Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF TIC INVENTION
POLYNUCLEOTIDE VACCINES EXPRESSING CODON OPTIMIZED HIV-1
POL AND MODIFIED HIV-1 POL
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit, under 35 U.S.C. ~119(e), of U.S.
provisional application 60/171,542, filed December 22, 1999.
STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not Applicable
15REFERENCE TO MICROFICHE APPENDIX
Not Applicable
FIELD OF THE INVENTION
The present invention relates to HIV Pol 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 Pol 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 Pol and derivatives of optimized HIV-
1
Pol, including constructs wherein protease, reverse transcriptase, RNAse H and
integrase activity of HIV-1 Pol is inactivated. 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 (A1DS) and related disorders. HIV-1
is an RNA virus of the Retroviridae family and exhibits the 5' LTR-gag pol-
erzv-
LTR 3' organization of all retroviruses. The integrated form of HIV-l, 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 fief 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 R5 (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-
HTV-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.
Larder, et al., (1987, Nature 327: 716-717) and~Larder, et al., (1989, Proc.
Natl. Acad. Sci. 86: 4803-4807) disclose site specific mutagenesis of HIV-1 RT
and
the effect such changes have on izz vitro activity and infectivity related to
interaction
with known inhibitors of RT.
Davies, et al. (1991, Science 252:, 88-9S) disclose the crystal structure of
the
RNase H domain of HIV-1 Pol.
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Schatz, et al. (1989, FEBS Lett. 257: 311-314) disclose that mutations
G1u478G1n and His539Phe in a complete HIV-1 RT/RNase H DNA fragment results
in defective RNase activity without effecting RT activity.
Mizrahi, et al. (1990, Nucl. Acids. Res. 18: pp. 5359-5353) disclose
additional
mutations Asp443Asn and Asp498Asn in the RNase region of the pol gene which
also
results in defective RNase activity. The authors note that the Asp498Asn
mutant was
difficult to characterize due to instability of this mutant protein.
Leavitt, et al. (1993, J. Biol. Chem. 268: 2113-2119) disclose several
mutations, including a Asp64Va1 mutation, which show differing effect on HIV-1
integrase (IN) activity.
Wiskerchen, et al. (1995, J. Virol. 69: 376-386) disclose singe and double
mutants, including mutation of aspartic acid residues which effect HIV-1
IN'and viral
replication functions. .
It would be of great import in the battle against AIDS 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 modified versions of the HIV-1 gene, pol.
SUIVEVIARY 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 cellular immune and humoral responses
upon
administration to the host, including primates and especially humans, and also
including a non-human mammal of commercial or domestic veterinary importance.
An effect of the cellular immune-directed vaccines of the present invention
should be
the lower transmission rate to previously uninfected individuals and/or
reduction in
the 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 Pol, wherein administration,
intracellular delivery and expression of the HIV-1 Pol 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 Pol, codon
optimized
versions of HIV-1 Pol fusion proteins, and codon optimized versions of HIV-1
Pol
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proteins and fusion protein, including but not limited to pol modifications
involving
residues within the catalytic regions responsible for RT, RNase and IN
activity within
the host cell.
A particular embodiment of the present invention relates to codon optimized
wt-pol DNA constructs wherein DNA sequences encoding the protease (PR)
activity
are deleted, leaving codon optimized "wild type" sequences which encode RT
(reverse transcriptase and RNase H activity) and IN integrase activity. The
nucleotide
sequence of a DNA molecule which encodes this protein is disclosed herein as
SEQ
ID NO:1 and the corresponding amino acid sequence of the expressed protein is
disclosed herein as SEQ ID N0:2.
The present invention preferably relates to a HIV-1 DNA pol construct which
is devoid of DNA sequences encoding any PR activity, as well as containing a
mutations) which at least partially, and preferably substantially, abolishes
RT, RNase
andlor IN activity. One type of HIV-1 pol mutant may include but is not
limited to a
mutated DNA molecule comprising at least one nucleotide substitution which
results
in a point mutation which effectively alters an active site within the RT,
RNase and/or
IN regions of the expressed protein, resulting in at least substantially
decreased
enzymatic activity for the RT, RNase H and/or IN functions of HIV-1 Pol. In a
preferred embodiment of this portion of the invention, a HIV-1 DNA pol
construct
contains a mutation or mutations within the Pol coding region which
effectively
abolishes RT, RNase H and IN activity. An especially preferable HIV-1 DNA pol
construct in a DNA molecule which contains at least one point mutation which
alters
the active site of the RT, RNase H and IN domains of Pol, such that each
activity is at
least substantially abolished. Such a HIV-1 Pol mutant will most likely
comprise at
least one point mutation in or around each catalytic domain responsible for
RT,
RNase H and IN activity, respectfully. To this end, an especially preferred
HIV-1
DNA pol construct is exemplified herein and contains nine codon substitution
mutations which results in an inactivated Pol protein (IA Pol: SEQ ID N0:4,
Figure
2A-C) which has no PR, RT, RNase or IN activity, wherein three such point
mutations reside within each of the RT, RNase and IN catalytic domains. Any
combination of the mutations disclosed herein may suitable and therefore may
be
utilized as an IA-Pol-based vaccine of the present invention. While addition
and
deletion mutations are contemplated and within the scope of the invention, the
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preferred mutation is a point mutation resulting in a substitution of the wild
type
amino acid with an alternative amino acid residue.
Another aspect of the present invention is to generate HIV-1 Pol-based
vaccine constructions which comprise a eukaryotic trafficking signal peptide
such as
the leader peptide from human tPA. To this end, the present invention relates
to a
DNA molecule which encodes a codon optimized wt-pol DNA construct wherein the
protease (PR) activity is deleted and a human tPA leader sequence is fused to
the 5'
end of the coding region. A DNA molecule which encodes this protein is
disclosed
herein as SEQ ID N0:5, the open reading frame disclosed herein as SEQ ID NO:6.
The present invention especially relates to a HIV-1 Pol mutant such as IA-Pol
(SEQ ID N0:4) which comprises a leader peptide, such as the human tPA leader,
at the
amino terminal portion of the protein, which may effect cellular trafficking
and hence,
immunogenicity of the expressed protein within the host cell. Any such HIV-1
DNA pol
mutant disclosed in the above paragraphs is suitable for fusion downstream of
a leader
peptide, including but by no means limited to the human tPA leader sequence.
Therefore, .
any such leader peptide-based HIV-1 pol mutant construct may include but is
not limited
to a mutated DNA molecule which effectively alters the catalytic activity of
the RT,
RNase andlor IN region of the expressed protein, resulting in at least
substantially
decreased enzymatic activity one or more of the RT, RNase H andlor IN
functions of
HIV-1 Pol. In a preferred embodiment of this portion of the invention, a
leader
peptide/HIV-1 DNA pol construct contains a mutation or mutations within the
Pol coding
region which effectively abolishes RT, RNase H and IN activity. An especially
preferable HIV-1 DNA pol construct is a DNA molecule which contains at least
one point
mutation which alters the active site and catalytic activity within the RT,
RNase H and IN
domains of Pol, such that each activity is at least substantially abolished,
and preferably
totally abolished. Such a HIV-1 Pol mutant will most likely comprise at least
one point
mutation in or around each catalytic domain responsible for RT, RNase H and IN
activity,
respectfully. An especially preferred embodiment of this portion of the
invention relates
to a human tPA leader fused to the IA-Pol protein comprising the nine
mutations shown
in Table 1. The DNA molecule is disclosed herein as SEQ ID N0:7 and the
expressed
tPA-IA Pol protein comprises a fusion junction as shown in Figure 3. The
complete
amino acid sequence of the expressed protein is set forth in SEQ ID N0:8.
The present invention also relates to a substantially purified protein
expressed
from the DNA polynucleotide vaccines of the present invention, especially the
purified
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proteins set forth below as SEQ ID NOs: 2, 4, 6, and 8. These purified
proteins may be
useful as protein-based HIV vaccines.
The present invention also relates to non-codon optimized versions of DNA
molecules and associated polynucleotides and associated DNA vaccines which
encode the various wild type and modified forms of the HIV Pol 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 Pol which are shown to promote a substantial cellular immune and humoral
immune responses 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, DNA
plasmid vectors of the present invention preferably comprise an antibiotic
resistance
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 exemplified
herein
include Vl, V1J (SEQ ID N0:13), VlJneo (SEQ ID N0:14), VlJns (Figure 1A, SEQ
ID N0:15), 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 N0:28.
The present invention especially relates to a DNA vaccine and a
pharmaceutically active vaccine composition which contains this DNA 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 codon optimized HIV-1 Pol (e.g. SEQ ID N0:2), codon optimized HIV-1
Pol fused to an amino terminal localized leader sequence (e.g. SEQ ID N0:6),
and
especially preferable, and the essence of the present invention, biologically
inactive
Pol proteins (IA Pol; e.g., SEQ m N0:4) devoid of significant PR, RT, RNase or
IN
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activity associated with wild type Pol and a concomitant construct which
contains a
leader peptide at the amino terminal region of the IA Pol protein. These
constructs
are ligated within an appropriate DNA plasmid vector, with or without a
nucleotide
sequence encoding a functional leader peptide. Preferred DNA vaccines of the
present invention comprise codon optimized DNA molecules encoding codon
optimized HIV-1 Pol and inactivated version of Pol, ligated in DNA vectors
disclosed
herein, 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 ID N0:28.
Therefore, the present invention relates to DNA vaccines which include, but
are in no way limited to VlJns-WTPoI (comprising the DNA molecule encoding WT
Pol, as set forth in SEQ m N0:2), VlJns-tPA-WTPoI, (comprising the DNA
molecule encoding tPA Pol, as set forth in SEQ ID N0:6), VlJns-IAPoI
(comprising
the DNA molecule encoding IA Pol, as set forth in SEQ ID NO:4), and VlJns-tPA-
IAPoI, (comprising the DNA molecule encoding tPA-IA Pol, as set forth in SEQ
ID
N0:8). Especially preferred are VlJns-IAPoI and VlJns-tPA-IAPol, as
exemplified
in Example Section 2.
The present invention also relates to HIV Pol 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 promoting an enhanced cellular and/or humoral response subsequent
to
inoculation. A preferred adjuvant is an aluminum phosphate-based adjuvant or a
calcium phosphate based adjuvant, 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 cellular responses to DNA vaccination.
As used herein, a DNA vaccine or DNA 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 pol
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genes of the present invention.
BRIEF DESCRIPTION OF THE FIGURES
Figure lA-B shows schematic representation of DNA vaccine expression
vectors VlJns (A) and VlJns-tPA (B) utilized for HIV-1 pol and HIV-1 modified
pol
constructs.
Figure 2A-C shows the nucleotide (SEQ ID N0:3) and amino acid sequence
(SEQ ID N0:4) of IA-Pol. Underlined codons and amino acids denote mutations,
as
listed in Table 1.
Figure 3 shows the codon optimized nucleotide and amino acid sequences
through the fusion junction of tPA-IA-Pol (contained within SEQ ID NOs: 7 and
8,
respectively). The underlined portion represents the NH2-terminal region of IA-
Pol.
Figure 4 shows generation of a humoral response (measured as the geometric
means of anti-RT endpoint titers) from mice immunized with one or two doses of
codon optimized VlJns-IApol and VlJns-tpa-IApol. A portion of mice that
received
30 ug of each plasmid was boosted at T=8 wks; sera from all mice were
collected at 4
wk post dose 2.
Figure 5 shows the number of IFN-gamma secreting cells per 10e6 cells
following stimulation with pools of either CD4+ (aa641-660, aa731-750) or CD8~
(aa201-220, aa311-330, aa571-590, aa781-800) specific peptides of splenocytes
(pool
of 5 spleenslcohort) from control mice and those vaccinated with increasing
single
dose of codon optimized VlJns-IApol or 30 ug of codon optimized VlJns-tpa-
IApol
(13 wks post dose 1). Mice (n=5) vaccinated with a second dose of 30 ug of
either
plasmid were analyzed in an Elispot assay at 6 wks post dose 2. Reported are
the
sums of the number of spots stimulated by each individual CD8+ peptides
because the
spots in the wells to which the pool was added are too dense to acquire
accurate
counts. The CD4+ cell counts are taken from the responses to the peptide pool.
Error
bars represent standard deviations for counts from triplicate wells per sample
per
antigen.
Figure 6A-C shows ELIspot analysis of peripheral blood cells collected from
rhesus macaques immunized three times (T=0, 4, 8 wks) with S mgs of codon
optimized HIV-1 Pol expressing plasmids. Antigen-specific IFN-gamma secretion
was stimulated by adding one of two pools consisting of 20-mer peptides
derived
from vaccine sequence (mpol-l, aal-420; mpol-2, aa411-850). (A) Frequencies of
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spot-forming cells (SFC) as a function of time for 3 monkeys (Tag No. 948008,
948013, 948033) vaccinated with VlJns-IApol. The reported values are corrected
for background responses without peptide restimulation. (B) Frequencies of
spot-
forming cells (SFC) as a function of time for 3 monkeys (Tag No. 920078,
920073,
948028) vaccinated with 5mgs of VlJns-tpa-IApol. (C) ELIspot responses were
also
measured from a monkey (920072) that did not receive any immunization.
Figure 7A-B show bulk CTL killing from rhesus macaques immunized with
colon optimized V lJns-IApol (A)or colon optimized V lJns-tpa-IApol (B) at 8
weeks
following the third vaccination. Restimulation was performed using recombinant
vaccinia virus expressing pol and target cells were prepared by pulsing with
the
peptide pools, mpol-1 and mpol-2.
Figure 8 shows detection of irz vitro pol expression from cell lysates of 293
cells transfected with 10 ug of various pol constructs. Bands were detected
using anti-
serum from an HIV-1 seropositive human subject. Equal amounts of total protein
were loaded for each lane. The lanes contain the lysates from cells
transfected with
the following: 1: mock; 2: VlJns-wt-pol; 3: VlJns-IApol (colon optimized);
4: VlJns-tpa-IApol (colon optimized); 5: VlJns-tpa-pol (colon optimized); 6:
V1R-
wt-pol (colon optimized); 7: blank; and 8: 80 ng RT.
Figure 9 shows the geometric mean anti-RT titers (GMT) plus the standard
errors of the geometric means for cohorts of 5 mice that received one (open
circles) or
two doses (solid circles) of l, 10, 100 ~,g of V1R-wt-pol (colon optimized) or
VlJns-
wt-pol. Sera from all animals were collected at 2 weeks post dose 2 (or 7 wks
post
dose 1) and assayed simultaneously. Statistical analyses were performed to
compare
cohorts that received the same amount and number of immunization of either
plasmids; p values (two-tail) less than 5% are above the bars the connect the
correlated cohorts to reflect statistically significant differences.
Figure 10 shows cellular immune responses in BALB/c mice vaccinated i.m.
with 1 (pdl) or 2 (pd2) doses of varying amounts of either wt-pol (virus
derived) or
wt-pol (colon optimized) plasmids. At 3 wks post dose 2, frequencies of IFN-~y-
secreting splenocytes are determined from pools of 5 spleens per cohort
against
mixtures of either CD4+ peptides (aa21-40, aa411-430, aa531-550, aa641-660,
aa731-
750, aa771-790) or CD8+ peptides (aa201-220, aa311-330) at 4 p,g/mL final
concentration per peptide.
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DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to synthetic DNA molecules and associated
DNA vaccines which elicit CTL and Th cellular immune responses upon
administration to the host, including primates and especially humans. An
effect of the
cellular immune-directed vaccines of the present invention should be a lower
transmission rate to previously uninfected individuals and/or reduction in the
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 Pol, wherein administration, intracellular
delivery and expression of the HIV-1 Pol gene of interest elicits a host CTL
and Th
response. The preferred synthetic DNA molecules of the present invention
encode
codon optimized wild type Pol (without Pro activity) and various codon
optimized
inactivated HIV-1 Pol proteins. The HIV-1 pol constructs disclosed herein are
especially preferred for pharmaceutical uses, especially for human
administration as a
DNA vaccine. The HIV-1 genome employs predominantly uncommon codons
compared to highly expressed human genes. Therefore, the pol 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 pol open reading frame, whether encoding full
length pol or a modification or fusion as described herein, wherein the codon
usage
has been optimized for expression in a mammal, especially a human.
