Hormone (BGH) transcription termination
S elements which control the expression of heterologous genes, was purified
from an
agarose electrophoresis gel. The ends of this DNA fragment were then "blunted"
using the T4 DNA polymerise enzyme in order to facilitate its ligation to
another
"blunt-ended" DNA fragment. pUClB was chosen to provide the "backbone" of the
expression vector. It is known to produce high yields of plasmid, is well-
characterized by sequence and function, and is of small size. The entire lac
operon
was removed from this vector by partial digestion with the HaeII restriction
enzyme.
The remaining plasmid was purified from an agarose electrophoresis gel, blunt-
ended
with the T4 DNA polymerise treated with calf intestinal alkaline phosphatase,
and
ligated to the CMVintA/BGH element described above. Plasmids exhibiting either
of
1S two possible orientations of the promoter elements within the pUC backbone
were
obtained. One of these plasmids gave much higher yields of DNA in E. coli and
was
designated V 1J. This vector's structure was verified by sequence analysis of
the
junction regions and was subsequently demonstrated to give comparable or
higher
expression of heterologous genes compared with V1. The nucleotide sequence of
V1J
is as follows:
TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA
CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG
TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC
ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGATTGG
2S CTATTGGCCA TTGCATACGT TGTATCCATA TCATAATATG TACATTTATA TTGGCTCATG
TCCAACATTA CCGCCATGTT GACATTGATT ATTGACTAGT TATTAATAGT AATCAATTAC
GGGGTCATTA GTTCATAGCC CATATATGGA GTTCCGCGTT ACATAACTTA CGGTAAATGG
CCCGCCTGGC TGACCGCCCA ACGACCCCCG CCCATTGACG TCAATAATGA CGTATGTTCC
CATAGTAACG CCAATAGGGA CTTTCCATTG ACGTCAATGG GTGGAGTATT TACGGTAAAC
3O TGCCCACTTG GCAGTACATC AAGTGTATCA TATGCCAAGT ACGCCCCCTA TTGACGTCAA
TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTATGGG ACTTTCCTAC
TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG GTGATGCGGT TTTGGCAGTA
CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC ACCCCATTGA
CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT GTCGTAACAA
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CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG
AGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCA
TAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGAT
TCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACCCCCTTGGC
S TTCTTATGCATGCTATACTGTTTTTGGCTTGGGGTCTATACACCCCCGCTTCCTCATGTT
ATAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTATTGACCACTCCC
CTATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACTCTCT
TTATTGGCTATATGCCAATACACTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTAC
AGGATGGGGTCTCATTTATTATTTACAAATTCACATATACAACACCACCGTCCCCAGTGC
1O CCGCAGTTTTTATTAAACATAACGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGG
ACATGGGCTCTTCTCCGGTAGCGGCGGAGCTTCTACATCCGAGCCCTGCTCCCATGCCTC
CAGCGACTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCA
CAGCACGATGCCCACCACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTC
TGAAAATGAGCTCGGGGAGCGGGCTTGCACCGCTGACGCATTTGGAAGACTTAAGGCAGC
IS GGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTGTTCTGATAAGAGTCAGAGGTAACTCC
CGTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGC
GCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTT
CTGCAGTCACCGTCCTTAGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCC
CCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAA
ZO ATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGG
GGCAGCACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGG
GCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCAGAAAGAAGC
AGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTTCTTAGTTCCAGCCC
CACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAAGTAC
2S TTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGAGTGG
GAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATG
TGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTG
CGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTA
TCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCC
3O AGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAG
CATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATAC
CAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACC
GGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGT
AGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCC
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GTTCAGCCCGACCGCTGCGCCTTATCCGGT TTGAGTCCAACCCGGTAAGA
AACTATCGTC
CACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTA
GGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTA
TTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGA
S TCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACG
CGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAG
TGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACC
TAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACT
TGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTT
1O CGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTA
CCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTA
TCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCC
GCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAAT
AGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGT
IS ATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTG
TGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCA
GTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTA
AGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGG
CGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACT
ZO TTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCG
CTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTT
ACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGA
ATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGC
ATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAA
ZS CAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATT
ATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC (SEQ
ID
N0:14).
VlJneo vector involved
- Construction
of vaccine
vector
V lJneo
expression
removal of the ampr gene and insertion of the kanr gene (neomycin
30 phosphotransferase). The ampr gene from the pUC backbone of V1J was removed
by
digestion with SspI and Eam110SI restriction enzymes. The remaining plasmid
was
purified by agarose gel electrophoresis, blunt-ended with T4 DNA polymerase,
and
then treated with calf intestinal alkaline phosphatase. The commercially
available
kanr gene, derived from transposon 903 and contained within the pUC4K plasmid,
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was excised using the PstI restriction enzyme, purified by agarose gel
electrophoresis,
and blunt-ended with T4 DNA polymerise. This fragment was ligated with the V1J
backbone and plasmids with the kanr gene in either orientation were derived
which
were designated as VlJneo #'s 1 and 3. Each of these plasmids was confirmed by
S restriction enzyme digestion analysis, DNA sequencing of the junction
regions, and
was shown to produce similar quantities of plasmid as V1J. Expression of
heterologous gene products was also comparable to V1J for these VlJneo
vectors.
VlJneo#3, referred to as VlJneo hereafter, was selected which contains the
kanr gene
in the same orientation as the ampr gene in V1J as the expression construct
and
provides resistance to neomycin, kanamycin and 6418. The nucleotide sequence
of
V lJneo is as follows:
TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA
CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG
TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC
IS ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGATTGG
CTATTGGCCA TTGCATACGT TGTATCCATA TCATAATATG TACATTTATA TTGGCTCATG
TCCAACATTA CCGCCATGTT GACATTGATT ATTGACTAGT TATTAATAGT AATCAATTAC
GGGGTCATTA GTTCATAGCC CATATATGGA GTTCCGCGTT ACATAACTTA CGGTAAATGG
CCCGCCTGGC TGACCGCCCA ACGACCCCCG CCCATTGACG TCAATAATGA CGTATGTTCC
ZO CATAGTAACG CCAATAGGGA CTTTCCATTG ACGTCAATGG GTGGAGTATT TACGGTAAAC
TGCCCACTTG GCAGTACATC AAGTGTATCA TATGCCAAGT ACGCCCCCTA TTGACGTCAA
TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTATGGG ACTTTCCTAC
TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG GTGATGCGGT TTTGGCAGTA
CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC ACCCCATTGA
2S CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT GTCGTAACAA
CTCCGCCCCA TTGACGCAAA TGGGCGGTAG GCGTGTACGG TGGGAGGTCT ATATAAGCAG
AGCTCGTTTA GTGAACCGTC AGATCGCCTG GAGACGCCAT CCACGCTGTT TTGACCTCCA
TAGAAGACAC CGGGACCGAT CCAGCCTCCG CGGCCGGGAA CGGTGCATTG GAACGCGGAT
TCCCCGTGCC AAGAGTGACG TAAGTACCGC CTATAGAGTC TATAGGCCCA CCCCCTTGGC
3O TTCTTATGCA TGCTATACTG TTTTTGGCTT GGGGTCTATA CACCCCCGCT TCCTCATGTT
ATAGGTGATG GTATAGCTTA GCCTATAGGT GTGGGTTATT GACCATTATT GACCACTCCC
CTATTGGTGA CGATACTTTC CATTACTAAT CCATAACATG GCTCTTTGCC ACAACTCTCT
TTATTGGCTA TATGCCAATA CACTGTCCTT CAGAGACTGA CACGGACTCT GTATTTTTAC
AGGATGGGGT CTCATTTATT ATTTACAAAT TCACATATAC AACACCACCG TCCCCAGTGC
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CCGCAGTTTTTATTAAACATAACGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGG
ACATGGGCTCTTCTCCGGTAGCGGCGGAGCTTCTACATCCGAGCCCTGCTCCCATGCCTC
CAGCGACTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCA
CAGCACGATGCCCACCACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTC
S TGAAAATGAGCTCGGGGAGCGGGCTTGCACCGCTGACGCATTTGGAAGACTTAAGGCAGC
GGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTGTTCTGATAAGAGTCAGAGGTAACTCC
CGTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGC
GCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTT
CTGCAGTCACCGTCCTTAGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCC
1O CCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAA
ATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGG
GGCAGCACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGG
GCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCAGAAAGAAGC
AGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTTCTTAGTTCCAGCCC
IS CACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAAGTAC
TTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGAGTGG
GAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATG
TGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTG
CGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTA
ZO TCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCC
AGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAG
CATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATAC
CAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACC
GGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGT
ZS AGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCC
GTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGA
CACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTA
GGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTA
TTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGA
3O TCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACG
CGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAG
TGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACC
TAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACT
TGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTT
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CGTTCATCCATAGTTGCCTGACTCCGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGA
AGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGA
GCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTT
TGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAA
S AGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGT
TACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAAT
TTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGA
GAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCG
ACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGT
1O GAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCT
TTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACC
AAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAA
GGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACA
ATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATC
IS GCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGA
GGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACG
CTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAG
ATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCA
TCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATA
2O ACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTT
TTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCCCCCCCCCCC
CATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATT
TAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC
TAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTT
2S CGTC (SEQ ID N0:15).
VIJns - The expression vector VIJns was generated by adding an SfiI site to
VlJneo to facilitate integration studies. A commercially available 13 base
pair SfiI
linker (New England BioLabs) was added at the KpnI site within the BGH
sequence
of the vector. V lJneo was linearized with KpnI, gel purified, blunted by T4
DNA
30 polymerase, and ligated to the blunt SfiI linker. Clonal isolates were
chosen by
restriction mapping and verified by sequencing through the linker. The new
vector
was designated V lJns. Expression of heterologous genes in V lJns (with SfiI)
was
comparable to expression of the same genes in VlJneo (with KpnI).