The synthetic pol gene disclosed herein comprises the coding sequences for
the reverse transcriptase (or RT which consists of a polymerase and RNase H
activity)
and integrase (IN). The protein sequence is based on that of Hxb2r, a clonal
isolate of
IIIB; this sequence has been shown to be closest to the consensus Glade B
sequence
with only 16 nonidentical residues out of 848 (Korber, et al., 1998, Human
retroviruses and AIDS, Los Alamos National Laboratory, Los Alamos, New
Mexico).
The skilled artisan will understand after review of this specification that
any available
HIV-1 or HIV-2 strain provides a potential template for the generation of HIV
pol
DNA vaccine constructs disclosed herein. It is further noted that the protease
gene is
excluded from the DNA vaccine constructs of the present invention to insure
safety
from any residual protease activity in spite of mutational inactivation. The
design of
the gene sequences for both wild-type (wt-pol) and inactivated pol (IA-pol)
incorporates the use of human preferred ("humanized") codons for each amino
acid
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residue in the sequence in order to maximize ifa vivo mammalian expression
(Lathe,
1985, J. Mol. Biol. 183:1-12). As can be discerned by inspecting the colon
usage in
SEQ ID NOs: 1, 3, 5 and 7, the following colon 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) colon optimization, see
WO 97/31115 (PCT/US97102294), which is hereby incorporated by reference. It is
intended that the skilled artisan may use alternative versions of colon
optimization or
may omit this step when generating HIV pol vaccine constructs within the scope
of
the present invention. Therefore, the present invention also relates to non-
colon
optimized versions of DNA molecules and associated DNA vaccines which encode
the various wild type and modified forms of the HIV Pol protein disclosed
herein.
However, colon optimization of these constructs is a preferred embodiment of
this
invention:
A particular embodiment of the present invention relates to colon optimized
wt-pol DNA constructs (herein, "wt-pol" or "wt-pol (colon optimized))" wherein
DNA sequences encoding the protease (PR) activity are deleted, leaving colon
optimized "wild type" sequences which encode RT (reverse transcriptase and
RNase
H activity) and IN integrase activity. A DNA molecule which encodes this
protein is
disclosed herein as SEQ ID NO:1, the open reading frame being contained from
an
initiating Met residue at nucleotides 10-12 to a termination colon from
nucleotides
2560-2562. SEQ m N0:1 is as follows:
AGATCTACCA TGGCCCCCAT CTCCCCCATT GAGACTGTGC CTGTGAAGCT GAAGCCTGGC
ATGGATGGCC CCAAGGTGAA GCAGTGGCCC CTGACTGAGG AGAAGATCAA GGCCCTGGTG
GAAATCTGCA CTGAGATGGA GAAGGAGGGC AAAATCTCCA AGATTGGCCC CGAGAACCCC
TACAACACCC CTGTGTTTGC CATCAAGAAG AAGGACTCCA CCAAGTGGAG GAAGCTGGTG
GACTTCAGGG AGCTGAACAA GAGGACCCAG GACTTCTGGG AGGTGCAGCT GGGCATCCCC
CACCCCGCTG GCCTGAAGAA GAAGAAGTCT GTGACTGTGC TGGATGTGGG GGATGCCTAC
3O TTCTCTGTGC CCCTGGATGA GGACTTCAGG AAGTACACTG CCTTCACCAT CCCCTCCATC
AACAATGAGA CCCCTGGCAT CAGGTACCAG TACAATGTGC TGCCCCAGGG CTGGAAGGGC
TCCCCTGCCA TCTTCCAGTC CTCCATGACC AAGATCCTGG AGCCCTTCAG GAAGCAGAAC
CCTGACATTG TGATCTACCA GTACATGGAT GACCTGTATG TGGGCTCTGA CCTGGAGATT
GGGCAGCACA GGACCAAGAT TGAGGAGCTG AGGCAGCACC TGCTGAGGTG GGGCCTGACC
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ACCCCTGACAAGAAGCACCAGAAGGAGCCCCCCTTCCTGTGGATGGGCTATGAGCTGCAC
CCCGACAAGTGGACTGTGCAGCCCATTGTGCTGCCTGAGAAGGACTCCTGGACTGTGAAT
GACATCCAGAAGCTGGTGGGCAAGCTGAACTGGGCCTCCCAAATCTACCCTGGCATCAAG
GTGAGGCAGCTGTGCAAGCTGCTGAGGGGCACCAAGGCCCTGACTGAGGTGATCCCCCTG
S ACTGAGGAGGCTGAGCTGGAGCTGGCTGAGAACAGGGAGATCCTGAAGGAGCCTGTGCAT
GGGGTGTACTATGACCCCTCCAAGGACCTGATTGCTGAGATCCAGAAGCAGGGCCAGGGC
CAGTGGACCTACCAAATCTACCAGGAGCCCTTCAAGAACCTGAAGACTGGCAAGTATGCC
AGGATGAGGGGGGCCCACACCAATGATGTGAAGCAGCTGACTGAGGCTGTGCAGAAGATC
ACCACTGAGTCCATTGTGATCTGGGGCAAGACCCCCAAGTTCAAGCTGCCCATCCAGAAG
IO GAGACCTGGGAGACCTGGTGGACTGAGTACTGGCAGGCCACCTGGATCCCTGAGTGGGAG
TTTGTGAACACCCCCCCCCTGGTGAAGCTGTGGTACCAGCTGGAGAAGGAGCCCATTGTG
GGGGCTGAGACCTTCTATGTGGATGGGGCTGCCAACAGGGAGACCAAGCTGGGCAAGGCT.
GGCTATGTGACCAACAGGGGCAGGCAGAAGGTGGTGACCCTGACTGACACCACCAACCAG
AAGACTGAGCTCCAGGCCATCTACCTGGCCCTCCAGGACTCTGGCCTGGAGGTGAACATT
IS GTGACTGACTCCCAGTATGCCCTGGGCATCATCCAGGCCCAGCCTGATCAGTCTGAGTCT
GAGCTGGTGAACCAGATCATTGAGCAGCTGATCAAGAAGGAGAAGGTGTACCTGGCCTGG
GTGCCTGCCCACAAGGGCATTGGGGGCAATGAGCAGGTGGACAAGCTGGTGTCTGCTGGC
ATCAGGAAGGTGCTGTTCCTGGATGGCATTGACAAGGCCCAGGATGAGCATGAGAAGTAC
CACTCCAACTGGAGGGCTATGGCCTCTGACTTCAACCTGCCCCCTGTGGTGGCTAAGGAG
ATTGTGGCCTCCTGTGACAAGTGCCAGCTGAAGGGGGAGGCCATGCATGGGCAGGTGGAC
TGCTCCCCTGGCATCTGGCAGCTGGACTGCACCCACCTGGAGGGCAAGGTGATCCTGGTG
GCTGTGCATGTGGCCTCCGGCTACATTGAGGCTGAGGTGATCCCTGCTGAGACAGGCCAG
GAGACTGCCTACTTCCTGCTGAAGCTGGCTGGCAGGTGGCCTGTGAAGACCATCCACACT
GACAATGGCTCCAACTTCACTGGGGCCACAGTGAGGGCTGCCTGCTGGTGGGCTGGCATC
ZS AAGCAGGAGTTTGGCATCCCCTACAACCCCCAGTCCCAGGGGGTGGTGGAGTCCATGAAC
AAGGAGCTGAAGAAGATCATTGGGCAGGTGAGGGACCAGGCTGAGCACCTGAAGACAGCT
GTGCAGATGGCTGTGTTCATCCACAACTTCAAGAGGAAGGGGGGCATCGGGGGCTACTCC
GCTGGGGAGAGGATTGTGGACATCATTGCCACAGACATCCAGACCAAGGAGCTCCAGAAG
CAGATCACCAAGATCCAGAACTTCAGGGTGTACTACAGGGACTCCAGGAACCCCCTGTGG
3O AAGGGCCCTGCCAAGCTGCTGTGGAAGGGGGAGGGGGCTGTGGTGATCCAGGACAACTCT
GACATCAAGGTGGTGCCCAGGAGGAAGGCCAAGATCATCAGGGACTATGGCAAGCAGATG
GCTGGGGATGACTGTGTGGCCTCCAGGCAGGATGAGGACTAAAGCCCGGGCAGATCT
{SEQ
ID N0:1).
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The
open
reading
frame
of
the
wild
type
pol
construct
disclosed
as
SEQ
)D
NO: l amino ds, as
contains aci disclosed SEQ
8$0 herein ID
N0:2,
as
follows:
Met Ala ProIle SerProIle GluThrVal ProValLys LeuLysPro
Gly Met AspGly ProLysVal LysGlnTrp ProLeuThr GluGluLys
$ Ile Lys AlaLeu_ValGluIle CysThrGlu MetGluLys GluGlyLys
Ile Ser LysIle GlyProGlu AsnProTyr AsnThrPro ValPheAla
Ile Lys LysLys AspSerThr LysTrpArg LysLeuVal AspPheArg
Glu Leu AsnLys ArgThrGln AspPheTrp GluValGln LeuGlyIle
Pro His ProAla GlyLeuLys LysLysLys SerValThr ValLeuAsp
Val Gly AspAla TyrPheSer ValProLeu AspGluAsp PheArgLys
Tyr Thr AlaPhe ThrIlePro SerIleAsn AsnGluThr ProGlyIle
Arg Tyr GlnTyr AsnValLeu ProGlnGly TrpLysGly SerProAla
Ile Phe GlnSer SerMetThr LysIleLeu GluProPhe ArgLysGln
Asn Pro AspIle ValIleTyr GlnTyrMet AspAspLeu TyrValGly
1$ Ser Asp LeuGlu IleG1yGln HisArgThr LysIleGlu GluLeuArg
Gln His LeuLeu ArgTrpGly LeuThrThr ProAspLys LysHisGln
Lys G1u ProPro PheLeuTrp MetGlyTyr GluLeuHis ProAspLys
Trp Thr ValGln ProIleVal LeuProGlu LysAspSer TrpThrVal
Asn Asp IleGln LysLeuVal GlyLysLeu AsnTrpAla SerGlnIle
Tyr Pro GlyIle LysValArg GlnLeuCys LysLeuLeu ArgGlyThr
Lys Ala LeuThr GluValIle ProLeuThr GluGluAla GluLeuGlu
Leu Ala GluAsn ArgGluIle LeuLysGlu ProValHis GlyValTyr
Tyr Asp ProSer LysAspLeu IleAlaGlu IleGlnLys GlnGlyGln
Gly Gln TrpThr TyrGlnIle TyrGlnGlu ProPheLys AsnLeuLys
2$ Thr Gly LysTyr AlaArgMet ArgGlyAla HisThrAsn AspValLys
Gln Leu ThrGlu AlaValGln LysIleThr ThrGluSer IleValIle
Trp Gly LysThr ProLysPhe LysLeuPro IleGlnLys GluThrTrp
Glu Thr TrpTrp ThrGluTyr TrpGlnAla ThrTrpIle ProGluTrp
Glu Phe ValAsn ThrProPro LeuValLys LeuTrpTyr GlnLeuGlu
Lys Glu ProIle ValGlyAla GluThrPhe TyrValAsp GlyAlaAla
Asn Arg GluThr LysLeuGly LysAlaGly TyrValThr AsnArgGly
Arg Gln LysVal ValThrLeu ThrAspThr ThrAsnGln LysThrGlu
Leu Gln AlaIle TyrLeuAla LeuGlnAsp SerGlyLeu GluValAsn
Ile Val ThrAsp SerGlnTyr A1aLeuGly IleIleGln AlaGlnPro
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Asp G1n Ser Glu Ser Glu Leu Val Asn Gln Ile Ile Glu Gln Leu Ile
Lys Lys Glu Lys Val Tyr Leu Ala Trp Val Pro Ala His Lys Gly Ile
Gly Gly Asn Glu Gln Val Asp Lys Leu Val Ser Ala Gly Ile Arg Lys
Val Leu Phe Leu Asp Gly Ile Asp Lys Ala Gln Asp Glu His Glu Lys
Tyr His Ser Asn Trp Arg Ala Met Ala Ser Asp Phe Asn Leu Pro Pro
Val Val Ala Lys Glu Ile Val Ala Ser Cys Asp Lys Cys Gln Leu Lys
Gly Glu Ala Met His Gly Gln Va1 Asp Cys Ser Pro Gly Ile Trp Gln
Leu Asp Cys Thr His Leu Glu Gly Lys Val Ile Leu Val Ala Val His
Val A1a Ser Gly Tyr Ile Glu Ala Glu Val Ile Pro Ala Glu Thr Gly
Gln Glu Thr Ala Tyr Phe Leu Leu Lys Leu Ala Gly Arg Trp Pro Val
Lys Thr Ile His Thr Asp Asn Gly Ser Asn Phe Thr Gly A1a Thr Val
Arg Ala Ala Cys Trp Trp Ala Gly Ile Lys Gln Glu Phe Gly Ile Pro
Tyr Asn Pro Gln Ser G1n Gly Val Val Glu Ser Met Asn Lys Glu Leu
Lys Lys Ile Ile Gly Gln Val Arg Asp Gln Ala Glu His Leu Lys Thr
Ala Val Gln Met Ala Val Phe Ile His Asn Phe Lys Arg Lys Gly Gly
Ile Gly Gly Tyr Ser Ala Gly Glu Arg Ile Val Asp Ile Ile A1a Thr
Asp Ile Gln Thr Lys Glu Leu G1n Lys Gln Ile Thr Lys Ile Gln Asn
Phe Arg Val Tyr Tyr Arg Asp Ser Arg Asn Pro Leu Trp Lys Gly Pro
Ala Lys Leu Leu Trp Lys Gly Glu Gly Ala Val Val Ile Gln Asp Asn
Ser Asp Ile Lys Val Val Pro Arg Arg Lys Ala Lys Ile Ile Arg Asp
Tyr Gly Lys Gln Met Ala Gly Asp Asp Cys Val Ala Ser Arg Gln Asp
Glu Asp (SEQ ID N0:2).
The present invention especially relates to a codon optimized HIV-1 DNA pol
construct wherein, in addition to deletion of the portion of the wild type
sequence
encoding the protease activity, a combination of active site residue mutations
are
introduced which are deleterious to HIV-1 pol (RT-RH-IN) activity of the
expressed
protein. Therefore, the present invention preferably relates to a HIV-1 DNA
pol
construct which is devoid of DNA sequences encoding any PR activity, as well
as
containing a mutations) which at least partially, and preferably
substantially,
abolishes RT, RNase andlor IN activity. One type of HIV-1 pol mutant may
include
but is not limited to a mutated DNA molecule comprising at least one
nucleotide
substitution which results in a point mutation which effectively alters an
active site
within the RT, RNase andlor IN regions of the expressed protein, resulting in
at least
substantially decreased enzymatic activity for the RT, RNase H and/or IN
functions of
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HIV-1 Pol. In a preferred embodiment of this portion of the invention, a HIV-1
DNA
pol construct contains a mutation or mutations within the Pol coding region
which
effectively abolishes RT, RNase H and IN activity. An especially preferable
HIV-1
DNA pol construct in a DNA molecule which contains at least one point mutation
which alters the active site of the RT, RNase H and IN domains of Pol, such
that each
activity is at least substantially abolished. Such a HIV-1 Pol mutant will
most likely
comprise at least one point mutation in or around each catalytic domain
responsible
for RT, RNase H and IN activity, respectfully. To this end, an especially
preferred
HIV-1 DNA pol construct is exemplified herein and contains nine codon
substitution
mutations which results in an inactivated Pol protein (IA Pol: SEQ ID N0:4,
Figure
2A-C) which has no PR, RT, RNase or IN activity, wherein three such point
mutations reside within each of the RT, RNase and IN catalytic domains.
Therefore,
an especially preferred exemplification is a DNA molecule which encodes IA-
pol,
which contains all nine mutations as shown below in Table 1. An additional
preferred
amino acid residue for substitution is Asp551, localized within the RNase
domain of
Pol. Any combination of the mutations disclosed herein may suitable and
therefore
may be utilized as an IA-Pol-based vaccine of the present invention. While
addition
and deletion mutations are contemplated and within the scope of the invention,
the
preferred mutation is a point mutation resulting in a substitution of the wild
type
amino acid with an alternative amino acid residue.
Table 1
wt as as residue mutant as enzyme function
Asp 112 Ala RT
Asp 187 Ala RT
Asp 188 Ala RT
Asp 445 Ala RNase H
Glu 480 Ala RNase H
Asp 500 Ala RNase H
Asp 626 Ala IN
Asp 678 Ala IN
Glu 714 Ala IN
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It is preferred
that point
mutations
be incorporated
into the
IApol
mutant
vaccines
of
the present to lessen the possibility
invention of altering epitopes
so as in and around
the active Pol.
sites)
of HIV-1
To this which codes
end, SEQ for
ID N0:3
discloses
the nucleotide
sequence
S a codon s shown
optimized in Table
pol in 1, disclosed
addition as
to the
nine mutation
follows, referred
and to herein
as "IApol":
AGATCTACCATGGCCCCCATCTCCCCCATT GAGACTGTGCCTGTGAAGCTGAAGCCTGGC
ATGGATGGCCCCAAGGTGAAGCAGTGGCCC CTGACTGAGGAGAAGATCAAGGCCCTGGTG
GAAATCTGCACTGAGATGGAGAAGGAGGGC AAAATCTCCAAGATTGGCCCCGAGAACCCC
IO TACAACACCCCTGTGTTTGCCATCAAGAAG AAGGACTCCACCAAGTGGAGGAAGCTGGTG
GACTTCAGGGAGCTGAACAAGAGGACCCAG GACTTCTGGGAGGTGCAGCTGGGCATCCCC
CACCCCGCTGGCCTGAAGAAGAAGAAGTCT GTGACTGTGCTGGCTGTGGGGGATGCCTAC
TTCTCTGTGCCCCTGGATGAGGACTTCAGG AAGTACACTGCCTTCACCATCCCCTCCATC
AACAATGAGACCCCTGGCATCAGGTACCAG TACAATGTGCTGCCCCAGGGCTGGAAGGGC
IS TCCCCTGCCATCTTCCAGTCCTCCATGACC AAGATCCTGGAGCCCTTCAGGAAGCAGAAC
CCTGACATTGTGATCTACCAGTACATGGCT GCCCTGTATGTGGGCTCTGACCTGGAGATT
GGGCAGCACAGGACCAAGATTGAGGAGCTG AGGCAGCACCTGCTGAGGTGGGGCCTGACC
ACCCCTGACAAGAAGCACCAGAAGGAGCCC CCCTTCCTGTGGATGGGCTATGAGCTGCAC
CCCGACAAGTGGACTGTGCAGCCCATTGTG CTGCCTGAGAAGGACTCCTGGACTGTGAAT
GACATCCAGAAGCTGGTGGGCAAGCTGAAC TGGGCCTCCCAAATCTACCCTGGCATCAAG
GTGAGGCAGCTGTGCAAGCTGCTGAGGGGC ACCAAGGCCCTGACTGAGGTGATCCCCCTG
ACTGAGGAGGCTGAGCTGGAGCTGGCTGAG AACAGGGAGATCCTGAAGGAGCCTGTGCAT
GGGGTGTACTATGACCCCTCCAAGGACCTG ATTGCTGAGATCCAGAAGCAGGGCCAGGGC
CAGTGGACCTACCAAATCTACCAGGAGCCC TTCAAGAACCTGAAGACTGGCAAGTATGCC
~S AGGATGAGGGGGGCCCACACCAATGATGTG AAGCAGCTGACTGAGGCTGTGCAGAAGATC
ACCACTGAGTCCATTGTGATCTGGGGCAAG ACCCCCAAGTTCAAGCTGCCCATCCAGAAG
GAGACCTGGGAGACCTGGTGGACTGAGTAC TGGCAGGCCACCTGGATCCCTGAGTGGGAG
TTTGTGAACACCCCCCCCCTGGTGAAGCTG TGGTACCAGCTGGAGAAGGAGCCCATTGTG
GGGGCTGAGACCTTCTATGTGGCTGGGGCT GCCAACAGGGAGACCAAGCTGGGCAAGGCT
3O GGCTATGTGACCAACAGGGGCAGGCAGAAG GTGGTGACCCTGACTGACACCACCAACCAG
AAGACTGCCCTCCAGGCCATCTACCTGGCC CTCCAGGACTCTGGCCTGGAGGTGAACATT
GTGACTGCCTCCCAGTATGCCCTGGGCATC ATCCAGGCCCAGCCTGATCAGTCTGAGTCT
GAGCTGGTGAACCAGATCATTGAGCAGCTG ATCAAGAAGGAGAAGGTGTACCTGGCCTGG
GTGCCTGCCCACAAGGGCATTGGGGGCAAT GAGCAGGTGGACAAGCTGGTGTCTGCTGGC
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ATCAGGAAGGTGCTGTTCCTGGATGGCATTGACAAGGCCCAGGATGAGCATGAGAAGTAC
CACTCCAACTGGAGGGCTATGGCCTCTGACTTCAACCTGCCCCCTGTGGTGGCTAAGGAG
ATTGTGGCCTCCTGTGACAAGTGCCAGCTGAAGGGGGAGGCCATGCATGGGCAGGTGGAC
TGCTCCCCTGGCATCTGGCAGCTGGCCTGCACCCACCTGGAGGGCAAGGTGATCCTGGTG
GCTGTGCATGTGGCCTCCGGCTACATTGAGGCTGAGGTGATCCCTGCTGAGACAGGCCAG
GAGACTGCCTACTTCCTGCTGAAGCTGGCTGGCAGGTGGCCTGTGAAGACCATCCACACT
GCCAATGGCTCCAACTTCACTGGGGCCACAGTGAGGGCTGCCTGCTGGTGGGCTGGCATC
AAGCAGGAGTTTGGCATCCCCTACAACCCCCAGTCCCAGGGGGTGGTGGCCTCCATGAAC
AAGGAGCTGAAGAAGATCATTGGGCAGGTGAGGGACCAGGCTGAGCACCTGAAGACAGCT
1O GTGCAGATGGCTGTGTTCATCCACAACTTCAAGAGGAAGGGGGGCATCGGGGGCTACTCC
GCTGGGGAGAGGATTGTGGACATCATTGCCACAGACATCCAGACCAAGGAGCTCCAGAAG
CAGATCACCAAGATCCAGAACTTCAGGGTGTACTACAGGGACTCCAGGAACCCCCTGTGG
AAGGGCCCTGCCAAGCTGCTGTGGAAGGGGGAGGGGGCTGTGGTGATCCAGGACAACTCT
GACATCAAGGTGGTGCCCAGGAGGAAGGCCAAGATCATCAGGGACTATGGCAAGCAGATG
IS GCTGGGGATGACTGTGTGGCCTCCAGGCAGGATGAGGACTAAAGCCCGGGCAGATCT (SEQ
ID
N0:3).
In order to produce the IA-pol DNA vaccine construction, inactivation of the
enzymatic functions was achieved by replacing a total of nine active-site
residues
from the enzyme subunits with alanine side-chains. As shown in Table 1, all
residues
20 that comprise the catalytic triad of the polymerase, namely Asp112, Asp187,
and
Asp188, were substituted with alanine (Ala) residues (Larder, et al., Nature
1987,
327: 716-717; Larder, et al., 1989, Proc. Natl. Acad. Sci. 1989, 86: 4803-
4807).
Three additional mutations were introduced at Asp445, G1u480 and Asp500 to
abolish
RNase H activity (Asp551 was left unchanged in this IA Pol construct), with
each
25 residue being substituted for an Ala residue, respectively (Davies, et al.,
1991,
Science 252:, 88-95; Schatz, et al., 1989, FEBS Lett. 257: 311-314; Mizrahi,
et al.,
1990, Nucl. Acids. Res. 18: pp. 5359-5353). HIV pol integrase function was
abolished through three mutations at Asp626, Asp678 and G1u714. Again, each of
these residues has been substituted with an Ala residue (Wiskerchen, et al.,
1995, J.
30 Virol. 69: 376-386; Leavitt, et al., 1993, J. Biol. Chem. 268: 2113-2119).
Amino
acid residue Pro3 of SEQ ll~ N0:4 marks the start of the RT gene. The complete
amino acid sequence of IA-Pol is disclosed herein as SEQ ID N0:4, as follows:
Met Ala Pro Ile Ser Pro Ile Glu Thr Val Pro Val Lys Leu Lys Pro
Gly Met Asp Gly Pro Lys Val Lys Gln Trp Pro Leu Thr Glu Glu Lys
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Ile Lys AlaLeuVal GluIleCys ThrGluMet GluLys GluGlyLys
Ile Ser LysIleGly ProGluAsn ProTyrAsn ThrPro Va1PheAla
Ile Lys LysLysAsp SerThrLys TrpArgLys LeuVal AspPheArg
Glu Leu AsnLysArg ThrGlnAsp PheTrpGlu ValGln LeuGlyIle
Pro His ProAlaGly LeuLysLys LysLysSer ValThr ValLeuAla
Val Gly AspAlaTyr PheSerVal ProLeuAsp GluAsp PheArgLys
Tyr Thr AlaPheThr IleProSer IleAsnAsn GluThr ProGlyIle
Arg Tyr GlnTyrAsn ValLeuPro GlnGlyTrp LysGly SerProAla
Ile Phe GlnSerSer MetThrLys TleLeuGlu ProPhe ArgLysGln
Asn Pro AspIleVal IleTyrGln TyrMetAla AlaLeu TyrValGly
Ser Asp LeuGluIle GlyGlnHis ArgThrLys IleGlu GluLeuArg
Gln His LeuLeuArg TrpGlyLeu ThrThrPro AspLys LysHisGln
Lys Glu ProProPhe LeuTrpMet GlyTyrGlu LeuHis ProAspLys
Trp Thr ValGlnPro IleValLeu ProGluLys AspSer TrpThrVal
Asn Asp IleGlnLys LeuValGly LysLeuAsn TrpAla SerGlnIle
Tyr Pro GlyIleLys ValArgGln LeuCysLys LeuLeu ArgGlyThr
Lys Ala LeuThrGlu ValIlePro LeuThrGlu GluAla GluLeuGlu
Leu Ala GluAsnArg GluIleLeu LysGluPro ValHis GlyValTyr
Tyr Asp ProSerLys AspLeuTle AlaGluIle GlnLys GlnGlyGln
Gly Gln TrpThrTyr GlnIleTyr GlnGluPro PheLys AsnLeuLys
Thr Gly LysTyrAla ArgMetArg GlyAlaHis ThrAsn AspValLys
Gln Leu ThrGluAla ValGlnLys IleThrThr GluSer IleValIle
Trp Gly LysThrPro LysPheLys LeuProIle .GlnLys GluThrTrp
Glu Thr TrpTrpThr GluTyrTrp GlnA1aThr TrpIle ProGluTrp
G1u Phe Va1AsnThr ProProLeu ValLysLeu TrpTyr GlnLeuGlu
Lys Glu ProIleVal GlyAlaGlu ThrPheTyr ValAla GlyAlaAla
Asn Arg GluThrLys LeuGlyLys AlaGlyTyr ValThr AsnArgGly
Arg Gln LysValVal ThrLeuThr AspThrThr AsnGln LysThrAla
Leu G1n AlaIleTyr LeuAlaLeu GlnAspSer GlyLeu GluValAsn
Ile Val ThrAlaSer GlnTyrAla LeuGlyIle IleGln AlaGlnPro
Asp Gln SerGluSer GluLeuVal AsnGlnIle IleGlu GlnLeuIle
Lys Lys GluLysVal TyrLeuAla TrpValPro AlaHis LysGlyIle
Gly Gly AsnGluGln ValAspLys LeuValSer AlaGly I1eArgLys
Val Leu PheLeuAsp GlyIleAsp LysAlaGln AspGlu HisGluLys
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Tyr His SerAsnTrp ArgAlaMet AlaSerAsp PheAsn LeuProPro
Val Val AlaLysGlu IleValAla SerCysAsp LysCys GlnLeuLys
Gly Glu AlaMetHis GlyGlnVal AspCysSer ProGly IleTrpGln
Leu Ala CysThrHis LeuGluGly LysValIle LeuVal AlaValHis
Val Ala SerGlyTyr IleGluAla GluValIle ProAla GluThrGly
Gln Glu ThrAlaTyr PheLeuLeu LysLeuAla GlyArg TrpProVal
Lys Thr IleHisThr AlaAsnGly SerAsnPhe ThrGly AlaThrVal
Arg Ala AlaCysTrp TrpAlaGly IleLysGln GluPhe GlyIlePro
Tyr Asn ProGlnSer GlnGlyVal ValA1aSer MetAsn LysGluLeu
Lys Lys IleIleGly GlnValArg AspGlnAla GluHis LeuLysThr
Ala Val GlnMetAla ValPheIle HisAsnPhe LysArg LysGlyGly
Ile Gly GlyTyrSer AlaGlyGlu ArgIleVal AspIle IleAlaThr
Asp Ile GlnThrLys GluLeuGln LysGlnIle ThrLys IleGlnAsn
Phe Arg ValTyrTyr ArgAspSer ArgAsnPro LeuTrp LysG1yPro
Ala Lys LeuLeuTrp LysGlyGlu GlyAlaVal ValIle GlnAspAsn
Ser Asp IleLysVal ValProArg ArgLysAla LysIle I1eArgAsp
Tyr Gly LysG1nMet AlaGlyAsp AspCysVal AlaSer ArgGlnAsp
G1u Asp (SEQ ID N0:4).
As noted above, it will be understood that any combination of the mutations
disclosed above may be suitable and therefore be utilized as an IA-pol-based
vaccine
of the present invention. For example, it may be possible to mutate only 2 of
the 3
residues within the respective reverse transcriptase, RNase H, and integrase
coding
regions while still abolishing these enzymatic activities. However, the IA-pol
construct described above and disclosed as SEQ >D N0:3, as well as the
expressed
protein (SEQ JD N0:4) is preferred. It is also preferred that at least one
mutation be
present in each of the three catalytic domains.
Another aspect of the present invention is to generate codon optimized HIV-1
Pol-based vaccine constructions which comprise a eukaryotic trafficking signal
peptide such as from tPA (tissue-type plasminogen activator) or by a leader
peptide
such as is found in highly expressed mammalian proteins such as immunoglobulin
leader peptides. Any functional leader peptide may be tested for efficacy.
However,
a preferred embodiment of the present invention is to provide for HIV-1 Pol
mutant
vaccine constructions as disclosed herein which also comprise a leader
peptide,
preferably a leader peptide from human tPA. In other words, a codon optimized
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HIV-1 Pol mutant such as IA-Pol (SEQ ID N0:4) may also comprise a leader
peptide
at the amino terminal portion of the protein, which may effect cellular
trafficking and
hence, immunogenicity of the expressed protein within the host cell. 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
cloning of the modified HIV-1 protein of interest results in a nucleotide
sequence
which encodes a modified HIV-1 tPA/Pol 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 Pol 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 Pol protein of interest,
including but not limited to a HIV-1 PoI protein which contains a leader
peptide. The
amino acid sequence of the human tPA leader utilized herein is as follows:
MDAMKRGLCCVLLLCGAVFVSPSEISS (SEQ ID N0:28). Therefore, another
aspect of the present invention is to generate HIV-1 Pol-based vaccine
constructions
which comprise a eukaryotic trafficking signal peptide such as from tPA. To
this end,
the present invention relates to a DNA molecule which encodes a codon
optimized
wt-pol DNA construct wherein the protease (PR) activity is deleted and a human
tPA
leader sequence is fused to the 5' end of the coding region. A DNA molecule
which
encodes this protein is disclosed herein as SEQ ID N0:5, the open reading
frame
disclosed herein as SEQ ID N0:6.