The nucleotide sequence of VlJns is as follows:
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TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA
CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG
TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC
ACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGATTGG
S CTATTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATG
TCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTAC
GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGG
CCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC
CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAAC
IO TGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA
TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTAC
TTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTA
CATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGA
CGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA
IS CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG
AGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCA
TAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGAT
TCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGACTCTATAGGCACACCCCTTTGGC
TCTTATGCATGCTATACTGTTTTTGGCTTGGGGCCTATACACCCCCGCTTCCTTATGCTA
ZO TAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTATTGACCACTCCCC
TATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACTATCTC
TATTGGCTATATGCCAATACTCTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTACA
GGATGGGGTCCCATTTATTATTTACAAATTCACATATACAACAACGCCGTCCCCCGTGCC
CGCAGTTTTTATTAAACATAGCGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGGA
ZS CATGGGCTCTTCTCCGGTAGCGGCGGAGCTTCCACATCCGAGCCCTGGTCCCATGCCTCC
AGCGGCTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCAC
AGCACAATGCCCACCACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTCT
GAAAATGAGCGTGGAGATTGGGCTCGCACGGCTGACGCAGATGGAAGACTTAAGGCAGCG
GCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTATTCTGATAAGAGTCAGAGGTAACTCCC
3O GTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCG
CGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTTC
TGCAGTCACCGTCCTTAGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCC
CTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAA
TGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGG
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GCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGG
CTCTATGGCCGCTGCGGCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCAGAAAG
AAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTTCTTAGTTCCA
GCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAA
S GTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGA
GTGGGAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAA
CATGTGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTC
GCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACG
GTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAA
IO GGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGA
CGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAG
ATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCT
TACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACG
CTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACC
IS CCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGT
AAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTA
TGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAAC
AGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTC
TTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGAT
ZO TACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGC
TCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTT
CACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTA
AACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCT
ATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGA
ZS AGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAG
GGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTG
CTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGC
AAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAG
TGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGC
3O AATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAA
GGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATT
CCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCA
AGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATT
TCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCA
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ACCAAACCGT TATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTA
AAAGGACAAT TACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCA
ACAATATTTT CACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGG
ATCGCAGTGG TGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGA
S AGAGGCATAA ATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCA
ACGCTACCTT TGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGA
TAGATTGTCG CACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCA
GCATCCATGT TGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTC
ATAACACCCC TTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATA
1O TTTTTATCTT GTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCCCCCCCC
CCCCATTATT GAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGT
ATTTAGAAAA ATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAC
GTCTAAGAAA CCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCC
TTTCGTC(SEQ ID N0:16).
1S The underlined nucleotides
of SEQ ID N0:16
represent the Sfil
site
introduced into ite of
the Kpn 1 s V lJneo.
VlJns-tPA - The V lJns-tPA in order
vaccine vector was constructed to fuse
an heterologous leader peptide sequence to the nef DNA constructs of the
present
invention. More specifically, the vaccine vector V lJns was modified to
include the
20 human tissue-specific plasminogen activator (tPA) leader. As an
exemplification, but
by no means a limitation of generating a nef DNA construct comprising an amino-
terminal leader sequence, plasmid V lJneo was modified to include the human
tissue-
specific plasminogen activator (tPA) leader. Two synthetic complementary
oligomers
were annealed and then legated into VlJneo which had been BgIII digested. The
2S sense and antisense oligomers were S' GATCACCATGGATGCAATGAAGAGAG
GGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCAG
CGA-3' (SEQ ID N0:17); and, S'-GATCTCGCTGGGCGAAACGAAGACTGC
TCCACACAGCAGCAGCACACAGCAGAGCCCTCTCTTCATTGCATCCAT
GGT-3' (SEQ ID N0:18). The Kozak sequence is underlined in the sense oligomer.
30 These oligomers have overhanging bases compatible for legation to BgIII-
cleaved
sequences. After legation the upstream BgIII site is destroyed while the
downstream
BgIII is retained for subsequent legations. Both the junction sites as well as
the entire
tPA leader sequence were verified by DNA sequencing. Additionally, in order to
conform with VlJns (=VlJneo with an SfiI site), an SfiI restriction site was
placed at
-3 S-
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the KpnI within
site the BGH
terminator
region
of V
lJneo-tPA
by blunting
the KpnI
site with DNA polymerise by ligationth an
T4 followed wi SfiI
linker
(catalogue
#1138, England
New Biolabs),
resulting
in V
lJns-tPA.
This
modification
was
verified
by restriction
digestion
and agarose
gel electrophoresis.
S The V lJns-tpa ce is as s:
vector follow
nucleotide
sequen
TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA
CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG
TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC
ACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGATTGG
1O CTATTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATG
TCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTAC
GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGG
CCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC
CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAAC
IS TGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA
TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTAC
TTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTA
CATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGA
CGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA
ZO CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG
AGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCA
TAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGAT
TCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGACTCTATAGGCACACCCCTTTGGC
TCTTATGCATGCTATACTGTTTTTGGCTTGGGGCCTATACACCCCCGCTTCCTTATGCTA
ZS TAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTATTGACCACTCCCC
TATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACTATCTC
TATTGGCTATATGCCAATACTCTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTACA
GGATGGGGTCCCATTTATTATTTACAAATTCACATATACAACAACGCCGTCCCCCGTGCC
CGCAGTTTTTATTAAACATAGCGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGGA
3O CATGGGCTCTTCTCCGGTAGCGGCGGAGCTTCCACATCCGAGCCCTGGTCCCATGCCTCC
AGCGGCTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCAC
AGCACAATGCCCACCACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTCT
GAAAATGAGCGTGGAGATTGGGCTCGCACGGCTGACGCAGATGGAAGACTTAAGGCAGCG
GCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTATTCTGATAAGAGTCAGAGGTAACTCCC
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GTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCG
CGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTTC
TGCAGTCACCGTCCTTAGATCACCATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTG
CTGCTGTGTGGAGCAGTCTTCGTTTCGCCCAGCGAGATCTGCTGTGCCTTCTAGTTGCCA
S GCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCAC
TGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTAT
TCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCA
TGCTGGGGATGCGGTGGGCTCTATGGCCGCTGCGGCCAGGTGCTGAAGAATTGACCCGGT
TCCTCCTGGGCCAGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGC
IO CCCTGGTTCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCT
TCAATCCCACCCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAAC
CAAACCTAGCCTCCAAGAGTGGGAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGG
GAGAGAAAATGCCTCCAACATGTGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGC
TTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCA
IS CTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTG
AGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCA
TAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAA
CCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCC
TGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGC
ZO GCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCT
GGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCG
TCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG
GATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTA
CGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGG
ZS AAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTT
TGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTT
TTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAG
ATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAAT
CTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACC
3O TATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGGGGGGGCG
CTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATC
ATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTT
GGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGAT
CTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTC
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AGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAA
AAACTCATCG
AGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAA
AGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCC
TGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCG
S TCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAAT
GGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCA
TCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGA
AATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGG
AACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGG
IO AATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATA
AAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCA
TCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCG
GGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCAT
TTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTT
IS TCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTT
ATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACA
ACGTGGCTTTCCCCCCCCCCCCATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGC
GGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCC
CGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAAT
ZO AGGCGTATCACGAGGCCCTTTCGTC ID N0:9).
(SEQ
The u nderlinedleotides
nuc of SEQ
ID N0:9
represent
the Sfi
1 site
introduced
into the 1 site eo while icized
Kpn of V the underlined/ital nucleotides
IJn represent
the
human tPA
leader
sequence.
V1 R - Vaccine onstructedobtain
vector to a minimum-sized
V 1R
was c
2S vaccine vector without unneeded DNA sequences, which still retained the
overall
optimized heterologous gene expression characteristics and high plasmid yields
that
V1J and VlJns afford. It was determined that (1) regions within the pUC
backbone
comprising the E. coli origin of replication could be removed without
affecting
plasmid yield from bacteria; (2) the 3'-region of the kanr gene following the
30 kanamycin open reading frame could be removed if a bacterial terminator was
inserted in its place; and, (3) 300 by from the 3'- half of the BGH terminator
could
be removed without affecting its regulatory function (following the original
KpnI
restriction enzyme site within the BGH element). V1R was constructed by using
PCR
to synthesize three segments of DNA from VlJns representing the CMVintA
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promoter/BGH terminator, origin of replication, and kanamycin resistance
elements,
respectively. Restriction enzymes unique for each segment were added to each
segment end using the PCR oligomers: SspI and XhoI for CMVintABGH; EcoRV
and BamHI for the kan r gene; and, BcII and SaII for the on r. These enzyme
sites
were chosen because they allow directional ligation of each of the PCR-derived
DNA
segments with subsequent loss of each site: EcoRV and SspI leave blunt-ended
DNAs which are compatible for ligation while BamHI and BcII leave
complementary
overhangs as do SaII and XhoI. After obtaining these segments by PCR each
segment
was digested with the appropriate restriction enzymes indicated above and then
ligated together in a single reaction mixture containing all three DNA
segments. The
5'-end of the on r was designed to include the T2 rho independent terminator
sequence that is normally found in this region so that it could provide
termination
information for the kanamycin resistance gene. The ligated product was
confirmed by
restriction enzyme digestion (>8 enzymes) as well as by DNA sequencing of the
ligation junctions. DNA plasmid yields and heterologous expression using viral
genes
within V 1R appear similar to V lJns. The net reduction in vector size
achieved was
1346 by (V lJns = 4.86 kb; V 1R = 3.52 kb). PCR oligomer sequences used to
synthesize V1R (restriction enzyme sites are underlined and identified in
brackets
following sequence) are as follows: (1) 5'-GGTACAAATATTGGCTATTGGC
CATTGCATACG-3' (SEQ >D N0:20) [SspI]; (2) 5'-CCACATCTCGAGGAA
CCGGGTCAATTCTTCAGCACC-3' (SEQ >D N0:21) [XhoI] (for CMVintA/BGH
segment); (3) 5'-GGTACAGATATCGGAAAGCCACGTTGTG TCTCAAAATC-3'
(SEQ >D N0:22) [EcoRV]; (4) 5'-CACATGGATCCGTAATGCTCTGCCAGTGT
TACAACC-3' (SEQ ID N0:23) [BamHI], (for kanamycin resistance gene segment)
(5) 5'-GGTACATG ATCACGTAGAAAAGATCAAAGGATCTTCTTG-3' (SEQ >D
N0:24) [BcII]; (6) 5'-CCACATGTCGACCCGTAAAAAGGCCGCGTTGCTGG-3'
(SEQ >D N0:25): [SaII], (for E. coli origin of replication).