To this end, the present invention relates to a DNA molecule which encodes a
codon optimized wt-pol DNA construct wherein the protease (PR) activity is
deleted
and a human tPA leader sequence is fused to the 5' end of the coding region (
herein,
"tPA-wt-pol"). A DNA molecule which encodes this protein is disclosed herein
as
SEQ ID N0:5, the open reading frame being contained from an initiating Met
residue
at nucleotides 8-10 to a termination codon from nucleotides 2633-2635. SEQ ID
N0:5 is as follows:
GATCACCATG GATGCAATGA AGAGAGGGCT CTGCTGTGTG CTGCTGCTGT GTGGAGCAGT
CTTCGTTTCG CCCAGCGAGA TCTCCGCCCC CATCTCCCCC ATTGAGACTG TGCCTGTGAA
GCTGAAGCCT GGCATGGATG GCCCCAAGGT GAAGCAGTGG CCCCTGACTG AGGAGAAGAT
CAAGGCCCTG GTGGAAATCT GCACTGAGAT GGAGAAGGAG GGCAAAATCT CCAAGATTGG
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CCCCGAGAAC CCCTACAACA CCCCTGTGTT TGCCATCAAG AAGAAGGACT CCACCAAGTG
GAGGAAGCTG GTGGACTTCA GGGAGCTGAA CAAGAGGACC CAGGACTTCT GGGAGGTGCA
GCTGGGCATC CCCCACCCCG CTGGCCTGAA GAAGAAGAAG TCTGTGACTG TGCTGGATGT
GGGGGATGCC TACTTCTCTG TGCCCCTGGA TGAGGACTTC AGGAAGTACA CTGCCTTCAC
S CATCCCCTCC ATCAACAATG AGACCCCTGG CATCAGGTAC CAGTACAATG TGCTGCCCCA
GGGCTGGAAG GGCTCCCCTG CCATCTTCCA GTCCTCCATG ACCAAGATCC TGGAGCCCTT
CAGGAAGCAG AACCCTGACA TTGTGATCTA CCAGTACATG GATGACCTGT ATGTGGGCTC
TGACCTGGAG ATTGGGCAGC ACAGGACCAA GATTGAGGAG CTGAGGCAGC ACCTGCTGAG
GTGGGGCCTG ACCACCCCTG ACAAGAAGCA CCAGAAGGAG CCCCCCTTCC TGTGGATGGG
ZO CTATGAGCTG CACCCCGACA AGTGGACTGT GCAGCCCATT GTGCTGCCTG AGAAGGACTC
CTGGACTGTG AATGACATCC AGAAGCTGGT GGGCAAGCTG AACTGGGCCT CCCAAATCTA
CCCTGGCATC AAGGTGAGGC AGCTGTGCAA GCTGCTGAGG GGCACCAAGG CCCTGACTGA
GGTGATCCCC CTGACTGAGG AGGCTGAGCT GGAGCTGGCT GAGAACAGGG AGATCCTGAA
GGAGCCTGTG CATGGGGTGT ACTATGACCC CTCCAAGGAC CTGATTGCTG AGATCCAGAA
IS GCAGGGCCAG GGCCAGTGGA CCTACCAAAT CTACCAGGAG CCCTTCAAGA ACCTGAAGAC
TGGCAAGTAT GCCAGGATGA GGGGGGCCCA CACCAATGAT GTGAAGCAGC TGACTGAGGC
TGTGCAGAAG ATCACCACTG AGTCCATTGT GATCTGGGGC AAGACCCCCA AGTTCAAGCT
GCCCATCCAG AAGGAGACCT GGGAGACCTG GTGGACTGAG TACTGGCAGG CCACCTGGAT
CCCTGAGTGG GAGTTTGTGA ACACCCCCCC CCTGGTGAAG CTGTGGTACC AGCTGGAGAA
GGAGCCCATT GTGGGGGCTG AGACCTTCTA TGTGGATGGG GCTGCCAACA.'GGGAGACCAA
GCTGGGCAAG GCTGGCTATG TGACCAACAG GGGCAGGCAG AAGGTGGTGA CCCTGACTGA
CACCACCAAC CAGAAGACTG AGCTCCAGGC CATCTACCTG GCCCTCCAGG ACTCTGGCCT
GGAGGTGAAC ATTGTGACTG ACTCCCAGTA TGCCCTGGGC ATCATCCAGG CCCAGCCTGA
TCAGTCTGAG TCTGAGCTGG TGAACCAGAT CATTGAGCAG CTGATCAAGA AGGAGAAGGT
2S GTACCTGGCC TGGGTGCCTG CCCACAAGGG CATTGGGGGC AATGAGCAGG TGGACAAGCT
GGTGTCTGCT GGCATCAGGA AGGTGCTGTT CCTGGATGGC ATTGACAAGG CCCAGGATGA
GCATGAGAAG TACCACTCCA ACTGGAGGGC TATGGCCTCT GACTTCAACC TGCCCCCTGT
GGTGGCTAAG GAGATTGTGG CCTCCTGTGA CAAGTGCCAG CTGAAGGGGG AGGCCATGCA
TGGGCAGGTG GACTGCTCCC CTGGCATCTG GCAGCTGGAC TGCACCCACC TGGAGGGCAA
3O GGTGATCCTG GTGGCTGTGC ATGTGGCCTC CGGCTACATT GAGGCTGAGG TGATCCCTGC
TGAGACAGGC CAGGAGACTG CCTACTTCCT GCTGAAGCTG GCTGGCAGGT GGCCTGTGAA
GACCATCCAC ACTGACAATG GCTCCAACTT CACTGGGGCC ACAGTGAGGG CTGCCTGCTG
GTGGGCTGGC ATCAAGCAGG AGTTTGGCAT CCCCTACAAC CCCCAGTCCC AGGGGGTGGT
GGAGTCCATG AACAAGGAGC TGAAGAAGAT CATTGGGCAG GTGAGGGACC AGGCTGAGCA
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CCTGAAGACA TTCAAGAGGA
GCTGTGCAGA AGGGGGGCAT
TGGCTGTGTT
CATCCACAAC
CGGGGGCTAC GCCACAGACA
TCCGCTGGGG TCCAGACCAA
AGAGGATTGT
GGACATCATT
GGAGCTCCAG GTGTACTACA GGGACTCCAG
AAGCAGATCA
CCAAGATCCA
GAACTTCAGG
GAACCCCCTG GGGGAGGGGG
TGGAAGGGCC CTGTGGTGAT
CTGCCAAGCT
GCTGTGGAAG
CCAGGACAAC GCCAAGATCA
TCTGACATCA TCAGGGACTA
AGGTGGTGCC
CAGGAGGAAG
TGGCAAGCAG CAGGATGAGG
ATGGCTGGGG ACTAAAGCCC
ATGACTGTGT
GGCCTCCAGG
GGGCAGATCT (SEQID
N0:5).
T he the ype o1 ct
open wild tPA-p constru disclosed
reading t as
frame SEQ
of
)D contains acids,
N0:5 875 disclosed
amino herein
as
SEQ
m
N0:6,
as
follows:
l~ Met Asp AlaMet LysArgGly LeuCysCys ValLeuLeu LeuCysGly
Ala Val PheVal SerProSer GluIleSer AlaProIle SerProIle
Glu Thr ValPro ValLysLeu LysProGly MetAspGly ProLysVal
Lys Gln TrpPro LeuThrGlu GluLysIle LysAlaLeu ValGluIle
Cys Thr GluMet GluLysGlu GlyLysIle SerLysIle GlyProGlu
Asn Pro TyrAsn ThrProVal PheAlaIle LysLysLys AspSerThr
Lys Trp ArgLys LeuValAsp PheArgGlu LeuAsnLys ArgThrGln
Asp Phe TrpGlu ValGlnLeu GlyIlePro HisProAla GlyLeuLys
Lys Lys LysSer ValThrVal LeuAspVal GlyAspAla TyrPheSer
Val Pro LeuAsp GluAspPhe ArgLysTyr ThrAlaPhe ThrIlePro
Ser Ile AsnAsn GluThrPro GlyIleArg TyrGlnTyr AsnValLeu
Pro Gln GlyTrp LysGlySer ProAlaIle PheGlnSer SerMetThr
Lys Ile LeuGlu ProPheArg LysGlnAsn ProAspIle ValIleTyr
Gln Tyr MetAsp AspLeuTyr ValGlySer AspLeuGlu IleGlyGln
His Arg ThrLys IleGluGlu LeuArgGln HisLeuLeu ArgTrpGly
~5 Leu Thr ThrPro AspLysLys HisGlnLys GluProPro PheLeuTrp
Met Gly TyrGlu LeuHisPro AspLysTrp ThrValGln ProTleVal
Leu Pro GluLys AspSerTrp ThrVa1Asn AspIleGln LysLeuVal
Gly Lys LeuAsn TrpAlaSer GlnIleTyr ProGlyIle LysValArg
Gln Leu CysLys LeuLeuArg GlyThrLys AlaLeuThr GluValIle
3~ Pro Leu ThrGlu GluAlaGlu LeuGluLeu AlaGluAsn ArgGluIle
Leu Lys GluPro ValHisGly ValTyrTyr AspProSer LysAspLeu
Ile Ala GluIle GlnLysGln GlyGlnGly GlnTrpThr TyrGlnIle
Tyr Gln GluPro PheLysAsn LeuLysThr GlyLysTyr AlaArgMet
Arg Gly AlaHis ThrAsnAsp ValLysGln LeuThrGlu AlaValGln
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Lys Ile Thr Thr Glu Ser Ile Val Ile Trp G1y Lys Thr Pro Lys Phe
Lys Leu Pro Ile Gln Lys Glu Thr Trp Glu Thr Trp Trp Thr Glu Tyr
Trp Gln Ala Thr Trp Ile Pro Glu Trp Glu Phe Val Asn Thr Pro Pro
Leu Val Lys Leu Trp Tyr Gln Leu Glu Lys Glu Pro Ile Val Gly Ala
Glu Thr Phe Tyr Val Asp Gly Ala Ala Asn Arg Glu Thr Lys Leu Gly
Lys Ala Gly Tyr Val Thr Asn Arg Gly Arg Gln Lys Val Val Thr Leu
Thr Asp Thr Thr Asn Gln Lys Thr Glu Leu Gln Ala Ile Tyr Leu Ala
Leu Gln Asp Ser Gly Leu Glu Val Asn Ile Val Thr Asp Ser Gln Tyr
Ala Leu Gly Ile I1e Gln A1a Gln Pro Asp Gln Ser Glu Ser Glu Leu
1~ Val Asn Gln Ile Ile Glu Gln Leu Ile Lys Lys Glu Lys Val Tyr Leu
A1a Trp Val Pro Ala His Lys Gly Ile Gly Gly Asn Glu G1n Val Asp
Lys Leu Val Ser Ala Gly Ile Arg Lys Val Leu Phe Leu Asp Gly Ile
Asp Lys Ala Gln Asp Glu His Glu Lys Tyr His Ser Asn Trp Arg Ala
Met Ala Ser Asp Phe Asn Leu Pro Pro Val Val Ala Lys Glu Ile Val
15 Ala Ser Cys Asp Lys Cys Gln Leu Lys Gly Glu Ala Met His Gly Gln
Val Asp Cys Ser Pro Gly Ile Trp Gln Leu Asp Cys Thr His Leu Glu
Gly Lys Val Ile Leu Val Ala Val His Val Ala Ser Gly Tyr Ile Glu
Ala Glu Val Ile Pro Ala Glu Thr Gly Gln Glu Thr Ala Tyr Phe Leu
Leu Lys Leu Ala Gly Arg Trp Pro Val Lys Thr Ile His Thr Asp Asn
Gly Ser Asn Phe Thr Gly Ala Thr Val Arg Ala A1a Cys Trp Trp Ala
Gly Ile Lys Gln Glu Phe Gly Ile Pro Tyr Asn Pro Gln Ser Gln Gly
Val Val Glu Ser Met Asn Lys Glu Leu Lys Lys Ile Ile Gly Gln Val
Arg Asp Gln Ala Glu His Leu Lys Thr Ala Val Gln Met Ala Val Phe
Ile His Asn Phe Lys Arg Lys Gly Gly Ile Gly Gly Tyr Ser Ala Gly
2~ Glu Arg Ile Val Asp Ile Ile Ala Thr Asp Ile Gln Thr Lys Glu Leu
Gln Lys Gln Ile Thr Lys Ile Gln Asn Phe Arg Val Tyr Tyr Arg Asp
Ser Arg Asn Pro Leu Trp Lys G1y Pro Ala Lys Leu Leu Trp Lys Gly
Glu Gly Ala Val Val Ile Gln Asp Asn Ser Asp Ile Lys Val Val Pro
Arg Arg Lys Ala Lys Ile Ile Arg Asp.Tyr Gly Lys Gln Met Ala Gly
30 Asp Asp Cys Val Ala Ser Arg Gln Asp Glu Asp (SEQ ID N0:6).
The present invention also relates to a codon optimized HIV-1 Pol mutant such
as IA-Pol (SEQ ID N0:4) which comprises a leader peptide at the amino terminal
portion of the protein, which may effect cellular trafficking and hence,
immunogenicity of the expressed protein within the host cell. Any such HIV-1
DNA
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pol mutant disclosed in the above paragraphs is suitable for fusion downstream
of a
leader peptide, such as a leader peptide including but not limited to the
human tPA
leader sequence. Therefore, any such leader peptide-based HIV-1 pol mutant
construct may include but is not limited to a mutated DNA molecule which
effectively
alters the catalytic activity of the RT, RNase and/or IN region of the
expressed protein,
resulting in at least substantially decreased enzymatic activity one or more
of the RT,
RNase H and/or IN functions of HIV-1 Pol. In a preferred embodiment of this
portion
of the invention, a leader peptide/HIV-1 DNA pol construct contains a mutation
or
mutations within the Pol coding region which effectively abolishes RT, RNase H
and
IN activity. An especially preferable HIV-1 DNA pol construct is a DNA
molecule
which contains at least one point mutation which alters the active site and
catalytic
activity within the RT, RNase H and IN domains of Pol, such that each activity
is at
least substantially abolished, and preferably totally abolished. Such a HIV-1
Pol
mutant will most likely comprise at least one point mutation in or around each
catalytic domain responsible for RT, RNase H and IN activity, respectfully. An
especially preferred embodiment of this portion of the invention relates to a
human ,
tPA leader fused to the IA-Pol protein comprising the nine mutations shown in
Table
1. The DNA molecule is disclosed herein as SEQ ID N0:7 and the expressed tPA-
IA
Pol protein comprises a fusion junction as shown in Figure 3. The complete
amino
acid sequence of the expressed protein is set forth in SEQ ID N0:8. To this
end; SEQ
ID N0:7 discloses the nucleotide sequence which codes for a human tPA leader
fused
to the IA Pol protein comprising the nine mutations shown in Table 1 (herein,
"tPA-
opt-IApol"). The open reading frame begins with the initiating Met
(nucleotides 8-10)
and terminates with a "TAA" codon at nucleotides 2633-2635. The nucleotide
sequence encoding tPA-IAPoI is also disclosed as follows:
GATCACCATG GATGCAATGA AGAGAGGGCT CTGCTGTGTG CTGCTGCTGT GTGGAGCAGT
CTTCGTTTCG CCCAGCGAGA TCTCCGCCCC CATCTCCCCC ATTGAGACTG TGCCTGTGAA
GCTGAAGCCT GGCATGGATG GCCCCAAGGT GAAGCAGTGG CCCCTGACTG AGGAGAAGAT
CAAGGCCCTG GTGGAAATCT GCACTGAGAT GGAGAAGGAG GGCAAAATCT CCAAGATTGG
3O CCCCGAGAAC CCCTACAACA CCCCTGTGTT TGCCATCAAG AAGAAGGACT CCACCAAGTG
GAGGAAGCTG GTGGACTTCA GGGAGCTGAA CAAGAGGACC CAGGACTTCT GGGAGGTGCA
GCTGGGCATC CCCCACCCCG CTGGCCTGAA GAAGAAGAAG TCTGTGACTG TGCTGGCTGT
GGGGGATGCC TACTTCTCTG TGCCCCTGGA TGAGGACTTC AGGAAGTACA CTGCCTTCAC
CATCCCCTCC ATCAACAATG AGACCCCTGG CATCAGGTAC CAGTACAATG TGCTGCCCCA
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GGGCTGGAAG GGCTCCCCTG CCATCTTCCA GTCCTCCATG ACCAAGATCC TGGAGCCCTT
CAGGAAGCAG AACCCTGACA TTGTGATCTA CCAGTACATG GCTGCCCTGT ATGTGGGCTC
TGACCTGGAG ATTGGGCAGC ACAGGACCAA GATTGAGGAG CTGAGGCAGC ACCTGCTGAG
GTGGGGCCTG ACCACCCCTG ACAAGAAGCA CCAGAAGGAG CCCCCCTTCC TGTGGATGGG
S CTATGAGCTG CACCCCGACA AGTGGACTGT GCAGCCCATT GTGCTGCCTG AGAAGGACTC
CTGGACTGTG AATGACATCC AGAAGCTGGT GGGCAAGCTG AACTGGGCCT CCCAAATCTA
CCCTGGCATC AAGGTGAGGC AGCTGTGCAA GCTGCTGAGG GGCACCAAGG CCCTGACTGA
GGTGATCCCC CTGACTGAGG AGGCTGAGCT GGAGCTGGCT GAGAACAGGG AGATCCTGAA
GGAGCCTGTG CATGGGGTGT ACTATGACCC CTCCAAGGAC CTGATTGCTG AGATCCAGAA
IO GCAGGGCCAG GGCCAGTGGA CCTACCAAAT CTACCAGGAG CCCTTCAAGA ACCTGAAGAC
TGGCAAGTAT GCCAGGATGA GGGGGGCCCA CACCAATGAT GTGAAGCAGC TGACTGAGGC
TGTGCAGAAG ATCACCACTG AGTCCATTGT GATCTGGGGC AAGACCCCCA AGTTCAAGCT
GCCCATCCAG AAGGAGACCT GGGAGACCTG GTGGACTGAG TACTGGCAGG CCACCTGGAT
CCCTGAGTGG GAGTTTGTGA ACACCCCCCC CCTGGTGAAG CTGTGGTACC AGCTGGAGAA
IS GGAGCCCATT GTGGGGGCTG AGACCTTCTA TGTGGCTGGG GCTGCCAACA GGGAGACCAA
GCTGGGCAAG GCTGGCTATG TGACCAACAG GGGCAGGCAG AAGGTGGTGA CCCTGACTGA
CACCACCAAC CAGAAGACTG CCCTCCAGGC CATCTACCTG GCCCTCCAGG ACTCTGGCCT
GGAGGTGAAC ATTGTGACTG CCTCCCAGTA TGCCCTGGGC ATCATCCAGG CCCAGCCTGA
TCAGTCTGAG TCTGAGCTGG TGAACCAGAT CATTGAGCAG CTGATCAAGA AGGAGAAGGT
ZO GTACCTGGCC TGGGTGCCTG CCCACAAGGG CATTGGGGGC AATGAGCAGG'TGGACAAGCT
GGTGTCTGCT GGCATCAGGA AGGTGCTGTT CCTGGATGGC ATTGACAAGG CCCAGGATGA
GCATGAGAAG TACCACTCCA ACTGGAGGGC TATGGCCTCT GACTTCAACC TGCCCCCTGT
GGTGGCTAAG GAGATTGTGG CCTCCTGTGA CAAGTGCCAG CTGAAGGGGG AGGCCATGCA
TGGGCAGGTG GACTGCTCCC CTGGCATCTG GCAGCTGGCC TGCACCCACC TGGAGGGCAA
2S GGTGATCCTG GTGGCTGTGC ATGTGGCCTC CGGCTACATT GAGGCTGAGG TGATCCCTGC
TGAGACAGGC CAGGAGACTG CCTACTTCCT GCTGAAGCTG GCTGGCAGGT GGCCTGTGAA
GACCATCCAC ACTGCCAATG GCTCCAACTT CACTGGGGCC ACAGTGAGGG CTGCCTGCTG
GTGGGCTGGC ATCAAGCAGG AGTTTGGCAT CCCCTACAAC CCCCAGTCCC AGGGGGTGGT
GGCCTCCATG AACAAGGAGC TGAAGAAGAT CATTGGGCAG GTGAGGGACC AGGCTGAGCA
3O CCTGAAGACA GCTGTGCAGA TGGCTGTGTT CATCCACAAC TTCAAGAGGA AGGGGGGCAT
CGGGGGCTAC TCCGCTGGGG AGAGGATTGT GGACATCATT GCCACAGACA TCCAGACCAA
GGAGCTCCAG AAGCAGATCA CCAAGATCCA GAACTTCAGG GTGTACTACA GGGACTCCAG
GAACCCCCTG TGGAAGGGCC CTGCCAAGCT GCTGTGGAAG GGGGAGGGGG CTGTGGTGAT
CCAGGACAAC TCTGACATCA AGGTGGTGCC CAGGAGGAAG GCCAAGATCA TCAGGGACTA
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TGGCAAGCAG ATGGCTGGGG ATGACTGTGT GGCCTCCAGG CAGGATGAGG ACTAAAGCCC
GGGCAGATCT (SEQ ID N0:7).
The open reading frame of the tPA-IA-pol construct disclosed as SEQ ID
N0:7 contains 875 amino acids, disclosed herein as tPA-IA-Pol and SEQ ID N0:8,
as
follows:
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
Ala Val Phe Val Ser Pro Ser Glu Ile Ser Ala Pro Ile Ser Pro Ile
Glu Thr Val Pro Val Lys Leu Lys Pro Gly Met Asp Gly Pro Lys Val
Lys Gln Trp Pro Leu Thr Glu Glu Lys Ile Lys Ala Leu Val Glu Ile
1~ Cys Thr Glu Met Glu Lys Glu Gly Lys Ile Ser Lys Ile Gly Pro Glu
Asn Pro Tyr Asn Thr Pro Val Phe Ala Ile Lys Lys Lys Asp Ser Thr
Lys Trp Arg Lys Leu Val Asp Phe Arg Glu Leu Asn Lys Arg Thr Gln
Asp Phe Trp Glu Val Gln Leu Gly Ile Pro His Pro Ala Gly Leu Lys
Lys Lys Lys Ser Val Thr Val Leu Ala Val Gly Asp Ala Tyr Phe Ser
Val Pro Leu Asp Glu Asp Phe Arg Lys Tyr Thr Ala Phe Thr Ile Pro
Ser Ile Asn Asn Glu Thr Pro G1y I1e Arg Tyr Gln Tyr Asn Val Leu
Pro Gln Gly Trp Lys Gly Ser Pro Ala Ile Phe Gln Ser Ser Met Thr
Lys Ile Leu Glu Pro Phe Arg Lys Gln Asn Pro Asp Ile Val Ile Tyr
Gln Tyr Met Ala Ala Leu Tyr Val Gly Ser Asp Leu Glu Ile Gly Gln
His Arg Thr Lys Ile Glu Glu Leu Arg Gln His Leu Leu Arg Trp Gly
Leu Thr Thr Pro Asp Lys Lys His Gln Lys Glu Pro Pro Phe Leu Trp
Met Gly Tyr Glu Leu His Pro Asp Lys Trp Thr Val Gln Pro Ile'Val
Leu Pro Glu Lys Asp Ser Trp Thr Val Asn Asp Ile Gln Lys Leu Val
Gly Lys Leu Asn Trp Ala Ser Gln Ile Tyr Pro Gly Ile Lys Val Arg
Gln Leu Cys Lys Leu Leu Arg Gly Thr Lys Ala Leu Thr Glu Val Ile
Pro Leu Thr Glu Glu Ala Glu Leu Glu Leu Ala Glu Asn Arg Glu Ile
Leu Lys Glu Pro Val His Gly Val Tyr Tyr Asp Pro Ser Lys Asp Leu
Ile Ala Glu Ile Gln Lys Gln Gly Gln Gly Gln Trp Thr Tyr Gln Ile
Tyr Gln Glu Pro Phe Lys Asn Leu Lys Thr Gly Lys Tyr Ala Arg Met
Arg Gly Ala His Thr Asn Asp Val Lys Gln Leu Thr Glu Ala Val Gln
Lys Ile Thr Thr Glu Ser Ile Val Ile Trp Gly Lys Thr Pro Lys Phe
Lys Leu Pro Ile Gln Lys Glu Thr Trp Glu Thr Trp Trp Thr Glu Tyr
Trp G1n Ala Thr Trp Ile Pro Glu Trp Glu Phe Val Asn Thr Pro Pro
Leu Val Lys Leu Trp Tyr Gln Leu Glu Lys Glu Pro Ile Val Gly Ala
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Glu Thr Phe Tyr Val Ala Gly Ala Ala Asn Arg Glu Thr Lys Leu Gly
Lys Ala Gly Tyr Val Thr Asn Arg Gly Arg Gln Lys Val Val Thr Leu
Thr Asp Thr Thr Asn Gln Lys Thr Ala Leu Gln Ala Ile Tyr Leu Ala
Leu Gln Asp Ser Gly Leu Glu Val Asn Ile Val Thr Ala Ser Gln Tyr
Ala Leu Gly Ile Ile Gln Ala Gln Pro Asp Gln Ser Glu Ser Glu Leu
Val Asn Gln Ile Ile Glu Gln Leu Ile Lys Lys Glu Lys Val Tyr Leu
Ala Trp Val Pro Ala His Lys Gly Ile Gly Gly Asn Glu Gln Val Asp
Lys Leu Val Ser Ala Gly Ile Arg Lys Val Leu Phe Leu Asp Gly Ile
Asp Lys Ala G1n Asp Glu His Glu Lys Tyr His Ser Asn Trp Arg Ala
Met Ala Ser Asp Phe Asn Leu Pro Pro Val Val Ala Lys Glu Ile Val
Ala Ser Cys Asp Lys Cys Gln Leu Lys Gly Glu Ala Met His Gly Gln
Val Asp Cys Ser Pro Gly I1e Trp Gln Leu Ala Cys Thr His Leu Glu
Gly Lys Val Ile Leu Val Ala Val His Val Ala Ser Gly Tyr Ile Glu
Ala Glu Val Ile Pro Ala Glu Thr Gly Gln Glu Thr Ala Tyr Phe Leu
1$ Leu Lys Leu Ala Gly Arg Trp Pro Val Lys Thr Ile His Thr Ala Asn
Gly Ser Asn Phe Thr Gly Ala Thr Val Arg Ala Ala Cys Trp Trp Ala
Gly Ile Lys Gln Glu Phe Gly Ile Pro Tyr Asn Pro Gln Ser Gln Gly
Val Val Ala Ser Met Asn Lys Glu Leu Lys Lys Ile Ile Gly Gln Val
Arg Asp Gln Ala Glu His Leu Lys Thr Ala Val Gln Met Ala Val Phe
Ile His Asn Phe Lys Arg Lys,Gly Gly I1e Gly Gly Tyr Ser Ala Gly
Glu Arg Ile Val Asp Ile Ile Ala Thr Asp Ile Gln Thr Lys Glu Leu
Gln Lys Gln Ile Thr Lys Ile Gln Asn Phe Arg Val Tyr Tyr Arg Asp
Ser Arg Asn Pro Leu Trp Lys Gly Pro Ala Lys Leu Leu Trp Lys Gly
Glu Gly Ala Val Val Ile Gln Asp Asn Ser Asp Ile Lys Val Val Pro
25 Arg Arg Lys Ala Lys Ile Ile Arg Asp Tyr Gly Lys Gln Met Ala Gly
Asp Asp Cys Val Ala Ser Arg Gln Asp Glu Asp (SEQ ID N0:8).
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 ll~ NOs: 2, 4, 6, and 8. These
purified
30 proteins may be useful as protein-based HIV vaccines.
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
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transcription, and a transcriptional terminator 3'to the HIV pol 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 pol 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
I5 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 which exemplify but in no way limit the present
invention are disclosed in PCT International Application No. PCT/US94/02751,
International Publication No. WO 94/21797, hereby incorporated by reference. .
A
first DNA expression vector 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 V 1 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 pol-based genes disclosed herein. The backbone of V1J is provided
by
pUClB. 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,
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blunt-ended with the T4 DNA polymerase, treated with calf intestinal alkaline
phosphatase, and ligated to the CMVintA/BGH element. In a preferred DNA
expression vector, the ampicillin resistance gene is removed from V1J and
replaced
with a neomycin resistance gene, to generate VlJneo. An especially preferred
DNA
expression vector is VlJns, which is the same as V1J except that a unique Sfi1
restriction site has been engineered into the single Kpn1 site at position
2114 of V1J-
neo. The incidence of Sfi1 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 Sfil digestion of extracted
genomic
DNA. Yet another preferred DNA expression vector used as the backbone to the
HIV-1 pol-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. The specific DNA vectors of the present
invention include but are not limited to V1, V1J (SEQ )D N0:13), VlJneo (SEQ ~
N0:14), VlJns (Figure 1A, SEQ ID N0:15), 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 ll~
N0:28.
The present invention especially relates to a DNA vaccine and a
pharmaceutically active vaccine composition which contains this DNA 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 Pol or biologically active Pol modifications or Pol-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 may comprise codon optimized DNA molecules encoding HIV-1
Pol or biologically active Pol modifications or Pol-containing fusion proteins
ligated
in DNA vectors V1, V1J (SEQ ID N0:14), VlJneo (SEQ ID N0:15), VlJns (Figure
1A, SEQ )D N0:16), V1R (SEQ ID N0:26), or any of the aforementioned vectors
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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 N0:28. To this end,
polynucleotide vaccine constructions include , VlJns-wtpol and V1R-wtpol
(comprising the DNA molecule encoding WT Pol, as set forth in SEQ ID N0:2),
VlJns-tPA-WTPoI, (comprising the DNA molecule encoding tPA Pol, as set forth
in
SEQ ID N0:6), VlJns-IAPoI (comprising the DNA molecule encoding IA Pol, as set
forth in SEQ ID NO:4), and VlJns-tPA-IAPoI, (comprising the DNA molecule
encoding tPA-IA Pol, as set forth in SEQ ID N0:8). Polynucleotide vaccine
constructions V 1R-wtpol, V lJns-IAPoI, and V lJns-tPA-IAPoI, are exemplified
in
Example Sections 3-5.
It will be evident upon review of the teaching within this specification that
numerous vector/Pol antigen constructs may be generated. While the exemplified
constructs are preferred, any number of vector/Pol antigen combinations are
within
the scope of the present invention, especially wild type or
modified/inactivated Pol
proteins which comprise at least one, preferably 5 or more and especially all
nine
mutations as shown in Table 1, with or without the inclusion of a leader
sequence
such as human tPA.
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
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exposure, as well as the 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% 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 and PCT International Publication
No. WO 97/40839, both of which are hereby incorporated by reference.
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. Another
adjuvant for use in the DNA vector vaccines of the present invention are one
or
more forms of an aluminum phosphate-based adjuvant wherein the aluminum
phosphate-based adjuvant possesses a molar PO4lA1 ratio of approximately 0.9.
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
cellular and humoral responses to DNA vaccination. These mineral-based
compounds for use as DNA vaccines adjuvants are disclosed in PCT International
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Application No. PCT/US98/02414, PCT International Publication No.
WO 98/35562, which is hereby incorporated by reference. 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°l0 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 invention in comparison
to
administration of a non-adjuvanted polynucleotide vaccine.
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
Pol-
based DNA vaccines disclosed herein are intramuscular injection, subcutaneous
administration and needle-free injection. An especially preferred method is
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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 Pol-
based
DNA vector vaccines of the present invention.
The aforementioned polynucleotides, when directly introduced into a
vertebrate in vivo, express the respective HIV-1 Pol protein within the animal
and in
turn induce a cellular immune response within the host to the expressed Pol
antigen.
To this end, the present invention also relates to methods of using the HIV-1
Pol-
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 pol-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 pol-
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 intracellular expression of these DNA pol-based vaccines. This
intracellular expression of the Pol-based immunogen induces a cellular immune
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.