The nucleotide sequence of vector V1R is as follows:
TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA
3O CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG
TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC
ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGATTGG
CTATTGGCCA TTGCATACGT TGTATCCATA TCATAATATG TACATTTATA TTGGCTCATG
TCCAACATTA CCGCCATGTT GACATTGATT ATTGACTAGT TATTAATAGT AATCAATTAC
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GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGG
CCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC
CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAAC
TGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA
S TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTAC
TTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTA
CATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGA
CGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA
CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG
IO AGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCA
TAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGAT
TCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACCCCCTTGGC
TTCTTATGCATGCTATACTGTTTTTGGCTTGGGGTCTATACACCCCCGCTTCCTCATGTT
ATAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTATTGACCACTCCC
IS CTATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACTCTCT
TTATTGGCTATATGCCAATACACTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTAC
AGGATGGGGTCTCATTTATTATTTACAAATTCACATATACAACACCACCGTCCCCAGTGC
CCGCAGTTTTTATTAAACATAACGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGG
ACATGGGCTCTTCTCCGGTAGCGGCGGAGCTTCTACATCCGAGCCCTGCTCCCATGCCTC
ZO CAGCGACTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCA
CAGCACGATGCCCACCACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTC
TGAAAATGAGCTCGGGGAGCGGGCTTGCACCGCTGACGCATTTGGAAGACTTAAGGCAGC
GGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTGTTCTGATAAGAGTCAGAGGTAACTCC
CGTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGC
ZS GCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTT
CTGCAGTCACCGTCCTTAGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCC
CCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAA
ATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGG
GGCAGCACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGG
3O GCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCAGAAAGAAGC
AGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTTCTTAGTTCCAGCCC
CACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAAGTAC
TTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGAGTGG
GAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATG
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TGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTG
CGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTA
TCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCC
AGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAG
S CATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATAC
CAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACC
GGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGT
AGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCC
GTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGA
1O CACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTA
GGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTA
TTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGA
TCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACG
CGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAG
IS TGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACC
TAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACT
TGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTT
CGTTCATCCATAGTTGCCTGACTCCGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGA
AGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGA
2O GCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTT
TGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAA
AGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGT
TACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAAT
TTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGA
ZS GAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCG
ACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGT
GAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCT
TTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACC
AAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAA
3O GGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACA
ATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATC
GCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGA
GGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACG
CTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAG
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ATTGTCGCAC CTGATTGCCC GACATTATCG CGAGCCCATT TATACCCATA TAAATCAGCA
TCCATGTTGG AATTTAATCG CGGCCTCGAG CAAGACGTTT CCCGTTGAAT ATGGCTCATA
ACACCCCTTG TATTACTGTT TATGTAAGCA GACAGTTTTA TTGTTCATGA TGATATATTT
TTATCTTGTG CAATGTAACA TCAGAGATTT TGAGACACAA CGTGGCTTTC CCCCCCCCCC
S CATTATTGAA GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT
TAGAAAAATA AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACGTC
TAAGAAACCA TTATTATCAT GACATTAACC TATAAAAATA GGCGTATCAC GAGGCCCTTT
CGTC (SEQ ID N0:26).
EXAMPLE 2
Codon Optimized HIV-1 Nef and HIV-1 Nef Derivatives as DNA Vector Vaccines
HIV-1 Nef Vaccine Vectors - Codon optimized nef gene coding for wt Nef
protein of HIV-1 jrfl isolate was assembled from complementary, overlapping
synthetic oligonucleotides by polymerise chain reaction (PCR). The PCR primers
1S used were designed in such that a BgIII site was included in the extension
of S' primer
and an SrfI site and a BgIII site in the extension of 3' primer. The PCR
product was
digested with BgIII and cloned into BgIII site of a human cytomeglovirus early
promoter-based expression vector, VlJns (Figure 1A). The proper orientation of
nef
fragment in the context of the expression cassette was determined by
asymmetric
restriction mapping. The resultant plasmid is VlJns/nef. The S' and 3'
nucleotide
sequence junctions of codon optimized VlJns/nef are shown in Figure 3A.
The mutant nef (G2A,LLAA) was also made from synthetic oligonucleotides.
To assist in cloning, a PstI site and an SrfI site were included in the
extensions of S'
and 3' PCR primers, respectively. The PCR product was digested with PstI and
SrfI,
2S and cloned into the PstI and Srfl sites of VlJns/nef, replacing the
original nef with
nef(G2A,LLAA) fragment. This resulted in VlJns/nef(G2A,LLAA). The S' and 3'
nucleotide sequence junctions of codon optimized VlJns/nef (G2A,LLAA) are
shown
in Figure 3B.
To construct the expression vector containing human tissue plasminogen
activator leader peptide and the nef fusion gene, i.e., VlJns/tPAnef, a
truncated nef
gene fragment, lacking the coding sequence for the five amino terminal
residues, was
first amplified by PCR using VlJns/nef as template. Both S' and 3' PCR primers
used
in this reaction contained a BgIII extension. The PCR amplified fragment was
then
digested with BgIII and cloned into BgIII site of the expression vector,
VlJns/tpa
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(Figure 1B). The ligation of the 3' end of tpa leader peptide coding sequence
to the 5'
end of the nef PCR product restored the BgIII site and yielded an in-frame
fusion of
the two genes. The 5' and 3' nucleotide sequence junctions of codon optimized
VlJns/tPAnef are shown in Figure 3C.
Construction of V lJns/tpanef(LLAA) was carried out by replacing the Bsu36-
SacII fragment of VlJns/tpanef, which contains the 3' half of the nef gene and
part of
the vector backbone, with the Bsu36-SacII fragment from V lJns/nef(G2A,LLAA).
The 5' and 3' nucleotide sequence junctions of codon optimized V lJns/tpanef
(LLAA)
are shown in Figure 3C.
All the nef constructs were verified by sequencing. The amino acid junctions
of these constructs is shown schematically in Figure 4.
Transfection and protein expression - 293 cells (adenovirus transformed
human embryonic kidney cell line 293) grown at approximately 30% confluence in
minimum essential medium (MEM; GIBCO, Grand Island, MD) supplemented with
10% fetal bovine serum (FBS; GIBCO) in a 100 mm culture dish, were transfected
with 4 ug gag expression vector, VlJns/gag, or a mixture of 4 ug gag
expression
vector and 4 ug nef expression vector by Lipofectin following manufacture's
protocol
(GIBCO). Twelve hours post-transfection, cells were washed once with 10 ml of
serum-free medium, Opti-MEM I (GIBCO) and replenished with 5 ml of Opti-MEM.
Following an additional 60 hr incubation, culture supernatants and cells were
collected separately and used for Western blot analysis.
Western blot analysis - Fifty microliter of samples were separated on a 10%
SDS-polyacrylamide gel (SDS-PAGE) under reducing conditions. The proteins were
blotted onto a piece of PVDF membrane, and reacted to a mixture of gag mAb
(#18;
Intracel, Cambridge, MA) and Nef mAbs (aa64-68, aa195-201; Advanced
Biotechnologies, Columbia, MD), both at 1:2000 dilution, and horseradish
peroxidase
(HRP)-conjugated goat anti-rabbit IgG (Zymed, San Francisco, CA). The protein
bands were visualized by ECL Western blotting detection reagents, according to
the
manufacture's protocol (Amersham, Arlington Heights, IL).
Enzyme-linked immunosorbent assay (ELISA) - 96-well Immulon II, round-
bottom plates were coated with 50 u1 of Nef protein at the concentration of
2ug/ml in
bicarbonate buffer, pH 9.8., per well at 4°C overnight. Plates were
washed three
times with PBS containing 0.05°lo Tween-20 (PBST), and blocked with
5°Io skim milk
in PBST (milk-PBST) at 24°C for 2 hr, and then incubated with serial
dilutions of
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testing samples in milk-PBST at 24°C for 2 hr. Plates were washed with
PBST three
times, and added with 50 u1 of HRP-conjugated goat anti-mouse IgG (Zymed) per
well and incubated at 24°C for 1 hr. This was followed by three washes,
and the
addition of 100 u1 of 1 mg/ml ABTS [(2,2'-amino-di-(3-ethylbenzthiozoline
sulfonate)] (KPL, Gaithersburg, MD) per well. After 1 hr at 24°C,
plates were read at
a wavelength of 405nm using an ELISA plate reader.
Enzyme-linked spot assay (Elispot) - Nitrocellulose membrane-backed 96 well
plates (MSHA plates; Millipore, Bedford, MA) were coated with 50 u1 of rat
anti-
mouse IFN-gamma mAb, capture antibody, (R4-6A2; PharMingen, San Diego, CA) at
a concentration of Sug/ml in PBS per well at 4°C overnight. Plates were
washed three
times with PBST and blocked with 10% FBS in RPMI-1640 (FBS-RPMI) at
37°C in
a C02 incubator for 2 to 4 hrs. Splenocytes were suspended in RPMI-1640 with
10%
FBS at 4 x 10G cells per ml. 100 u1 cells were added to each well and plates
were
incubated at 37°C for 20 hrs. Each sample was tested in triplicate
wells. After
incubation, plates were rinsed briefly with distilled water and washed three
times with
PBST. Fifty u1 of biotinylated rat anti-mouse IFN-y mAb, detecting antibody
(XMG1.2; PharMingen), diluted in 1% BSA in PBST at a concentration of 2 ug/ml
was then added to each well. Plates were incubated at 24°C for 2 hr,
followed by
washes with PBST. Fifty u1 of streptavidin-conjugated alkaline phosphatase
(KPL) at
a dilution of 1:1000 in FBS-RPMI was added to each well. The plates were
incubated
at 24C for an additional one hr. Following extensive wash with BPST, 100u1
BCIT/NBT substrate (KPL) was added for 15 min, and color reaction was stopped
by
washing the plate with tap water. Plates were air-dried and spots were
countered using
a dissection microscope.
Cytotoxic T cell (CTL) assay - Splenocytes from immunized mouse were co-
cultured with syngenic peptide-pulsed, irradiated naive splenocytes for 7
days. EL-4
cells were incubated at 37°C for 1 hr with or without 20ug/ml of a
designated peptide
in the presence of sodium SICr-chromate and used as target cells. For the
assay, 104
target cells were added to a 96-well plate along with different numbers of
splenocytes
cells. Plates were incubated at 37°C for 4 hr. After incubation,
supernatants were
collected and counted in a Wallac gamma-counter. Specific lysis was calculated
as
([experimental release - spontaneous release]/maximum release- spontaneous
release]) x 100%. Spontaneous release was determined by incubating target
cells in
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medium alone, and maximum release was determined by incubating target cells in
2.5% TritonX-100. The assay was performed with triplicate samples.
Animal experiments - Female mice (Charles River Laboratories, Wilmington,
MA), 6 to 10 weeks old, were injected in quadriceps with 100 u1 of DNA in PBS.
Two weeks after immunization, spleens from individual mice were collected and
used
for CTL and Elispot assays.
Results (DNA Vector Vaccine Construction) - The exemplified Nef protein
sequence is based on HIV-1 Glade B jrfl isolate. A codon-optimized nef gene
was
chosen for vaccine construction and for use as the parental gene for other
exemplified
constructs. Figure 2A-B show the comparison of coding sequence of wt nef(jrfl)
and
the codon optimized nef(jrfl). Two forms of myristylation site mutations were
constructed; one contains a Gly2Ala change and the other a human tissue
plasminogen activator (tpa) leader sequence was fused to sixth residue, Ser,
of Nef
(tpanef). The dileucine motif mutation was made by introducing both Leu174A1a
and
Leu175A1a changes. Figure 4 shows the schematic depiction of the Nef and Nef
mutants. For in vitro expression and in vivo immunogenicity studies, the nef
genes
were cloned into expression vector, VlJns. The resultant plasmids containing
wt nef,
tpanef, tpanef with dileucine motif mutation, and nef mutant with the Gly2Ala
myristylation site and dileucine motif mutations were named as V lJns/nef,
VlJns/tpanef, VlJns/tpanef(LLAA) and VlJns/(G2A,LLAA), respectively.
Results - Expression and Western blotting analysis - To evaluate the
expression of the codon optimized nef constructs, adenovirus-transformed human
kidney 293 cells were cotransfected with individual nef plasmids and a gag
expression
vector, VlJns/gag. 72 hours post transfection, cells and medium were collected
separately and analyzed by Western blotting, using both Nef- and Gag-specific
mAbs.
The results are shown in Figure 5. Cells transfected with V lJns/gag only
revealed a
single distinct band of approximately 55 Kd, whereas the cells cotransfected
with gag
and nef plasmids revealed, in addition to the 55 Kd band, a major 30 Kd band
and
several minor bands. This pattern is consistent with that the 55 Kd species
represents
Gag polypeptide and the 30 Kd and other minor species are the Nef-related
products.
Therefore, all the nef constructs were expressed in the transfected cells.
When
measured against the relatively constant Gag signal as a reference, four nef
genes
seem to be expressed at different levels, with the following descending order,
tpanef,
nef, tpanef(LLAA) and nef(G2A, LLAA). With the exception of nef(G2A,LLAA),
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products of nef, tpanef, tpanef(LLAA) could be detected in both cellular and
medium
fractions.
Mapping of Nef specific CD8 and CD4 epitopes in mice - There was no
information available with respect to the properties of Nef(jrfl) in eliciting
cell-
mediated immune responses in mice. Therefore, to characterize immunogenicity
of
Nef and Nef mutants exemplified herein, CD8 and CD4 epitopes were mapped. An
overlapping set of overlapping nef peptides that encompass the entire 216 as
Nef
polypeptide were generated. A total 21 peptides were made, which include
twenty
20mers and one l6mer. Three strains of mice, Balb/c, C3H and C57BL/6, were
immunized with plasmid VlJns/Nef; splenocytes from immunized and naive mice
were isolated and assessed for Nef specific INF-gamma secreting cells (SFC) by
the
Elispot assay. Figure 6 shows where Elispot assays were performed against
separate
pools of the Nef peptides. All three strains of immunized mice responded to
the Nef
plasmid immunization; each developed positive Nef peptide-specific INF-'y
SFCs.
Based on this, further studies were carried out with fractionated CD8 and CD4
cells
against individual peptides. The results are shown in Figure 7A-C. In Balb/c
mice
(Figure 7A), four Nef peptides, namely, aal l-30, aa61-80, aa191-210 and aa200-
216,
were found to be able to induce significant numbers of CD4 SFCs. In C57BL/6
mice
(Figure 7B), only one peptide, ie., aa81-100, elicited significant numbers of
CD4
SFCs. Compared to Balb/c and C57BL/6 mice, C3H mice (Figure 7C) showed no
dominant CD4 SFC responses with particular peptides; instead, there were
modest
number of SFCs in response to an array of peptides, including aa21-40, aa31-
50,
aa121-140 aa131-150, aa181-200 and aa191-210. With respect to CD8 cells,
significant SFC responses were detected with a single peptide, ie., aa51-70,
in
C57BL/6 mice only.
The results from Elispot assay suggested that Nef peptide aa51-70 contained
an H-2b restricted CD8 cell epitope. In order to ascertain whether this CD8
epitope
also represents the cytotoxic T cell (CTL) epitope, a conventional CTL assay
was
carried out. The peptide aa51-70 (Figure 8A) induced low level of specific
killings
only. Peptides longer than 9 amino acids of a typical CTL epitope often have
lower
binding affinity to MHC class I molecule. It was contemplated that the low
specific
killings observed with peptide aa51-70 could be potentially resulted from the
low
binding affinity of this 20 amino acid peptide. Therefore, two shortened
peptides,
namely, aa60-68 and aa58-70, were synthesized and tested in CTL assays. While
the
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peptide aa60-68 failed to elicit any specific killings (Figure 8B), the
peptide aa58-70
exhibited a drastic increase of specific killing as compared to its longer
counterpart,
peptide aa61-80 (Figure 8C). For example, the percentage of specific killings
induced
by peptide aa58-70 at an effector/target ratio of 5 to 1 was comparable to
that induced
by peptide aa51-80 at an effector/target ratio of 45. Thus, between peptide
aa58-70
and peptide aa51-70, the former was almost ten-fold more effective in terms of
inducing Nef-specific killing. The results from CTL assay therefore confirmed
that
the CD8 epitope detected by the Elispot assay was indeed a CTL epitope. To
further
map the minimum amino acid sequence for the Nef CTL epitope, additional 5
peptides were synthesized and analyzed by Elispot assay, which mapped the CTL
epitope to Nef aa58-66, as shown in Table 1.
TABLE 1
Nef peptides** INF-y SFC*/106 splenocytes
Nef58-70 TAATNADCAWLEA 85
Nef59-69 AATNADCAWLE 1
Nef58-68 TAATNADCAWL 69
Nef58-67 TAATNADCAW 66
Nef58-66 TAATNADCA 92
Medium 1
* Average of duplicate samples.
** Amino acid sequence of all peptides contained within SEQ >D N0:2.
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Results (Evaluation of Immunogenicity of nef Mutants in Mice) - Having
identified H-2b restricted CTL and CD4 cell epitopes, the immunogenicity of
the
different codon optimized nef constructs in C57BL/6 mice was examined. This
was
performed in two separate experiments with identical immunization regimens.
The
first experiment involved nef, tpanef(LLAA) and nef(G2A,LLAA) and the second
experiment involved nef, tpanef, tpanef(LLAA) and nef(G2A,LLAA). Mice were
immunized with plasmids containing these respective codon optimized nef genes.
Two weeks post immunization, splenocytes from individual mice were isolated
and
analyzed by Elispot assay for Nef-specific CD8 and CD4 IFN-gamma SFCs using
Nef
peptide aa58-66 and aa81-100, respectively. The results are shown in Figure 9A-
B.
In the experiment 1 (Figure 9A), among the three groups tested, the mice
receiving
the codon optimized tpanef(LLAA) construct developed the highest CD8 and CD4
cell responses; comparing between tpanef(LLAA) and the nef, the former
elicited
about 40-fold higher CD8 SFCs and 10-fold higher CD4 SFCs. In contrast to
tpanef(LLAA), nef(G2A,LLAA) mutant was poorly immunogenic; mice receiving
this mutant had barely detectable CD8 and CD4 SFCS, under conditions tested.
Similar response profiles between the three mutants were also observed in the
experiment 2 (Figure 9B), except that the overall CD8 response of mice
receiving
tpanef(LLAA) was approximately 10-folder higher in experiment 2 than that
observed
in experiment 1. The tPAnef mutant showed comparable responses as that of
tpanef(LLAA). The results therefore showed that both codon optimized tpanef
and
tpanef(LLAA) had significantly enhanced immunogenicity.
Results (Evaluation of Immunogenicity of nef Mutants in Rhesus Monkeys) -
Monkeys were immunized with 5 mg of indicated codon optimized plasmids at
week 0, 4, and 8. Four weeks after each immunization , peripheral blood
mononuclear cells were collected and tested for Nef-specific INF-gamma
secreting
cells as described for the mice studies in this Example section. The results
are shown
in Table 2. As with the mouse study, tpanef(LLAA) shows significantly enhanced
immunogenicity when compared to tPAnef.
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TABLE 2
Nef specific
INF-gamma
secreting
cells/million
PBMC
A
al
Vaccine Week Week Week Week
0 4 8 12
No.
Medium Medium Medium Medium nef
nef nef nef
VlJns- 1 7439 30208 6 148 89 559
TpaNef 2 1 3 2845 1344 13 146
(LLAA)
3 5 5 1445 1111 14 35
V lJns-nef1 0 1 2433 1643 6 34
2 289 3135 1334 24 80
3 1 0 1631 1838 13 185
Control1 1 3 1633 1616 18 13
Monkeys were immunized with 5 mg of indicated plasmids at week 0, 4 and 8.
Four weeks after each immunization, peripheral blood mononuclear
cells were collected and tested for the Nef-specific IFN-gamma secreting
cells.
A codon-optimized nef gene coding for HIV-1 jrfl isolate Nef polypeptide was
synthesized. The resultant synthetic nef gene was well expressed in the in
vitro
transfected cells. Using this synthetic gene as parental molecule, nef mutants
involving myristylation site and dileucine motif mutations were constructed.
Two
forms of myristylation site mutation were made, one involving a single Gly2Ala
change and the other by fusing human plasminogen activator(tpa) leader peptide
with
the N-terminus of Nef polypeptide. The dileucine motif mutation was generated
by
Leu174A1a and Leu175A1a changes. The resultant nef constructs were named as
nef,
tpanef, tpanef(LLAA) and nef(G2A,LLAA). The addition of tpa leader peptide
sequence resulted in significantly increased expression of the nef gene in
vitro; in
contrast, either Gly2Ala mutation or dileucine mutation reduced the nef gene
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expression. In an effort to characterize immunogenicity of nef and nef
mutants,
experiments were carried out to map nef CTL and Th epitopes in mice. A single
CTL
epitope and a dominant Th epitope, both restricted by H-2b, were identified.
Consequently, C57BL/6 mice were immunized with different nef constructs by DNA
immunization means, and splenocytes from immunized mice were determined for
Nef-specific CTL and Th responses using Elisopt assay and the defined T cell
epitopes. The results showed that tpanef and tpanef(LLAA) were significantly
more
immunogenic than nef in terms of eliciting both CTL and Th responses.
Therefore, these aforementioned polynucleotides, when directly introduced
into a vertebrate in vivo, including mammals such as primates and humans,
should
express the respective HIV-1 Nef protein within the animal and in turn induce
at least
a cytotoxic T lymphocyte (CTL) response within the host to the expressed Nef
antigen.
The present invention is not to be limited in scope by the specific
embodiments described herein. Indeed, various modifications of the invention
in
addition to those described herein will become apparent to those skilled in
the art
from the foregoing description. Such modifications are intended to fall within
the
scope of the appended claims.
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SEQUENCE LISTING
<110> APPLICANT: Merck & Co., Inc.