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EXAMPLE 1
Vaccine Vectors
VI - Vaccine vector Vl 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-IE1 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'-
CTATAT
AAGCAGAGCTCGTTTAG-3' (SEQ )D NO:10); 3' primer: 5'-GTAGCAAA
GATCTAAGGACGGTGACTGCAG-3' (SEQ ID NO:11). The primers used to
remove the SacI site are: sense primer, 5'-GTATGTGTCTGAAAATGAGCG
TGGAGATTGGGCTCGCAC-3' (SEQ ID N0:12) and the antisense primer,
5'-GTGCGAGCCCAATCTCCACGCTCATTTTCAGAC ACATAC-3' (SEQ ID
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 V 1 in order to place them
within a
more defined context, create a more compact vector, and to improve plasmid
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
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 polymerase enzyme in order to facilitate its ligation to
another
"blunt-ended" DNA fragment. pUCl8 was chosen to provide the "backbone" of the
expression vector. It is known to produce high yields of plasmid, is well-
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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 polymerase treated with calf intestinal alkaline phosphatase,
and
S ligated to the CMVintABGH element described above. Plasmids exhibiting
either of
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 V1J. 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
1S 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
ZS 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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CCGCAGTTTTTATTAAACAT CTCCACGCGAATCTCGGGTACGTGTTCCGG
AACGTGGGAT
ACATGGGCTCTTCTCCGGTAGCGGCGGAGCTTCTACATCCGAGCCCTGCTCCCATGCCTC
CAGCGACTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCA
CAGCACGATGCCCACCACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTC
S TGAAAATGAGCTCGGGGAGCGGGCTTGCACCGCTGACGCATTTGGAAGACTTAAGGCAGC
GGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTGTTCTGATAAGAGTCAGAGGTAACTCC
CGTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGC
GCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTT
CTGCAGTCACCGTCCTTAGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCC
IO 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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CGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTA
CCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTA
TCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCC
GCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAAT
AGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGT
ATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTG
TGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCA
GTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTA
AGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGG
1O CGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACT
TTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCG
CTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTT
ACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGA
ATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGC
ATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAA
CAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATT
ATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC (SEQ
ID
N0:14).
V1 JfZeo vector involved
- Construction
of vaccine
vector
V lJneo
expression
removal of the ampr gene and insertion of the kanr gene (neomycin
phosphotransferase). The ampr gene from the pUC backbone of V1J was removed by
digestion with SspI and Eam1105I 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,
was excised using the PstI restriction enzyme, purified by agarose gel
electrophoresis,
and blunt-ended with T4 DNA polymerase. This fragment was ligated with the V
1J
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
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
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provides
resistance
to neomycin,
kanamycin
and 6418.
The nucleotide
sequence
of
V lJneo follows:
is as
TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA
CAGCTTGTCTGTAAGCGGAT~GCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG
S TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC
ACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGATTGG
CTATTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATG
TCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTAC
GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGG
1O CCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC
CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAAC
TGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA
TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTAC
TTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTA
1S CATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGA
CGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA
CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG
AGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCA
TAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGAT
ZO TCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACCCCCTTGGC
TTCTTATGCATGCTATACTGTTTTTGGCTTGGGGTCTATACACCCCCGCTTCCTCATGTT
ATAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTATTGACCACTCCC
CTATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACTCTCT
TTATTGGCTATATGCCAATACACTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTAC
ZS AGGATGGGGTCTCATTTATTATTTACAAATTCACATATACAACACCACCGTCCCCAGTGC
CCGCAGTTTTTATTAAACATAACGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGG
ACATGGGCTCTTCTCCGGTAGCGGCGGAGCTTCTACATCCGAGCCCTGCTCCCATGCCTC
CAGCGACTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCA
CAGCACGATGCCCACCACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTC
3O TGAAAATGAGCTCGGGGAGCGGGCTTGCACCGCTGACGCATTTGGAAGACTTAAGGCAGC
GGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTGTTCTGATAAGAGTCAGAGGTAACTCC
CGTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGC
GCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTT
CTGCAGTCACCGTCCTTAGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCC
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CCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAA
ATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGG
GGCAGCACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGG
GCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCAGAAAGAAGC
S AGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTTCTTAGTTCCAGCCC
CACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAAGTAC
TTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGAGTGG
GAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATG
TGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTG
1O CGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTA
TCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCC
AGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAG
CATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATAC
CAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACC
IS GGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGT
AGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCC
GTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGA
CACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTA
GGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTA
ZO TTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGA
TCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACG
CGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAG
TGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACC
TAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACT
ZS TGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTT
CGTTCATCCATAGTTGCCTGACTCCGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGA
AGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGA
GCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTT
TGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAA
3O AGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGT
TACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAAT
TTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGA
GAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCG
ACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGT
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GAGAAATCAC CATGAGTGAC GACTGAATCCGGTGAGAATG GCAAAAGCTTATGCATTTCT
TTCCAGACTT GTTCAACAGG CCAGCCATTACGCTCGTCAT CAAAATCACTCGCATCAACC
AAACCGTTAT TCATTCGTGA TTGCGCCTGAGCGAGACGAA ATACGCGATCGCTGTTAAAA
GGACAATTAC AAACAGGAAT CGAATGCAACCGGCGCAGGA ACACTGCCAGCGCATCAACA
S ATATTTTCAC CTGAATCAGG ATATTCTTCTAATACCTGGA ATGCTGTTTTCCCGGGGATC
GCAGTGGTGA GTAACCATGC ATCATCAGGAGTACGGATAA AATGCTTGATGGTCGGAAGA
GGCATAAATT CCGTCAGCCA GTTTAGTCTGACCATCTCAT CTGTAACATCATTGGCAACG
CTACCTTTGC CATGTTTCAG AAACAACTCTGGCGCATCGG GCTTCCCATACAATCGATAG
ATTGTCGCAC CTGATTGCCC GACATTATCGCGAGCCCATT TATACCCATATAAATCAGCA
1O TCCATGTTGG AATTTAATCG CGGCCTCGAGCAAGACGTTT CCCGTTGAATATGGCTCATA
ACACCCCTTG TATTACTGTT TATGTAAGCAGACAGTTTTA TTGTTCATGATGATATATTT
TTATCTTGTG CAATGTAACA TCAGAGATTTTGAGACACAA CGTGGCTTTCCCCCCCCCCC
CATTATTGAA GCATTTATCA GGGTTATTGTCTCATGAGCG GATACATATTTGAATGTATT
TAGAAAAATA AACAAATAGG GGTTCCGCGCACATTTCCCC GAAAAGTGCCACCTGACGTC
15 TAAGAAACCA TTATTATCAT GACATTAACCTATAAAAATA GGCGTATCACGAGGCCCTTT
CGTC (SEQ 2D N0:15).
V1 Jns - The expression vector
VIJns was generated by adding
an SfiI site to
VlJneo to facilitate integrationA commercially 3 base pair
studies. available 1 SfiI
linker (New England BioLabs)
was added at the KpnI site
within the BGH sequence
20 of the vector. VlJneo was
linearized with KpnI, gel
purified, blunted by T4 DNA
polymerase, and ligated to linker. Clonal
the blunt SfiI isolates were
chosen by
restriction mapping and verifiedencing through
by sequ the linker. The
new vector
was designated VlJns. Expression
of heterologous genes in
VlJns (with SfiI) was
comparable to expression of
the same genes in VlJneo
(with KpnI).
25 The nucleotide sequence of
VlJns is as follows:
TCGCGCGTTT CGGTGATGAC GGTGAAAACCTCTGACACAT GCAGCTCCCGGAGACGGTCA
CAGCTTGTCT GTAAGCGGAT GCCGGGAGCAGACAAGCCCG TCAGGGCGCGTCAGCGGGTG
TTGGCGGGTG TCGGGGCTGG CTTAACTATGCGGCATCAGA GCAGATTGTACTGAGAGTGC
ACCATATGCG GTGTGAAATA CCGCACAGATGCGTAAGGAG AAAATACCGCATCAGATTGG
3O CTATTGGCCA TTGCATACGT-TGTATCCATATCATAATATG TACATTTATATTGGCTCATG
TCCAACATTA CCGCCATGTT GACATTGATTATTGACTAGT TATTAATAGTAATCAATTAC
GGGGTCATTA GTTCATAGCC CATATATGGAGTTCCGCGTT ACATAACTTACGGTAAATGG
CCCGCCTGGC TGACCGCCCA ACGACCCCCGCCCATTGACG TCAATAATGACGTATGTTCC
CATAGTAACG CCAATAGGGA CTTTCCATTGACGTCAATGG GTGGAGTATTTACGGTAAAC
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TGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA
TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTAC
TTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTA
CATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGA
S CGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA
CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG
AGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCA
TAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGAT
TCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGACTCTATAGGCACACCCCTTTGGC
IO TCTTATGCATGCTATACTGTTTTTGGCTTGGGGCCTATACACCCCCGCTTCCTTATGCTA
TAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTATTGACCACTCCCC
TATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACTATCTC
TATTGGCTATATGCCAATACTCTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTACA
GGATGGGGTCCCATTTATTATTTACAAATTCACATATACAACAACGCCGTCCCCCGTGCC
IS CGCAGTTTTTATTAAACATAGCGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGGA
CATGGGCTCTTCTCCGGTAGCGGCGGAGCTTCCACATCCGAGCCCTGGTCCCATGCCTCC
AGCGGCTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCAC
AGCACAATGCCCACCACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTCT
GAAAATGAGCGTGGAGATTGGGCTCGCACGGCTGACGCAGATGGAAGACTTAAGGCAGCG
ZO GCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTATTCTGATAAGAGTCAGAGGTAACTCCC
GTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCG
CGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTTC
TGCAGTCACCGTCCTTAGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCC
CTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAA
25,TGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGG
GCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGG
CTCTATGGCCGCTGCGGCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCAGAAAG
AAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTTCTTAGTTCCA
GCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAA
3O GTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGA
GTGGGAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAA
CATGTGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTC
GCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACG
GTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAA
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GGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGA
CGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAG
ATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCT
TACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACG
S CTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACC
CCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGT
AAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTA
TGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAAC
AGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTC
IO TTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGAT
TACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGC
TCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTT
CACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTA
AACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCT
IS ATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGA
AGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAG
GGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTG
CTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGC
AAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAG
ZO TGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGC
AATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAA
GGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATT
CCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCA
AGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATT
ZS TCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCA
ACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTA
AAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCA
ACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGG
ATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGA
3O AGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCA
ACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGA
TAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCA
GCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTC
ATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATA
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TTTTTATCTT GTGCAATGTA ACATCAGAGA TTTTGAGACA CAACGTGGCT TTCCCCCCCC
CCCCATTATT GAAGCATTTA TCAGGGTTAT TGTCTCA'~GA GCGGATACAT ATTTGAATGT
ATTTAGAAAA ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT GCCACCTGAC
GTCTAAGAAA CCATTATTAT CATGACATTA ACCTATAAAA ATAGGCGTAT CACGAGGCCC
TTTCGTC(SEQ ID N0:16).
The underlined nucleotides of SEQ ID N0:16 represent the Sfi 1 site
introduced into the I~pn 1 site of VlJneo.
V1 JrZS-tPA - The vaccine vector V lJns-tPA was constructed in order to fuse
an heterologous leader peptide sequence to the pol DNA constructs of the
present
invention. More specifically, the vaccine vector VlJns was modified to include
the
human tissue-specific plasminogen activator (tPA) leader. As an
exemplification, but
by no means a limitation of generating a pol DNA construct comprising an amino-
terminal leader sequence, plasmid VlJneo was modified to include the human
tissue-
specific plasminogen activator (tPA) leader. Two synthetic complementary
oligomers
were annealed and then ligated into VlJneo which had been BgIII digested. The
sense and antisense oligomers were 5'-GATCACCATGGATGCAATGAAGAG
AGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCAG
CGA-3' (SEQ ID N0:17); and, 5'-GATCTCGCTGGGCGAAACGAAGACTGCTCC
ACACAGCAGCAGCACACAGCAGAGCCCTCTCTTCATTGCATCCATGGT-3'
(SEQ ID N0:18). The Kozak sequence is underlined in the sense oligomer. These
oligomers have overhanging bases compatible for ligation to BgIII-cleaved
sequences.
After ligation the upstream BgIII site is destroyed while the downstream BgIII
is
retained for subsequent ligations. 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
the KpnI
site within the BGH terminator region of VlJneo-tPA by blunting the KpnI site
with
T4 DNA polymerise followed by ligation with an SfiI linker (catalogue #1138,
New
England Biolabs), resulting in VlJns-tPA. This modification was verified by
restriction digestion and agarose gel electrophoresis.
The VlJns-tpa vector nucleotide sequence 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
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CTATTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATG
TCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTAC
GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGG
CCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC
S CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAAC
TGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA
TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTAC
TTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTA
CATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGA
IO CGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA
CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG
AGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCA
TAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGAT
TCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGACTCTATAGGCACACCCCTTTGGC
IS TCTTATGCATGCTATACTGTTTTTGGCTTGGGGCCTATACACCCCCGCTTCCTTATGCTA
TAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTATTGACCACTCCCC
TATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACTATCTC
TATTGGCTATATGCCAATACTCTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTACA
GGATGGGGTCCCATTTATTATTTACAAATTCACATATACAACAACGCCGTCCCCCGTGCC
ZO CGCAGTTTTTATTAAACATAGCGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGGA
CATGGGCTCTTCTCCGGTAGCGGCGGAGCTTCCACATCCGAGCCCTGGTCCCATGCCTCC
AGCGGCTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCAC
AGCACAATGCCCACCACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTCT
GAAAATGAGCGTGGAGATTGGGCTCGCACGGCTGACGCAGATGGAAGACTTAAGGCAGCG
ZS GCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTATTCTGATAAGAGTCAGAGGTAACTCCC
GTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCG
CGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTTC
TGCAGTCACCGTCCTTAGATCACCATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTG
CTGCTGTGTGGAGCAGTCTTCGTTTCGCCCAGCGAGATCTGCTGTGCCTTCTAGTTGCCA
3O GCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCAC
TGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTAT
TCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCA
TGCTGGGGATGCGGTGGGCTCTATGGCCGCTGCGGCCAGGTGCTGAAGAATTGACCCGGT
TCCTCCTGGGCCAGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGC
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CCCTGGTTCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCT
TCAATCCCACCCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAAC
CAAACCTAGCCTCCAAGAGTGGGAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGG
GAGAGAAAATGCCTCCAACATGTGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGC
S TTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCA
CTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTG
AGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCA
TAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAA
CCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCC
IO TGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGC
GCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCT
GGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCG
TCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG
GATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTA
IS CGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGG
AAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTT
TGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTT
TTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAG
ATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAAT
ZO CTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACC
TATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGGGGGGGCG
CTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATC
ATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTT
GGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGAT
2S CTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTC
AGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCG
AGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAA
AGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCC
TGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCG
3O TCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAAT
GGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCA
TCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGA
AATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGG
AACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGG
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AATGCTGTTT TCCCGGGGAT CGCAGTGGTG AGTAACCATG CATCATCAGG AGTACGGATA
AAATGCTTGA TGGTCGGAAG AGGCATAAAT TCCGTCAGCC AGTTTAGTCT GACCATCTCA
TCTGTAACAT CATTGGCAAC GCTACCTTTG CCATGTTTCA GAAACAACTC TGGCGCATCG
GGCTTCCCAT ACAATCGATA GATTGTCGCA CCTGATTGCC CGACATTATC GCGAGCCCAT
S TTATACCCAT ATAAATCAGC ATCCATGTTG GAATTTAATC GCGGCCTCGA GCAAGACGTT
TCCCGTTGAA TATGGCTCAT AACACCCCTT GTATTACTGT TTATGTAAGC AGACAGTTTT
ATTGTTCATG ATGATATATT TTTATCTTGT GCAATGTAAC ATCAGAGATT TTGAGACACA
ACGTGGCTTT CCCCCCCCCC CCATTATTGA AGCATTTATC AGGGTTATTG TCTCATGAGC
GGATACATAT TTGAATGTAT TTAGAAAAAT AAACAAATAG GGGTTCCGCG CACATTTCCC
1O CGAAAAGTGC CACCTGACGT CTAAGAAACC ATTATTATCA TGACATTAAC CTATAA.AA.AT
AGGCGTATCA CGAGGCCCTT TCGTC (SEQ ID N0:9).