<120> TITLE: POLYNUCLEOTIDE VACCINES EXPRESSING CODON
OPTIMIZED HIV-1 NEF AND MODIFIED HIV-1 NEF
<130> DOCKET/FILE REFERENCE: 20602Y
<160> NUMBER OF SEQUENCES: 30
<170> SOFTWARE: FastSEQ for Windows Version 4.0
<210> SEQ ID N0:1
<211> LENGTH: 671
<212> TYPE: DNA
<213> ORGANISM:Human Immunodeficiency Virus - 1
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (12)...(662)
<400> SEQ ID N0:1
gatctgccac c atg ggc ggc aag tgg tcc aag agg tcc gtg ccc ggc tgg 50
Met Gly Gly Lys Trp Ser Lys Arg Ser Val Pro Gly Trp
1 5 10
tcc acc gtg agg gag agg atg agg agg gcc gag ccc gcc gcc gac agg 98
Ser Thr Val Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Arg
15 20 25
gtg agg agg acc gag ccc gcc gcc gtg ggc gtg ggc gcc gtg tcc agg 146
Val Arg Arg Thr Glu Pro Ala Ala Val Gly Val Gly Ala Val Ser Arg
30 35 40 45
gac ctg gag aag cac ggc gcc atc acc tcc tcc aac acc gcc gcc acc 194
Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Ala Thr
50 55 60
aac gcc gac tgc gcc tgg ctg gag gcc cag gag gac gag gag gtg ggc 242
Asn Ala Asp Cys Ala Trp Leu Glu Ala Gln Glu Asp Glu Glu Val Gly
65 70 75
ttc ccc gtg agg ccc cag gtg ccc ctg agg ccc atg acc tac aag ggc 290
Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Gly
80 85 90
gcc gtg gac ctg tcc cac ttc ctg aag gag aag ggc ggc ctg gag ggc 338
Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly
95 100 105
ctg atc cac tcc cag aag agg cag gac atc ctg gac ctg tgg gtg tac 386
Leu Ile His Ser Gln Lys Arg Gln Asp Ile Leu Asp Leu Trp Val Tyr
110 115 120 125
cac acc cag ggc tac ttc ccc gac tgg cag aac tac acc ccc ggc ccc 434
His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro
1
CA 02393861 2002-06-05
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130 135 140
ggc atc agg ttc ccc ctg acc ttc ggc tgg tgc ttc aag ctg gtg ccc 482
Gly Ile Arg Phe Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu Val Pro
145 150 155
gtg gag ccc gag aag gtg gag gag gcc aac gag ggc gag aac aac tgc 530
Val Glu Pro Glu Lys Val Glu Glu Ala Asn Glu Gly Glu Asn Asn Cys
160 165 170
ctg ctg cac ccc atg tcc cag cac ggc atc gag gac ccc gag aag gag 578
Leu Leu His Pro Met Ser Gln His Gly Ile Glu Asp Pro Glu Lys Glu
175 180 185
gtg ctg gag tgg agg ttc gac tcc aag ctg gcc ttc cac cac gtg gcc 626
Val Leu Glu Trp Arg Phe Asp Ser Lys Leu Ala Phe His His Val Ala
190 195 200 205
agg gag ctg cac ccc gag tac tac aag gac tgc taa agcccgggc 671
Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys
210 215
<210> SEQ ID N0:2
<211> LENGTH: 216
<212> TYPE: PRT
<213> ORGANISM:Human Immunodeficiency Virus - 1
<400> SEQ ID N0:2
Met Gly Gly Lys Trp Ser Lys Arg Ser Val Pro Gly Trp Ser Thr Val
1 5 10 15
Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Arg Val Arg Arg
20 25 30
Thr Glu Pro Ala Ala Val Gly Val Gly Ala Val Ser Arg Asp Leu Glu
35 40 45
Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Ala Thr Asn Ala Asp
50 55 60
Cys Ala Trp Leu Glu Ala Gln Glu Asp Glu Glu Val Gly Phe Pro Val
65 70 75 80
Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Gly Ala Val Asp
85 90 95
Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile His
100 105 110
Ser Gln Lys Arg Gln Asp Ile Leu Asp Leu Trp Val Tyr His Thr Gln
115 120 125
Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Ile Arg
130 135 140
Phe Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu Val Pro Val Glu Pro
145 150 155 160
Glu Lys Val Glu Glu Ala Asn Glu Gly Glu Asn Asn Cys Leu Leu His
165 170 175
Pro Met Ser Gln His Gly Ile Glu Asp Pro Glu Lys Glu Val Leu Glu
180 185 190
Trp Arg Phe Asp Ser Lys Leu Ala Phe His His Val Ala Arg Glu Leu
195 200 205
His Pro Glu Tyr Tyr Lys Asp Cys
210 215
<210> SEQ ID N0:3
<211> LENGTH: 719
2
CA 02393861 2002-06-05
WO 01/43693 PCT/US00/34162
<212> TYPE: DNA
<213> ORGANISM:Human Immunodeficiency Virus - 1
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (2)...(715)
<400> SEQ ID N0:3
c atg gat gca atg aag aga ggg ctc tgc tgt gtg ctg ctg ctg tgt gga 49
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
gca gtc ttc gtt tcg ccc agc gag atc tcc tcc aag agg tcc gtg ccc 97
Ala Val Phe Val Ser Pro Ser Glu Ile Ser Ser Lys Arg Ser Val Pro
20 25 30
ggc tgg tcc acc gtg agg gag agg atg agg agg gcc gag ccc gcc gcc 145
Gly Trp Ser Thr Val Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala
35 40 45
gac agg gtg agg agg acc gag ccc gcc gcc gtg ggc gtg ggc gcc gtg 193
Asp Arg Val Arg Arg Thr Glu Pro Ala Ala Val Gly Val Gly Ala Val
50 55 60
tcc agg gac ctg gag aag cac ggc gcc atc acc tcc tcc aac acc gcc 241
Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala
65 70 75 80
gcc acc aac gcc gac tgc gcc tgg ctg gag gcc cag gag gac gag gag 289
Ala Thr Asn Ala Asp Cys Ala Trp Leu Glu Ala Gln Glu Asp Glu Glu
85 90 95
gtg ggc ttc ccc gtg agg ccc cag gtg ccc ctg agg ccc atg acc tac 337
Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr
100 105 110
aag ggc gcc gtg gac ctg tcc cac ttc ctg aag gag aag ggc ggc ctg 385
Lys Gly Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu
115 120 125
gag ggc ctg atc cac tcc cag aag agg cag gac atc ctg gac ctg tgg 433
Glu Gly Leu Ile His Ser Gln Lys Arg Gln Asp Ile Leu Asp Leu Trp
130 135 140
gtg tac cac acc cag ggc tac ttc ccc gac tgg cag aac tac acc ccc 481
Val Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro
145 150 155 160
ggc ccc ggc atc agg ttc ccc ctg acc ttc ggc tgg tgc ttc aag ctg 529
Gly Pro Gly Ile Arg Phe Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu
165 170 175
gtg ccc gtg gag ccc gag aag gtg gag gag gcc aac gag ggc gag aac 577
Val Pro Val Glu Pro Glu Lys Val Glu Glu Ala Asn Glu Gly Glu Asn
180 185 190
aac tgc ctg ctg cac ccc atg tcc cag cac ggc atc gag gac ccc gag 625
Asn Cys Leu Leu His Pro Met Ser Gln His Gly Ile Glu Asp Pro Glu
195 200 205
aag gag gtg ctg gag tgg agg ttc gac tcc aag ctg gcc ttc cac cac 673
3
CA 02393861 2002-06-05
WO 01/43693 PCT/US00/34162
Lys Glu Val Leu Glu Trp Arg Phe Asp Ser Lys Leu Ala Phe His His
210 215 220
gtg gcc agg gag ctg cac ccc gag tac tac aag gac tgc taa 715
Val Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys
225 230 235
agcc 719
<210> SEQ ID N0:4
<211> LENGTH: 237
<212> TYPE: PRT
<213> ORGANISM:Human Immunodeficiency Virus - 1
<400> SEQ ID N0:4
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser Glu Ile Ser Ser Lys Arg Ser Val Pro
20 25 30
Gly Trp Ser Thr Val Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala
35 40 45
Asp Arg Val Arg Arg Thr Glu Pro Ala Ala Val Gly Val Gly Ala Val
50 55 60
Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala
65 70 75 80
Ala Thr Asn Ala Asp Cys Ala Trp Leu Glu Ala Gln Glu Asp Glu Glu
85 90 95
Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr
100 105 110
Lys Gly Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu
115 120 125
Glu Gly Leu Ile His Ser Gln Lys Arg Gln Asp Ile Leu Asp Leu Trp
130 135 140
Val Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro
145 150 155 160
Gly Pro Gly Ile Arg Phe Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu
165 170 175
Val Pro Val Glu Pro Glu Lys Val Glu Glu Ala Asn Glu Gly Glu Asn
180 185 190
Asn Cys Leu Leu His Pro Met Ser Gln His Gly Ile Glu Asp Pro Glu
195 200 205
Lys Glu Val Leu Glu Trp Arg Phe Asp Ser Lys Leu Ala Phe His His
210 215 220
Val Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys
225 230 235
<210> SEQ ID N0:5
<211> LENGTH: 671
<212> TYPE: DNA
<213> ORGANISM:Human Immunodeficiency Virus - 1
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (12)...(662)
<400> SEQ ID N0:5
gatctgccac c atg gcc ggc aag tgg tcc aag agg tcc gtg ccc ggc tgg 50
Met Ala Gly Lys Trp Ser Lys Arg Ser Val Pro Gly Trp
1 5 10
tcc acc gtg agg gag agg atg agg agg gcc gag ccc gcc gcc gac agg 98
4
CA 02393861 2002-06-05
WO 01/43693 PCT/US00/34162
Ser Thr Val Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Arg
15 20 25
gtg agg agg acc gag ccc gcc gcc gtg ggc gtg ggc gcc gtg tcc agg 146
Val Arg Arg Thr Glu Pro Ala Ala Val Gly Val Gly Ala Val Ser Arg
30 35 40 45
gac ctg gag aag cac ggc gcc atc acc tcc tcc aac acc gcc gcc acc 194
Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Ala Thr
50 55 60
aac gcc gac tgc gcc tgg ctg gag gcc cag gag gac gag gag gtg ggc 242
Asn Ala Asp Cys Ala Trp Leu Glu Ala Gln Glu Asp Glu Glu Val Gly
65 70 75
ttc ccc gtg agg ccc cag gtg ccc ctg agg ccc atg acc tac aag ggc 290
Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Gly
80 85 90
gcc gtg gac ctg tcc cac ttc ctg aag gag aag ggc ggc ctg gag ggc 338
Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly
95 100 105
ctg atc cac tcc cag aag agg cag gac atc ctg gac ctg tgg gtg tac 386
Leu Ile His Ser Gln Lys Arg Gln Asp Ile Leu Asp Leu Trp Val Tyr
110 115 120 125
cac acc cag ggc tac ttc ccc gac tgg cag aac tac acc ccc ggc ccc 434
His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro
130 135 140
ggc atc agg ttc ccc ctg acc ttc ggc tgg tgc ttc aag ctg gtg ccc 482
Gly Ile Arg Phe Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu Val Pro
145 150 155
gtg gag ccc gag aag gtg gag gag gcc aac gag ggc gag aac aac tgc 530
Val Glu Pro Glu Lys Val Glu Glu Ala Asn Glu Gly Glu Asn Asn Cys
160 165 170
gcc gcc cac ccc atg tcc cag cac ggc atc gag gac ccc gag aag gag 578
Ala Ala His Pro Met Ser Gln His Gly Ile Glu Asp Pro Glu Lys Glu
175 180 185
gtg ctg gag tgg agg ttc gac tcc aag ctg gcc ttc cac cac gtg gcc 626
Val Leu Glu Trp Arg Phe Asp Ser Lys Leu Ala Phe His His Val Ala
190 195 200 205
agg gag ctg cac ccc gag tac tac aag gac tgc taa agcccgggc 671
Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys
210 215
<210> SEQ ID N0:6
<211> LENGTH: 217
<212> TYPE: PRT
<213> ORGANISM:Human Immunodeficiency Virus - 1
<400> SEQ ID N0:6
Met Ala Gly Lys Trp Ser Lys Arg Ser Val Pro Gly Trp Ser Thr Val
CA 02393861 2002-06-05
WO 01/43693 PCT/US00/34162
1 5 10 15
Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Arg Val Arg Arg
20 25 30
Thr Glu Pro Ala Ala Val Gly Val Gly Ala Val Ser Arg Asp Leu Glu
35 40 45
Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Ala Thr Asn Ala Asp
50 55 60
Cys Ala Trp Leu Glu Ala Gln Glu Asp Glu Glu Val Gly Phe Pro Val
65 70 75 80
Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Gly Ala Val Asp
85 90 95
Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile His
100 105 110
Ser Gln Lys Arg Gln Asp Ile Leu Asp Leu Trp Val Tyr His Thr Gln
115 120 125
Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Ile Arg
130 135 140
Phe Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu Val Pro Val Glu Pro
145 150 155 160
Glu Lys Val Glu Glu Ala Asn Glu Gly Glu Asn Asn Cys Ala Ala His
165 170 175
Pro Met Ser Gln His Gly Ile Glu Asp Pro Glu Lys Glu Val Leu Glu
180 185 190
Trp Arg Phe Asp Ser Lys Leu Ala Phe His His Val Ala Arg Glu Leu
195 200 205
His Pro Glu Tyr Tyr Lys Asp Cys Ser
210 215
<210> SEQ ID N0:7
<211> LENGTH: 720
<212> TYPE: DNA
<213> ORGANISM:Human Immunodeficiency Virus - 1
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (2)...(715)
<400> SEQ ID N0:7
c atg gat gca atg aag aga ggg ctc tgc tgt gtg ctg ctg ctg tgt gga 49
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
gca gtc ttc gtt tcg ccc agc gag atc tcc tcc aag agg tcc gtg ccc 97
Ala Val Phe Val Ser Pro Ser Glu Ile Ser Ser Lys Arg Ser Val Pro
20 25 30
ggc tgg tcc acc gtg agg gag agg atg agg agg gcc gag ccc gcc gcc 145
Gly Trp Ser Thr Val Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala
35 40 45