V1R - Vaccine vector V1R was constructed to obtain a minimum-sized
vaccine vector without unneeded DNA sequences, which still retained the
overall
optimized heterologous gene expression characteristics and high plasmid yields
that
15 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 karzr gene following the
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
20 be removed without affecting its regulatory function (following the
original I~pnI
restriction enzyme site within the BGH element). V1R was constructed by using
PCR
to synthesize three segments of DNA from VlJns representing the CMVintA
promoter/BGH terminator, origin of replication, and kanamycin resistance
elements,
respectively. Restriction enzymes unique for each segment were added to each
25 segment end using the PCR oligomers: SspI and XhoI for CMVintA/BGH; EcoRV
and BamHI for the kan r gene; and, BcII and SalI 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
30 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
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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 V1R appear similar to VlJns. The net reduction in vector size achieved
was
1346 by (VlJns = 4.86 kb; V1R = 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'-GGTACAAATATTGGCTATTGG
CCATTGCATACG-3' (SEQ ID N0:19) [SspI]; (2) 5'-CCACATCTCGAGGAAC
CGGGTCAATTCTTCAGCACC-3' (SEQ ID N0:20) [XhoI] (for CMVintA/BGH
segment); (3) 5'-GGTACAGATATCGGAAAGCCACGTTGTG TCTCAAAATC-3'
(SEQ ID NO:21) [EcoRV]; (4) 5'-CACATGGATCCGTAAT GCTCTGCCAGTGTT
ACAACC-3' (SEQ ID N0:2) [BamHI], (for kanamycin resistance gene segment) (5)
5'-GGTACATG ATCACGTAGAAAAGATCA AAGGATCTTCTTG-3' (SEQ ID
N0:23) [BcII]; (6) 5'-CCACATGTCGACCCGTAAA AAGGCCGCGTTGCTGG-3'
(SEQ ID N0:24): [SaII], (for E. coli origin of replication).
The nucleotide sequence of vector V1R is as follows:
TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA
CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG
TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC
2O 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
2S 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
3O 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
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TTCTTATGCATGCTATACTGTTTTTGGCTTGGGGTCTATACACCCCCGCTTCCTCATGTT
ATAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTATTGACCACTCCC
CTATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACTCTCT
TTATTGGCTATATGCCAATACACTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTAC
S AGGATGGGGTCTCATTTATTATTTACAAATTCACATATACAACACCACCGTCCCCAGTGC
CCGCAGTTTTTATTAAACATAACGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGG
ACATGGGCTCTTCTCCGGTAGCGGCGGAGCTTCTACATCCGAGCCCTGCTCCCATGCCTC
CAGCGACTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCA
CAGCACGATGCCCACCACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTC
IO TGAAAATGAGCTCGGGGAGC'GGGCTTGCACCGCTGACGCATTTGGAAGACTTAAGGCAGC
GGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTGTTCTGATAAGAGTCAGAGGTAACTCC
CGTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGC
GCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTT
CTGCAGTCACCGTCCTTAGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCC
IS CCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAA
ATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGG
GGCAGCACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGG
GCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCAGAAAGAAGC
AGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTTCTTAGTTCCAGCCC
ZO CACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAAGTAC
TTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGAGTGG
GAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATG
TGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTG
CGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTA
~,STCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCC
AGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAG
CATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATAC
CAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACC
GGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGT
3O AGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCC
GTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGA
CACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTA
GGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTA
TTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGA
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TCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACG
CGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAG
TGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACC
TAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACT
TGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTT
CGTTCATCCATAGTTGCCTGACTCCGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGA
AGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGA
GCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTT
TGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAA
IO AGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGT
TACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAAT
TTATTCATATCAGGATTATCAATACCATATTTTTGAAA.AAGCCGTTTCTGTAATGAAGGA
GAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCG
ACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAA.A.AATAAGGTTATCAAGT
IS GAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCT
TTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACC
AAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAA
GGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACA
ATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATC
ZO GCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGA
GGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACG
CTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAG
ATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCA
TCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATA
Z5 ACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTT
TTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCCCCCCCCCCC
CATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATT
TAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC
TAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTT
3O CGTC (SEQ
ID N0:25).
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EXAMPLE 2
Codon Optimized HIV-1 Pol and HIV-1 IA Pol Derivatives as DNA Vector Vaccines
Synthesis of WT optpol and IA-opt pol Gefae - Construction of both genes were
conducted by Midland Certified Reagent Company (Midland, TX) following
established strategies. Ten double stranded oligonucleotides, ranging from 159
to 340
bases long and encompassing the entire pol gene, were synthesized by solid
state
methods and cloned separately into pUC 18. For the wt-pol gene, the fragments
are as
follows:
BgIII#1-Ec1136II half site at 282 = pJS6A1-7
PmII half site at #285 - Ec1136II half site at #597 = pJS6B2-5
SspI half site at #600 - Ec1136II half site at #866 = pJS6C1-4
SmaI half site at #869 -ApaI #1095 = pJS6D1-4
ApaI #1095 - KpnI #1296 = pJS6E1-4
KpnI #1296 - XcmI #1636 = pJS6F1-5
XcmI #1636 - NsiI #1847 ~ = pJS6G1-2
NsiI #1847 - BcII half site at #2174 = pJS6Hl-14
BcII half site at #2174 - SacI #2333 = pJS6Il-2
SacI #2333 - BgIII #2577 = pJS6Jl-1
EcoRI and HindIII sequences were added upstream of each 5' end and downstream
of
each 3' end, respectively, to allow cloning into the EcoRI-HifzdIII sites of
pUCl8.
The next stage of the synthesis was to consolidate these cassettes into three
roughly equal fragments (alpha, beta, gamma) and was performed as follows:
Alpha: The SspI-HindIII small fragment of pJS6C1-4 was transferred into the
Ec1136II-HindIII sites of pJS6B2-5 to give pJS6BC1-1. Into the EcoRI-PnadI
sites of
this plasmid was inserted the EcoRI-Ec1136II small fragment of pJS6A1-7 to
give
pJS6a1-8.
Beta: The EcoRI-ApaI small fragment of pJS6D1-4 was inserted into the
corresponding sites of pJS6E1-2 to give pJS6DE1-2. Also, the EcoRI-XcmI small
fragment of pJS6F1-5 was inserted into the corresponding sites of pJS6Gl-2 to
give
pJS6FG1-1. Then the EcoRI-KpnI small fragment of pJS6DE1-2 was inserted into
the corresponding sites of pJS6FG1-1 to give pJS6(31-1.
Gamma: The SacI-HindIII small fragment of pJS6J1-1 was inserted into the
corresponding sites of pJS6Il-2 to give pJS6IJ1-1. This plasmid was propagated
through E. coli SCS110 (dam-ldcfri-) to permit subsequent cleavage at the BcII
site.
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The BcII-HindIII small fragment of the unmethylated pJS6IJl-1 was inserted
into the
BglII-HindIII sites of pJS6H1-14 to give pJS6x1-1.
The wt-pol alpha, beta, gamma were ligated into the entire sequence as
follows:
The EcoRI-Ec1136II small fragment of pJS6cc1-8 was inserted into the EcoRI-
SmaI
sites of pJS6(31-1 to give pJS6oc(32-1.
Into the NsiI-HindIII sites of this plasmid was inserted the NsiI-HindIII
small
fragment of pJS6x1-1 to give pUCl8-wt-pol. This final plasmid was completely
resequenced in both strands.
To construct the entire IA-pol gene, only 3 new small fragments were
synthesized:
PzzzlI half site at #285 - Ec1136II half site at #597 = pJS7B 1-1
KpnI #1296 -XcmI #1636 = pJS7F1-2
NsiI #1847 - BgIII half site at #2174 = pJS7H1-5
These were then used in the same reconstruction strategy as described above to
give
pUC 18-IA-pol.
Expressiozz Vector Cofzstruction - pUC 18-wt-pol and pUC 18-IA-pol were
digested with BgIII in order to isolate fragments containing the entire pol
genes. V 1R,
VlJns, VlJns-tpa (Shiver, et al., 1995, Immune responses to HIV gp120 elicited
by
DNA vaccination. In Vaccizzes 95 (eds. Chanock, R. M., Brown, F., Ginsberg,
H.S.,
& Norrby, E.) @ pp. 95-98; Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, New York; see also Example Section 1) were digested with BgIII. The
cut
vectors were then treated with calf intestinal alkaline phosphatase. Both wt-
pol and
IA-pol genes were ligated into cut V1R using T4 DNA ligase (16 °C,
overnight).
Competent DHSa cells were transformed with aliquots of the ligation mixtures.
Colonies were screened by restriction digestion of amplified plasmid isolates.
Following a similar strategy, the BgIII fragment containing the IA-pol was
subcloned
into the BgIII site of VlJns. To ligate the IA-pol gene into VlJns-tpa, the IA-
pol
gene was PCR-amplified from V 1R-IA-pol using pfu polymerise and the following
pair of primers: 5'-GGTACAAGATCTCCGCCCCCATCTCCCCCATTGAGA-3'
(SEQ ID N0:26), and 5'-CCACATAGATCTGCCCGGGCTTTAGTCCTCATC-3'
(SEQ ID N0:27). The upstream primer was designed to remove the initiation met
codon and place the pol gene in frame with the tpa leader coding sequence from
VlJns-tpa. The PCR product was purified from the agarose gel slab using Sigma
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DNA Purification spin columns. The purified products were digested with BgIII
and
subcloned into the BgIII site of VlJns-tpa.
Results - The codon humanized wt- and IA-pol genes were constructed via
stepwise ligation of 10 synthetic dsDNA fragments (Ferretti, et al., 1986,
Proc. Natl.
Acad. Sci. USA 83: 599-603). For expression in mammalian systems, the IA-pol
gene
was subcloned into V1R, VlJns, and VlJns-tpa. All these vectors place the gene
under the control of the human cytomegalovirus/intron A hybrid promoter
(hCMVIA). The DNA sequence of the IA-pol gene and the expressed protein
product
are shown in Figure 2A-B. Subcloning into VlJns-tpa attaches the leader
sequence
from human tissue-specific plasminogen activator (tpa) to the N-terminus of
the IA-
pol (Pennica, et al., 1983, Nature 301: 214-221) to allow secretion of the
protein. The
sequences of the tpa leader and the fusion junction are shown in Figure 3.
EXAMPLE 3
HIV-1 POL Vaccine - Rodent Studies
Materials - E. coli DHSa strain, penicillin, streptomycin, ACK lysis buffer,
hepes, L-glutamine, RPMI1640, and ultrapure CsCI were obtained from Gibco/BRL
(Grand Island, NY). Fetal bovine serum (FBS) was purchased from Hyclone.
Kanamycin, Tween 20, bovine serum albumin, hydrogen peroxide (30%),
concentrated sulfuric acid, (3-mercaptoethanol ((3-ME ), and concanavalin A
were
obtained from Sigma (St. Louis, MO). Female balb/c mice at 4-6 wks of age were
obtained from Taconic Farms (Germantown, NY). 0.3-mL insulin syringes were
purchased from Myoderm. 96-well flat bottomed Maxisorp plates were obtained
form
NLJNC (Rochester, NY). HIV-line RT p66 recombinant protein was obtained from
Advanced Biotechnologies, Inc. (Columbia, MD). 20-mer peptides were
synthesized
by Research Genetics (Huntsville, AL). Horseradish peroxidase (HRP)-conjugated
rabbit anti-mouse IgGl was obtained from ZYMED (San Francisco, CA). 1,2-
phenylenediamine dihydrochloride (OPD) tablets was obtained from DAKO
(Norway). Purified rat anti-mouse IFN-gamma (IgGl, clone R4-6A2), biotin-
conjugated rat anti-mouse IFN-gamma (IgGl, clone XMG 1.2), and strepavidin-
alkaline phosphatase conjugate were purchased from PharMingen (San Diego, CA).
1-STEP NBT/BCIP dye was obtained from Pierce Chemicals (Rockford, IL). 96-well
Multiscreen membrane plate was purchased from Millipore (France). Cell
strainer
was obtained from Becton-Dickinson (Franklin Lakes, NJ).
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Plasfnid Preparation - E. coli DHSa cells expressing the pol plasmids were
grown to saturation in LB broth supplemented with 100 ug/mL kanamycin. Plasmid
were purified by standard CsCI method and solubilized in saline at
concentrations
greater than 5 mg/mL until further use.
Vaccination - The plasmids were prepared in phosphate-buffered saline and
administered into balb/c by needle injection (28-1/2G insulin syringe) of 50
uL
aliquot into each quad muscle. VlJns-IApol was administered at 0.3, 3, 30 ug
dose
and for comparison, VlJns-tpa-IApol was given at 30 ug dose. Immunizations
were
conducted at T=0 and T=8 wks (for select animals from the 30-ug dose cohorts).
ELISA Assay - At T=12 wks, blood samples were collected by making an
incision of a tail vein and the serum separated. Anti-RT titers were obtained
following standard secondary antibody-based ELISA. Briefly, Maxisorp plates
were
coated by overnight incubation with 100 uL of 1 ug/mL HIV-1 RT protein (in
PBS).
The plates were washed with PBS/0.05% Tween 20 and incubated for approx. 2h
with
200 uL/well of blocking solution (PBS/0.05% tween/1% BSA). The blocking
solution was decanted; 100 uL aliquot of serially diluted serum samples were
added
per well and incubated for 2 h at room temperature. The plates were washed and
100
uL of 1/1000-diluted HRP-rabbit anti-mouse IgG were added with 1 h incubation.
The plates were washed thoroughly and soaked with 100 uL OPD/H202 solution
.for
15 min. The reaction was quenched by adding 100 uL of 0.5M H2S04 per well.
OD4~2 readings were recorded.
ELlspot - Spleens were collected from 5 mice/cohort at T=13-14 wks and
pooled into a tube of 8-mL R10 medium (RPMI1640, 10% FBS, 2mM L-glutamine,
100U/mL Penicillin, 100 u/mL streptomycin, 10 mM Hepes, 50 uM (3-ME).
Multiscreen opaque plates were coated with 100p,1/well of capture mAb
(purified R4-
6A2 diluted in PBS to 5p,g/ml) at 4°C overnight. The plates were washed
with
PBS/Pen/Strep in hood and blocked with 200~,1/well of complete R10 medium for
37°C for at least 2 hrs. The mouse spleens were ground on steel mesh,
collected into
15m1 tubes and centrifuged at 1200rpm for l0min. The pellet was treated in ACK
buffer (4m1 of lysis buffer per spleen) for 5min at room temperature to lyse
red blood
cells. The cell pellet was centrifuged as before, resuspended in K-medium (5m1
per
mouse spleen), filtered through a cell strainer and counted using a
hemacytometer.
Block medium was decanted from the plates and 100~,1/well of cell samples
(5.Ox10e5
cells per well) plus antigens were added. Pol-specific CD4+ cells were
stimulated
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using a mixture of previously identified two epitope-containing peptides
(aa641-660,
aa731-750). Antigen-specific CD8+ cells were stimulated using a pool of four
peptide epitope-containing peptides (aa201-220, aa311-330, aa571-590, aa781-
800)
or with individual peptides. A final concentration of 4 ug/mL per peptide was
used.
Each splenocyte sample is tested for IFN-gamma secretion by adding the
mitogen,
concanavalin A. Plates were incubated at 37°C, 5°7o COZ for 20-
24 h. The plates
were washed with PBS/0.05% Tween 20 and soaked with 100 uL/well of 5 ug/mL
biotin-conjugated rat anti-mouse IFN- mAb (clone XMG1.2) at 4°C
overnight. The
plates were washed and soaked with 100 uL/well 1/2500 dilution of strepavidin-
AP
(in PBS/0.005°Io Tween/5%FCS) for 30 min at 37 °C. Following a
wash, spots were
developed by incubating with 100~.1/well 1-step NBT/BCIP for 6-10 min. The
plates
were washed with water and allowed to air dry. The number of spots in each
wells
were determined using a dissecting microscope and normalized to 10e6 cells.