gac agg gtg agg agg acc gag ccc gcc gcc gtg ggc gtg ggc gcc gtg 193
Asp Arg Val Arg Arg Thr Glu Pro Ala Ala Val Gly Val Gly Ala Val
50 55 60
tcc agg gac ctg gag aag cac ggc gcc atc acc tcc tcc aac acc gcc 241
Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala
65 70 75 80
gcc acc aac gcc gac tgc gcc tgg ctg gag gcc cag gag gac gag gag 289
Ala Thr Asn Ala Asp Cys Ala Trp Leu Glu Ala Gln Glu Asp Glu Glu
6
CA 02393861 2002-06-05
WO 01/43693 PCT/US00/34162
85 90 95
gtg ggc ttc ccc gtg agg ccc cag gtg ccc ctg agg ccc atg acc tac 337
Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr
100 105 110
aag ggc gcc gtg gac ctg tcc cac ttc ctg aag gag aag ggc ggc ctg 385
Lys Gly Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu
115 120 125
gagggcctgatc cactcccag aagaggcag gacatcctg gacctgtgg 433
GluGlyLeuIle HisSerGln LysArgGln AspIleLeu AspLeuTrp
130 135 140
gtgtaccacacc cagggctac ttccccgac tggcagaac tacaccccc 481
ValTyrHisThr GlnGlyTyr PheProAsp TrpGlnAsn TyrThrPro
145 150 155 160
ggccccggcatc aggttcccc ctgaccttc ggctggtgc ttcaagctg 529
GlyProGlyIle ArgPhePro LeuThrPhe GlyTrpCys PheLysLeu
165 170 175
gtgcccgtggag cccgagaag gtggaggag gccaacgag ggcgagaac 577
ValProValGlu ProGluLys ValGluGlu AlaAsnGlu GlyGluAsn
180 185 190
aactgcgccgcc caccccatg tcccagcac ggcatcgag gaccccgag 625
AsnCysAlaAla HisProMet SerGlnHis GlyIleGlu AspProGlu
195 200 205
aaggaggtgctg gagtggagg ttcgactcc aagctggcc ttccaccac 673
LysGluValLeu GluTrpArg PheAspSer LysLeuAla PheHisHis
210 215 220
gtggccagggag ctgcacccc gagtactac aaggactgc taa 715
ValAlaArgGlu LeuHisPro GluTyrTyr LysAspCys
225 230 235
agccc 720
<210> SEQ ID N0:8
<211> LENGTH: 237
<212> TYPE: PRT
<213> ORGANISM:Human Immunodeficiency Virus - 1
<400> SEQ ID N0:8
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser Glu Ile Ser Ser Lys Arg Ser Val Pro
20 25 30
Gly Trp Ser Thr Val Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala
35 40 45
Asp Arg Val Arg Arg Thr Glu Pro Ala Ala Val Gly Val Gly Ala Val
50 55 60
Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala
65 70 75 80
Ala Thr Asn Ala Asp Cys Ala Trp Leu Glu Ala Gln Glu Asp Glu Glu
85 90 95
Val Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr
100 105 110
7
CA 02393861 2002-06-05
WO 01/43693 PCT/US00/34162
Lys Gly Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu
115 120 125
Glu Gly Leu Ile His Ser Gln Lys Arg Gln Asp Ile Leu Asp Leu Trp
130 135 140
Val Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro
145 150 155 160
Gly Pro Gly Ile Arg Phe Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu
165 170 175
Val Pro Val Glu Pro Glu Lys Val Glu Glu Ala Asn Glu Gly Glu Asn
180 185 190
Asn Cys Ala Ala His Pro Met Ser Gln His Gly Ile Glu Asp Pro Glu
195 200 205
Lys Glu Val Leu Glu Trp Arg Phe Asp Ser Lys Leu Ala Phe His His
210 215 220
Val Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys
225 230 235
<210> SEQ ID N0:9
<211> LENGTH: 4945
<212> TYPE: DNA
<213> ORGANISM: E. coli
<400> SEQ ID N0:9
tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtca 60
cagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtg 120
ttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgc 180
accatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcagattgg 240
ctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatg 300
tccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaattac 360
ggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatgg 420
cccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcc 480
catagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaac 540
tgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaa 600
tgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctac 660
ttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagta 720
catcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattga 780
cgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaa 840
ctccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcag 900
agctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctcca 960
tagaagacaccgggaccgatccagcctccgcggccgggaacggtgcattggaacgcggat 1020
tccccgtgccaagagtgacgtaagtaccgcctatagactctataggcacacccctttggc 1080
tcttatgcatgctatactgtttttggcttggggcctatacacccccgcttccttatgcta 1140
taggtgatggtatagcttagcctataggtgtgggttattgaccattattgaccactcccc 1200
tattggtgacgatactttccattactaatccataacatggctctttgccacaactatctc 1260
tattggctatatgccaatactctgtccttcagagactgacacggactctgtatttttaca 1320
ggatggggtcccatttattatttacaaattcacatatacaacaacgccgtcccccgtgcc 1380
cgcagtttttattaaacatagcgtgggatctccacgcgaatctcgggtacgtgttccgga 1440
catgggctcttctccggtagcggcggagcttccacatccgagccctggtcccatgcctcc 1500
agcggctcatggtcgctcggcagctccttgctcctaacagtggaggccagacttaggcac 1560
agcacaatgcccaccaccaccagtgtgccgcacaaggccgtggcggtagggtatgtgtct 1620
gaaaatgagcgtggagattgggctcgcacggctgacgcagatggaagacttaaggcagcg 1680
gcagaagaagatgcaggcagctgagttgttgtattctgataagagtcagaggtaactccc 1740
gttgcggtgctgttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgcg 1800
cgcgccaccagacataatagctgacagactaacagactgttcctttccatgggtcttttc 1860
tgcagtcaccgtccttagatcaccatggatgcaatgaagagagggctctgctgtgtgctg 1920
ctgctgtgtggagcagtcttcgtttcgcccagcgagatctgctgtgccttctagttgcca 1980
gccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccac 2040
tgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctat 2100
tctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggca 2160
tgctggggatgcggtgggctctatggccgctgcggccaggtgctgaagaattgacccggt 2220
tcctcctgggccagaaagaagcaggcacatccccttctctgtgacacaccctgtccacgc 2280
8
CA 02393861 2002-06-05
WO 01/43693 PCT/US00/34162
ccctggttcttagttccagccccactcataggacactcatagctcaggagggctccgcct 2340
tcaatcccacccgctaaagtacttggagcggtctctccctccctcatcagcccaccaaac 2400
caaacctagcctccaagagtgggaagaaattaaagcaagataggctattaagtgcagagg 2460
gagagaaaatgcctccaacatgtgaggaagtaatgagagaaatcatagaatttcttccgc 2520
ttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctca 2580
ctcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtg 2640
agcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttcca 2700
taggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaa 2760
cccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcc 2820
tgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggc 2880
gctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagct 2940
gggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcg 3000
tcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacag 3060
gattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaacta 3120
cggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcgg 3180
aaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttt 3240
tgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatctt 3300
ttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgag 3360
attatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaat 3420
ctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacc 3480
tatctcagcgatctgtctatttcgttcatccatagttgcctgactcggggggggggggcg 3540
ctgaggtctgcctcgtgaagaaggtgttgctgactcataccaggcctgaatcgccccatc 3600
atccagccagaaagtgagggagccacggttgatgagagctttgttgtaggtggaccagtt 3660
ggtgattttgaacttttgctttgccacggaacggtctgcgttgtcgggaagatgcgtgat 3720
ctgatccttcaactcagcaaaagttcgatttattcaacaaagccgccgtcccgtcaagtc 3780
agcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcg 3840
agcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaa 3900
agccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcc 3960
tggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcg 4020
tcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaat 4080
ggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtca 4140
tcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacga 4200
aatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcagg 4260
aacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctgg 4320
aatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggata 4380
aaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctca 4440
tctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcg 4500
ggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccat 4560
ttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtt 4620
tcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagtttt 4680
attgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgagacaca 4740
acgtggctttccccccccccccattattgaagcatttatcagggttattgtctcatgagc 4800
ggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccc 4860
cgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaat 4920
aggcgtatcacgaggccctttcgtc 4945
<210> SEQ ID N0:10
<211> LENGTH: 23
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide
<400> SEQ ID N0:10
ctatataagc agagctcgtt tag 23
<210> SEQ ID N0:11
<211> LENGTH: 30
9
CA 02393861 2002-06-05
WO 01/43693 PCT/US00/34162
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide
<400> SEQ ID N0:11
gtagcaaaga tctaaggacg gtgactgcag 30
<210> SEQ ID N0:12
<211> LENGTH: 39
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide
<400> SEQ ID N0:12
gtatgtgtct gaaaatgagc gtggagattg ggctcgcac 39
<210> SEQ ID N0:13
<211> LENGTH: 39
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide
<400> SEQ ID N0:13
gtgcgagccc aatctccacg ctcattttca gacacatac 39
<210> SEQ ID N0:14
<211> LENGTH: 4432
<212> TYPE: DNA
<213> ORGANISM: E. coli
<400> SEQ ID N0:14
tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtca 60
cagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtg 120
ttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgc 180
accatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcagattgg 240
ctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatg 300
tccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaattac 360
ggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatgg 420
cccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcc 480
catagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaac 540
tgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaa 600
tgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctac 660
ttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagta 720
catcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattga 780
cgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaa 840
ctccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcag 900
agctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctcca 960
tagaagacaccgggaccgatccagcctccgcggccgggaacggtgcattggaacgcggat 1020
tccccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacccccttggc 1080
ttcttatgcatgctatactgtttttggcttggggtctatacacccccgcttcctcatgtt 1140
ataggtgatggtatagcttagcctataggtgtgggttattgaccattattgaccactccc 1200
ctattggtgacgatactttccattactaatccataacatggctctttgccacaactctct 1260
ttattggctatatgccaatacactgtccttcagagactgacacggactctgtatttttac 1320
aggatggggtctcatttattatttacaaattcacatatacaacaccaccgtccccagtgc 1380
ccgcagtttttattaaacataacgtgggatctccacgcgaatctcgggtacgtgttccgg 1440
CA 02393861 2002-06-05
WO 01/43693 PCT/US00/34162
acatgggctcttctccggtagcggcggagcttctacatccgagccctgctcccatgcctc 1500
cagcgactcatggtcgctcggcagctccttgctcctaacagtggaggccagacttaggca 1560
cagcacgatgcccaccaccaccagtgtgccgcacaaggccgtggcggtagggtatgtgtc 1620
tgaaaatgagctcggggagcgggcttgcaccgctgacgcatttggaagacttaaggcagc 1680
ggcagaagaagatgcaggcagctgagttgttgtgttctgataagagtcagaggtaactcc 1740
cgttgcggtgctgttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgc 1800
gcgcgccaccagacataatagctgacagactaacagactgttcctttccatgggtctttt 1860
ctgcagtcaccgtccttagatctgctgtgccttctagttgccagccatctgttgtttgcc 1920
cctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaa 1980
atgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtgg 2040
ggcagcacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgg 2100
gctctatgggtacccaggtgctgaagaattgacccggttcctcctgggccagaaagaagc 2160
aggcacatccccttctctgtgacacaccctgtccacgcccctggttcttagttccagccc 2220
cactcataggacactcatagctcaggagggctccgccttcaatcccacccgctaaagtac 2280
ttggagcggtctctccctccctcatcagcccaccaaaccaaacctagcctccaagagtgg 2340
gaagaaattaaagcaagataggctattaagtgcagagggagagaaaatgcctccaacatg 2400
tgaggaagtaatgagagaaatcatagaatttcttccgcttcctcgctcactgactcgctg 2460
cgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggtta 2520
tccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggcc 2580
aggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgag 2640
catcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagatac 2700
caggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttacc 2760
ggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgt 2820
aggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacccccc 2880
gttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaaga 2940
cacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgta 3000
ggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagta 3060
tttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttga 3120
tccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacg 3180
cgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcag 3240
tggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacc 3300
tagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaact 3360
tggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctattt 3420
cgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggctta 3480
ccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagattta 3540
tcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatcc 3600
gcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaat 3660
agtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggt 3720
atggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttg 3780
tgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgca 3840
gtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgta 3900
agatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcgg 3960
cgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaact 4020
ttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccg 4080
ctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatctttt 4140
actttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaaggga 4200
ataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagc 4260
atttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaa 4320
caaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcta.agaaaccatt4380
attatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc 4432
<210> SEQ ID N0:15
<211> LENGTH: 4864
<212> TYPE: DNA
<213> ORGANISM: E. coli
<400> SEQ ID N0:15
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240
11
CA 02393861 2002-06-05
WO 01/43693 PCT/US00/34162
ctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatg 300
tccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaattac 360
ggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatgg 420
cccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcc 480
catagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaac 540
tgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaa 600
tgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctac 660
ttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagta 720
catcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattga 780
cgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaa 840
ctccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcag 900
agctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctcca 960
tagaagacaccgggaccgatccagcctccgcggccgggaacggtgcattggaacgcggat 1020
tccccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacccccttggc 1080
ttcttatgcatgctatactgtttttggcttggggtctatacacccccgcttcctcatgtt 1140
ataggtgatggtatagcttagcctataggtgtgggttattgaccattattgaccactccc 1200
ctattggtgacgatactttccattactaatccataacatggctctttgccacaactctct 1260
ttattggctatatgccaatacactgtccttcagagactgacacggactctgtatttttac 1320
aggatggggtctcatttattatttacaaattcacatatacaacaccaccgtccccagtgc 1380
ccgcagtttttattaaacataacgtgggatctccacgcgaatctcgggtacgtgttccgg 1440
acatgggctcttctccggtagcggcggagcttctacatccgagccctgctcccatgcctc 1500
cagcgactcatggtcgctcggcagctccttgctcctaacagtggaggccagacttaggca 1560
cagcacgatgcccaccaccaccagtgtgccgcacaaggccgtggcggtagggtatgtgtc 1620
tgaaaatgagctcggggagcgggcttgcaccgctgacgcatttggaagacttaaggcagc 1680
ggcagaagaagatgcaggcagctgagttgttgtgttctgataagagtcagaggtaactcc 1740
cgttgcggtgctgttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgc 1800
gcgcgccaccagacataatagctgacagactaacagactgttcctttccatgggtctttt 1860
ctgcagtcaccgtccttagatctgctgtgccttctagttgccagccatctgttgtttgcc 1920
cctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaa 1980
atgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtgg 2040
ggcagcacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgg 2100
gctctatgggtacccaggtgctgaagaattgacccggttcctcctgggccagaaagaagc 2160
aggcacatccccttctctgtgacacaccctgtccacgcccctggttcttagttccagccc 2220
cactcataggacactcatagctcaggagggctccgccttcaatcccacccgctaaagtac 2280
ttggagcggtctctccctccctcatcagcccaccaaaccaaacctagcctccaagagtgg 2340
gaagaaattaaagcaagataggctattaagtgcagagggagagaaaatgcctccaacatg 2400
tgaggaagtaatgagagaaatcatagaatttcttccgcttcctcgctcactgactcgctg 2460
cgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggtta 2520
tccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggcc 2580
aggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgag 2640
catcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagatac 2700
caggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttacc 2760
ggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgt 2820
aggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacccccc 2880
gttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaaga 2940
cacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgta 3000
ggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagta 3060
tttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttga 3120
tccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacg 3180
cgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcag 3240
tggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacc 3300
tagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaact 3360
tggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctattt 3420
cgttcatccatagttgcctgactccggggggggggggcgctgaggtctgcctcgtgaaga 3480
aggtgttgctgactcataccaggcctgaatcgccccatcatccagccagaaagtgaggga 3540
gccacggttgatgagagctttgttgtaggtggaccagttggtgattttgaacttttgctt 3600
tgccacggaacggtctgcgttgtcgggaagatgcgtgatctgatccttcaactcagcaaa 3660
agttcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccagtgt 3720
tacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaat 3780
ttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaagga 3840
gaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccg 3900
12
CA 02393861 2002-06-05
WO 01/43693 PCT/US00/34162
actcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagt 3960
gagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttct 4020
ttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaacc 4080
aaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaa 4140
ggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaaca 4200
atattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatc 4260
gcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaaga 4320
ggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacg 4380
ctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatag 4440
attgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagca 4500
tccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttgaatatggctcata 4560
acaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatattt 4620
ttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctttccccccccccc 4680
cattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatt 4740
tagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc 4800
taagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggcccttt 4860
cgtc 4864
<210> SEQ ID N0:16
<211> LENGTH: 4867
<212> TYPE: DNA
<213> ORGANISM: E. coli
<400> ID N0:16
SEQ
tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtca 60
cagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtg 120
ttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgc 180
accatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcagattgg 240
ctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatg 300
tccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaattac 360
ggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatgg 420
cccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcc 480
catagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaac 540
tgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaa 600
tgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctac 660
ttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagta 720
catcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattga 780
cgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaa 840
ctccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcag 900
agctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctcca 960
tagaagacaccgggaccgatccagcctccgcggccgggaacggtgcattggaacgcggat 1020
tccccgtgccaagagtgacgtaagtaccgcctatagactctataggcacacccctttggc 1080
tcttatgcatgctatactgtttttggcttggggcctatacacccccgcttccttatgcta 1140
taggtgatggtatagcttagcctataggtgtgggttattgaccattattgaccactcccc 1200
tattggtgacgatactttccattactaatccataacatggctctttgccacaactatctc 1260
tattggctatatgccaatactctgtccttcagagactgacacggactctgtatttttaca 1320
ggatggggtcccatttattatttacaaattcacatatacaacaacgccgtcccccgtgcc 1380
cgcagtttttattaaacatagcgtgggatctccacgcgaatctcgggtacgtgttccgga 1440
catgggctcttctccggtagcggcggagcttccacatccgagccctggtcccatgcctcc 1500
agcggctcatggtcgctcggcagctccttgctcctaacagtggaggccagacttaggcac 1560
agcacaatgcccaccaccaccagtgtgccgcacaaggccgtggcggtagggtatgtgtct 1620
gaaaatgagcgtggagattgggctcgcacggctgacgcagatggaagacttaaggcagcg 1680
gcagaagaagatgcaggcagctgagttgttgtattctgataagagtcagaggtaactccc 1740
gttgcggtgctgttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgcg 1800
cgcgccaccagacataatagctgacagactaacagactgttcctttccatgggtcttttc 1860
tgcagtcaccgtccttagatctgctgtgccttctagttgccagccatctgttgtttgccc 1920
ctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaa 1980
tgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggg 2040
gcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtggg 2100
ctctatggccgctgcggccaggtgctgaagaattgacccggttcctcctgggccagaaag 2160
aagcaggcacatccccttctctgtgacacaccctgtccacgcccctggttcttagttcca 2220
13
CA 02393861 2002-06-05
WO 01/43693 PCT/US00/34162
gccccactcataggacactcatagctcaggagggctccgccttcaatcccacccgctaaa 2280
gtacttggagcggtctctccctccctcatcagcccaccaaaccaaacctagcctccaaga 2340
gtgggaagaaattaaagcaagataggctattaagtgcagagggagagaaaatgcctccaa 2400
catgtgaggaagtaatgagagaaatcatagaatttcttccgcttcctcgctcactgactc 2460
gctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacg 2520
gttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaa 2580
ggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctga 2640
cgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaag 2700
ataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgct 2760
taccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacg 2820
ctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacc 2880
ccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggt 2940
aagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggta 3000
tgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaac 3060
agtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctc 3120
ttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagat 3180
tacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgc 3240
tcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatctt 3300
cacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagta 3360
aacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtct 3420
atttcgttcatccatagttgcctgactcggggggggggggcgctgaggtctgcctcgtga 3480
agaaggtgttgctgactcataccaggcctgaatcgccccatcatccagccagaaagtgag 3540
ggagccacggttgatgagagctttgttgtaggtggaccagttggtgattttgaacttttg 3600
ctttgccacggaacggtctgcgttgtcgggaagatgcgtgatctgatccttcaactcagc 3660
aaaagttcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccag 3720
tgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgc 3780
aatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaa 3840
ggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgatt 3900
ccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatca 3960
agtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatt 4020
tctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatca 4080
accaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgtta 4140
aaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatca 4200
acaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccgggg 4260
atcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcgga 4320
agaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggca 4380
acgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcga 4440
tagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatca 4500
gcatccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttgaatatggctc 4560
ataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatata 4620
tttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctttcccccccc 4680
ccccattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgt 4740
atttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgac 4800
gtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccc 4860
tttcgtc 4867
<210> SEQ ID N0:17
<211> LENGTH: 78
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide
<400> SEQ ID N0:17
gatcaccatg gatgcaatga agagagggct ctgctgtgtg ctgctgctgt gtggagcagt 60
cttcgtttcg cccagcga 78
<210> SEQ ID N0:18
<211> LENGTH: 78
14
CA 02393861 2002-06-05
WO 01/43693 PCT/US00/34162
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide
<400> SEQ ID N0:18
gatctcgctg ggcgaaacga agactgctcc acacagcagc agcacacagc agagccctct 60
cttcattgca tccatggt 78
<210> SEQ ID N0:19
<211> LENGTH: 27
<212> TYPE: PRT
<213> ORGANISM: Homo sapien
<400> SEQ ID N0:19
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser Glu Ile Ser Ser
20 25
<210> SEQ ID N0:20
<211> LENGTH: 33
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide
<400> SEQ ID N0:20
ggtacaaata ttggctattg gccattgcat acg 33
<210> SEQ ID N0:21
<211> LENGTH: 36
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide
<400> SEQ ID N0:21
ccacatctcg aggaaccggg tcaattcttc agcacc 36
<210> SEQ ID N0:22
<211> LENGTH: 38
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide
<400> SEQ ID N0:22
ggtacagata tcggaaagcc acgttgtgtc tcaaaatc 38
<210> SEQ ID N0:23
<211> LENGTH: 36
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide
CA 02393861 2002-06-05
WO 01/43693 PCT/US00/34162
<400> SEQ ID N0:23
cacatggatc cgtaatgctc tgccagtgtt acaacc 36
<210> SEQ ID N0:24
<211> LENGTH: 39
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide
<400> SEQ ID N0:24
ggtacatgat cacgtagaaa agatcaaagg atcttcttg 39
<210> SEQ ID N0:25
<211> LENGTH: 35
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: oligonucleotide
<400> SEQ ID N0:25
ccacatgtcg acccgtaaaa aggccgcgtt gctgg 35
<210> SEQ ID N0:26
<211> LENGTH: 4864
<212> TYPE: DNA
<213> ORGANISM: E. coli
<400> SEQ ID N0:26
tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtca 60
cagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtg 120
ttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgc 180
accatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcagattgg 240
ctattggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatg 300
tccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaattac 360
ggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatgg 420
cccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcc 480
catagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaac 540
tgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaa 600
tgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctac 660
ttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagta 720
catcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattga 780
cgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaa 840
ctccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcag 900
agctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctcca 960
tagaagacaccgggaccgatccagcctccgcggccgggaacggtgcattggaacgcggat 1020
tccccgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacccccttggc 1080
ttcttatgcatgctatactgtttttggcttggggtctatacacccccgcttcctcatgtt 1140
ataggtgatggtatagcttagcctataggtgtgggttattgaccattattgaccactccc 1200
ctattggtgacgatactttccattactaatccataacatggctctttgccacaactctct 1260
ttattggctatatgccaatacactgtccttcagagactgacacggactctgtatttttac 1320
aggatggggtctcatttattatttacaaattcacatatacaacaccaccgtccccagtgc 1380
ccgcagtttttattaaacataacgtgggatctccacgcgaatctcgggtacgtgttccgg 1440
acatgggctcttctccggtagcggcggagcttctacatccgagccctgctcccatgcctc 1500
cagcgactcatggtcgctcggcagctccttgctcctaacagtggaggccagacttaggca 1560
cagcacgatgcccaccaccaccagtgtgccgcacaaggccgtggcggtagggtatgtgtc 1620
tgaaaatgagctcggggagcgggcttgcaccgctgacgcatttggaagacttaaggcagc 1680
ggcagaagaagatgcaggcagctgagttgttgtgttctgataagagtcagaggtaactcc 1740
16
CA 02393861 2002-06-05
WO 01/43693 PCT/US00/34162
cgttgcggtgctgttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgc 1800
gcgcgccaccagacataatagctgacagactaacagactgttcctttccatgggtctttt 1860
ctgcagtcaccgtccttagatctgctgtgccttctagttgccagccatctgttgtttgcc 1920
cctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaa 1980
atgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtgg 2040
ggcagcacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgg 2100
gctctatgggtacccaggtgctgaagaattgacccggttcctcctgggccagaaagaagc 2160
aggcacatccccttctctgtgacacaccctgtccacgcccctggttcttagttccagccc 2220
cactcataggacactcatagctcaggagggctccgccttcaatcccacccgctaaagtac 2280
ttggagcggtctctccctccctcatcagcccaccaaaccaaacctagcctccaagagtgg 2340
gaagaaattaaagcaagataggctattaagtgcagagggagagaaaatgcctccaacatg 2400
tgaggaagtaatgagagaaatcatagaatttcttccgcttcctcgctcactgactcgctg 2460
cgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggtta 2520
tccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggcc 2580
aggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgag 2640
catcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagatac 2700
caggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttacc 2760
ggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgt 2820
aggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacccccc 2880
gttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaaga 2940
cacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgta 3000
ggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagta 3060
tttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttga 3120
tccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacg 3180
cgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcag 3240
tggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacc 3300
tagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaact 3360
tggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctattt 3420
cgttcatccatagttgcctgactccggggggggggggcgctgaggtctgcctcgtgaaga 3480
aggtgttgctgactcataccaggcctgaatcgccccatcatccagccagaaagtgaggga 3540
gccacggttgatgagagctttgttgtaggtggaccagttggtgattttgaacttttgctt 3600
tgccacggaacggtctgcgttgtcgggaagatgcgtgatctgatccttcaactcagcaaa 3660
agttcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccagtgt 3720
tacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaat 3780
ttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaagga 3840
gaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccg 3900
actcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagt 3960
gagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttct 4020
ttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaacc 4080
aaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaa 4140
ggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaaca 4200
atattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatc 4260
gcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaaga 4320
ggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacg 4380
ctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatag 4440
attgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagca 4500
tccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttgaatatggctcata 4560
acaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatattt 4620
ttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctttccccccccccc 4680
cattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatt 4740
tagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc 4800
taagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggcccttt 4860
cgtc 4864
<210> SEQ ID N0:27
<211> LENGTH: 139
<212> TYPE: DNA
<213> ORGANISM:E. coli / HIV-1
<400> SEQ ID N0:27
catgggtctt ttctgcagtc accgtccttg agatctgcca ccatgggcgg caagtggtcc 60
17
CA 02393861 2002-06-05
WO 01/43693 PCT/US00/34162
aagaggtccg tgccccaccc cgagtactac aaggactgct aaagcccggg cagatctgct 120
gtgccttcta gttgccagc 139
<210> SEQ ID N0:28
<211> LENGTH: 139
<212> TYPE: DNA
<213> ORGANISM:E. coli / HIV-1
<400> SEQ ID N0:28
catgggtctt ttctgcagtc accgtccttg agatctgcca ccatggccgg caagtggtcc 60
aagaggtccg tgccccaccc cgagtactac aaggactgct aaagcccggg cagatctgct 120
gtgccttcta gttgccagc 139
<210> SEQ ID N0:29
<211> LENGTH: 203
<212> TYPE: DNA
<213> ORGANISM:E. coli / HIV-1
<400> SEQ ID N0:29
catgggtctt ttctgcagtc accgtcctta tatctagatc accatggatg caatgaagag 60
agggctctgc tgtgtgctgc tgctgtgtgg agcagtcttc gtttcgccca gcgagatctc 120
ctccaagagg tccgtgcccc accccgagta ctacaaggac tgctaaagcc cgggcagatc 180
tgctgtgcct tctagttgcc agc 203
<210> SEQ ID N0:30
<211> LENGTH: 651
<212> TYPE: DNA
<213> ORGANISM:Human Immunodificiency Virus - 1
<400> SEQ ID N0:30
atgggtggcaagtggtcaaaacgtagtgtgcctggatggtctactgtaagggaaagaatg 60
agacgagctgagccagcagcagatagggtgagacgaactgagccagcagcagtaggggtg 120
ggagcagtatctcgagacctggaaaaacatggagcaatcacaagtagcaatacagcagct 180
accaatgctgattgtgcctggctagaagcacaagaggatgaggaagtgggttttccagtc 240
agacctcaggtacctttaagaccaatgacttacaagggagctgtagatcttagccacttt 300
ttaaaagaaaaggggggactggaagggctaattcactcacagaaaagacaagatatcctt 360
gatctgtgggtctaccacacacaaggctacttccctgattggcagaactacacaccaggg 420
ccaggaatcagatttccattgacctttggatggtgcttcaagctagtaccagttgagcca 480
gaaaaggtagaagaggccaatgaaggagagaacaactgcttgttacaccctatgagccag 540
catgggatagaggacccggagaaggaagtgttagagtggaggtttgacagcaagctagca 600
tttcatcacgtggcccgagagctgcatccggagtactacaaggactgctga 651
18