Results - Single vaccination of balb/c mice with VlJns-IApol is able to induce
antigen-specific antibody (Figure 4) and T cell (Figure 5) responses in a dose
response manner. IFN-gamma secretion from splenocytes can be detected from 3
and
30 ug cohort following stimulation with pools of peptides that contain CD4+
and
CD8+ T cell epitopes. These epitopes were identified by (1) screening 20-mer
peptides that encompass the entire pol sequence and overlap by 10 amino acid
for
ability to stimulate IFN-gamma secretion from vaccinee splenocytes, and (2)
determining the T cell type (CD4+ or CD8+) by depleting either population in
an
Elispot assay. Addition of tpa leader sequence to the pol gene is able to
induce
comparable, if not slightly higher, frequencies of pol-specific CD4+ and CD8+
cells.
A second immunization with either VlJns-IApol and VlJns-tpa-IApol resulted in
effective boosting of the immune responses.
EXAMPLE 4
HIV-1 Pol Vaccine - Non Human Primate Studies
Materials - E. coli DH5oc strain, penicillin, streptomycin, and ultrapure CsCl
were obtained from Gibco/BRL (Grand Island, NY). Kanamycin and
phytohemagluttinin (PHA-M) were obtained from Sigma (St. Louis, MO). 20-mer
peptides were synthesized by SynPep (Dublin, CA) and Research Genetics
(Huntsville, AL). 96-well Multiscreen Immobilon-P membrane plates were
obtained
from Millipore (France). Strepavidin-alkaline phosphatase conjugate were
purchased
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form Pharmingen (San Diego, CA). 1-Step NBT/BCIP dye was obtained form Pierce
Chemicals (Rockford, IL). Rat anti-human IFN-gamma mAb and biotin-conjugated
anti-human IFN-gamma reagent were obtained from R&D Systems (Minneapolis,
MN). Dynabeads M-450 anti-human CD4 were obtained from Dynal (Norway).
HIVp24 antigen assay was purchased from Coulter Corporation (Miami, FL). HIV-
lIIiB RT p66 recombinant protein was obtained from Advanced Biotechnologies,
Inc.
(Columbia, MD). Plastic 8 well strips/plates, flat bottom, Maxisorp, are
obtained
from NUNC (Rochester, NY). HIV+ human serum 9711234 was obtained from
Biological Specialty Corp.
Plasfnid Preparation - E. coli DHSoc cells expressing the pol plasmids were
grown to saturation in LB supplemented with 100 ug/mL kanamycin. Plasmid were
purified by standard CsCI method and solubilized in saline at concentrations
greater
than 5 mg/mL until further use.
Vaccination - Cohorts of 3 rhesus macaques (approx. 5-10 kg) were
vaccinated with 5 mg dose of either VlJns-IApol or VlJns-tpa-IApol. The
vaccine
was administered by needle injection of two 0.5 mL aliquots of 5 mg/mL plasmid
solution (in phosphate-buffered saline, pH 7.2) into both deltoid muscles.
Prior to
vaccination, the monkeys were chemically restraint with i.m. injection of 10
mg/kg
ketamine. The animals were immunized 3x at 4 week intervals (T=0, 4, 8 wks).
Sample Collection - Blood samples were collected at T = 0, 4, 8, 12, 16, 18
wks; sera and PBMCs were isolated using established protocols.
ELlspot Assay - Immobilon-IP plates were coated with 100 uL/well of rat anti-
human IFN-gamma mAb at 15 ug/mL at 4 °C overnight. The plates are then
washed
with PBS and block by adding 200 uL/well of R10 medium. 4x10e5 peripheral
blood
cells were plated per well and to each well, either media or one of the pol
peptide
pools (final concentration of 4 ug/mL per peptide) or PHA, a known mitogen, is
added to a final volume of 100 uL. Duplicate wells were set up per sample per
antigen and stimulation was performed for 20-24 h at 37 °C. The plates
are then
washed; biotinylated anti-human IFN-gamma reagent is added (0.1 ug/mL, 100 uL
per well) and allowed to incubate for overnight at 4 °C. The plates are
again washed
and 100 uL of 1:2500 dilution of the strepavidin-alkaline phosphatase reagent
(in
PBS/0.005% Tween/5% FCS) is added and allowed to incubate for 2 h at ambient
room temperature. After another wash, spots are developed by incubating with
100
uL/well of 1-step NBTBCIP for 6-10 min. CD4- T cell depletion was performed by
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adding 1 bead particle/10 cell of Dynabeads M450 anti-human CD4, prewashed
with
PBS, and incubating on the shaker at 4 °C for 30 min. The beads are
fractionated
magnetically and the unbound cells collected and quantified before plating
onto the
ELISpot assay plates ( at 4x10e5 cells per well).
CTL Assay - Procedures for establishing bulk CTL culture with fresh or
cryopreserved peripheral blood mononuclear cells (PBMC) are as follows. Twenty
percent total PBMC were infected in 0.5 ml volume with recombinant vaccinia
virus,
Vac-tpaPol, respectively, at multiplicity of infection (moi) of 5 for 1 hr at
37°C, and
then combined with the remaining PBMC sample. The cells were washed once in 10
ml R-10 medium, and plated in a 12 well plate at approximately 5 to 10 x 10~
cells/well in 4 ml R-10 medium. Recombinant human IL-7 was added to the
culture
at the concentration of 330 U/ml. Two or three days later, one milliliter of R-
10
containing recombinant human IL-2 (100 U/ml) was added to each well. And twice
weekly thereafter, two milliliters of cultured media were replaced with 2 ml
fresh R-
10 medium with rhlL-2 (100 U/ml). The lymphocytes were cultured at 37°C
in the
presence of 5% COZ for approximately 2 weeks, and used in cytotoxicity assay
as
described below. The effector cells harvested from bulk CTL cultures were
tested
against autologous B lymphoid cell lines (BLCL) sensitized with peptide pools.
To
prepare for the peptide-sensitized targets, the BLCL cells were washed once
with
R-10 medium, enumerated, and pulsed with peptide pool (about 4 to 8 /tg/ml
concentration for each individual peptide) in 1 ml volume overnight. A mock
target
was prepared by pulsing cells with peptide-free DMSO diluent to match the DMSO
concentration in the peptide-pulsed targets. The cells were enumerated the
next
morning, and 1 x 10~ cells were resuspended in 0.5 ml R-10 medium. Five to ten
microliters of Na51Cr04 were added to the tubes at the same time, and the
cells were
incubated for 1 to 2 hr 37°C. The cells were then washed 3 times and
resuspended at
5x104 cells/ml in R-10 medium to be used as target cells. The cultured
lymphocytes
were plated with target cells at designated effector to target (E:T) ratios in
triplicates
in 96-well plates, and incubated at 37°C for 4 hours in the presence of
5% C02. A
sample of 30 ~,1 supernatant from each well of cell mixture was harvested onto
a well
of a Lumaplate-96 (Packard Instrument, Meriden, CT), and the plate was allowed
to
air dry overnight. The amount of SICr in the well was determined through beta-
particle emission, using a plate counter from Packard Instrument. The
percentage of
specific lysis was calculated using the formula as: % specific lysis = (E-S) l
(M-S).
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The symbol E represents the average cpm released from target cells in the
presence of
effector cells, S is the spontaneous cpm released in the presence of medium
only, and
M is the maximum cpm released in the presence of 2% Triton X-100.
ELISA Assay - The pol-specific antibodies in the monkeys were measured in a
competitive RT EIA assay, wherein sample activity is determined by the ability
to
block RT antigen from binding to coating antibody on the plate well. Briefly,
Maxisorp plates were coated with saturating amounts of pol positive human
serum
(97111234). 250 uL of each sample is incubated with 15 uL of 266 ng/mL RT
recombinant protein (in RCM 563, 1 % BSA, 0.1 % tween, 0.1 % NaN3) and 20 uL
of
lysis buffer (Coulter p24 antigen assay kit) for 15 min at room temperature.
Similar
mixtures are prepared using serially diluted samples of a standard and a
negative
control which defines maximum RT binding. 200 uL/well of each sample and
standard were added to the washed plate and the plate incubated 16-24 h at
room
temperature. Bound RT is quantified following the procedures described
in:Coulter
p24 assay kit and reported in milliMerck units per mL arbitrarily defined by
the
chosen standard.
Results - Repeated vaccinations with V lJns-IApol induced in 1 of 3 monkeys
(948033) significant levels of antigen-specific T cell activation (Figure 6A-C
and
Table 2) and CTL killing of peptide-pulsed autologous cells (Figure 7A-B). A
significant CD8+ component to the T cell responses in this animal was
confirmed by
peptide-stimulation of CD4-depleted PBMCs in an ELIspot assay (Table 2).
Immunization with VlJns-tpa-IApol produced T cell responses from all 3
vaccinees (Figures 6A-C, Figure 7A-B; Table 2). Two (920078, 948028) exhibited
bulk CTL activity and detectable CD8+ components as measured by Elispot
analyses
of CD4-depleted PBMCs. For the third monkey (920073), the activated T cells
were
largely CD4+ (Table 2). Table 3 shows the time course data on the frequency of
IFN-gamma secreting cells (SFC/million cells) upon antigen-specific
stimulation for
monkeys vaccinated 3x with either VlJns-IApol or VlJns-tpa-IApol (5 mg dose).
At
T=18 wks, CD4-cell depletion were performed; the reported values are the
number of
spots per million of fractionated cells and are not corrected for the
resultant
enrichment of CD8+ T cells. PBMCs were stimulated with peptide pools that
represent either IA pol protein (mpol-1, mpol-2) or wt Pol (wtpol-1, wtpol-2).
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TABLE 2
Vaxine Animd Anti T ~ T~1 T~ T=12 T=18
N0. wk Wk Wk Wk Wk
Dose1 Dase2Dose3
VlJrs-IPpd 948008 r~ltxn 1 15 6 11 11 11
rr~ rrpd-1 3 ffl 28 61 20 15
rrpd-2 0 25 21 19 28 16
v~pd-1 49 20 53 18
w#pd-2 34 24 24 T9
948013 rnedun 0 14 6 9 18 11
rrpd-1 0 9 63 25 34 9
rpd-2 1 15 24 36 24 15
wtpd-1 9 50 33 18
wipd-2 6 21 29 25
948033 rnedun 4 15 11 14 13 8
rrpd-1 3 29 86 51 41 24
t'~pd-2 0 24 25 43 ffl 64
wipd-1 30 38 b0 53
v~pd-2 48 46 86 61
VlJrs-tpc~IPpd920078 rned~xn 0 24 13 11 14 11
5 rr~ rrpd-1 3 110 120 119 155 11
rrpd-2 1 221 130 561 289 145
~nlpd-1 115 53 70 116
wfpd-2 218 2C~ 490 194
9210073rnedun 0 13 3 15 15 6
rrpd-1 0 36 51 113 90 14
rrpd-2 0 29 16 83 115 34
wtpd-1 20 35 100 74
wtpd-2 25 16 79 6T
948028 rrecdxn 0 18 11 18 19 9
rrpd-1 1 30 24 29 30 28
rrpd-2 1 24 23 66 ~ 95
wtpd-1 23 25 34 29
wtpd-2 26 28 71 40
five 920072 rnedun 1 19 3 38 9 4
rrpd-1 0 24 11 25 4 6
t7pd-2 1 24 5 28 6 5
~n~pd-1 i8 13 20 6
v~pd-2 23 14 33 14
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For the Elispot assay, antigen specific stimulation were performed by using
pools of 20-mer peptide pools based on the vaccine sequence. The vaccine pol
sequence differs from the wild-type HIV-I sequence by 9 point mutations,
thereby
affecting 16 of the 20-mer peptides in the pool. Comparable responses were
observed
in the vaccinees when these peptides are replaced with those using the wild-
type
sequences.
Four of the vaccinees gave anti-RT titers above background after 3 dosages of
the plasmids (Table 2).
TABLE 3
Anti-RT levels in Rhesus Macaques Vaccinated 3x (4 week intervals) with
5 mgs of VlJns-IApol or VlJns-tpa-IApol expressed in mMUImL.
Vctxir~IVbrtk T~Wk T~1 T~ T=12 T=16
DCSE DCSE DCSE
1 2 3
VlJns-I , 5
948008 IUD <10 <10 15 14
948013 M <10 <10 <10 <10
948033 f~D <10 <10 25 19
VlJr~s~# ,
5
920078 f~D <10 <10 35 17
920073 M <10 <10 <10 <10
_948028 f~D <10 <10 20 63
EXAMPLE 5
Effect of Codon Optimization on In Vivo Expression and
Cellular Immune Response of wt-pol
Materials arcd Methods - Extraction of virus-derived pol gefae - The gene for
RT-1N
(wt-pol; a non-codon optimized wild type pol gene derived directly from the
HIV IIIB
genome) was extracted and amplified from the HIV IIIB genome using two
primers,
5'-CAG GCG AGA TCT ACC ATG GCC CCC ATT AGC CCT ATT GAG ACT
GTA-3' (SEQ )D N0:29) and 5'-CAG GCG AGA TCT GCC CGG GCT TTA ATC
CTC ATC CTG TCT ACT TGC CAC-3' (SEQ ID N0:30 ), containing BgIII sites.
The reaction contained 200 nmol of each primer, 2.5 U of pfu Turbo DNA
polymerase (Stratagene, La Jolla, CA), 0.2 mM of each dNTPs, and the template
DNA in lOmM KCl, lOmM (NH4)2504, 20mM Tris-HCl pH 8.75, 2mM MgS04,
0.1% TritonX-100, O.lmg/ml bovine serum albumin (BSA). Thermocycling
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conditions were as follows: 20 cycles of 1 min at 95 °C, 1 min at 56
°C, and 4 rains at
72 °C with 15-min capping at 72 °C. The digested PCR fragment
was subcloned into
the BgIII site of the expression plasmid VlJns (Shiver, et al., 1995, Immune
responses
to HIV gp120 elicited by DNA vaccination. In Chanock, R. M., Brown, F.,
Ginsberg,
H. S., and Norrby, E. (Eds.) Vaccines 95. Cold Spring Harbor Laboratory Press,
Cold
Spring Harbor, New York, pp 95-98; see also Example section 1 herein)
expression
plasmid following similar procedures as described above. The ligation mixtures
were
then used to transform competent E. coli DH5 cells and screened by PCR
amplification of individual colonies. Sequence of the entire gene insert was
confirmed. All plasmid constructs for animal immunization were purified by
CsCI
method (Sambrook, et al., 1989, Fritsch and Maniatis, T. (Eds) Molecular
cloning: a
laboratory mafzual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor).
In vitro expression in mammalian cells - 1.5x106 293 cells were transfected
with 1 or 10 ~.g of V1R-wt-pol (codon optimized) and VlJns-wt-pol (virus
derived)
using the Cell Phect kit and incubated for 48 h at 37 °C, 5% COZ, 90%
humidity.
Supernatants and cell lysates were prepared and assayed for protein content
using
Pierce Protein Assay reagent (Rockford, IL). Aliquots containing equal amounts
of
total protein were loaded unto 10-20% Tris glycine gel (Novex, San Diego, CA)
along
with the appropriate molecular weight markers. The pol product was detected
using
anti-serum from a seropositive patient (Scripps Clinic, San Diego, CA) diluted
1:1000
and the bands developed using goat anti-human IgG-HRP (Bethyl, Montgomery,.
TX)
at 1:2000 dilution and standard ECL reagent kit (Pharmacia LKB Biotechnology,
Uppsala, Sweden).
Ultrasensitive RT activity assay of pol cofzstructs - RT activities from codon
optimized wt-pol and IA pol plasmids were analyzed by the Product-Enhanced
Reverse Transcriptase (PERT) assay using Perkin Elmer 7700, Taqman technology
(Arnold, et al., 1999, One-step fluorescent probe product-enhanced reverse
transcriptase assay. In McClelland, M., Pardee, A. (Eds.) Expression genetics:
accelerated and high-throughput methods. Biotechniques Books, Natick, MA, pp.
201-210). Background levels for this assay were determined using 1:100,000
dilution
of lysates from mock (chemical treatment only, no vector) transfected 293
cells. This
background range is set as RT/reaction tube of 0.00 to 56.28 which is taken
from the
mean value of 13.80 +/- 3 standard deviations (sd=14.16). Any individual value
>56.28 would be considered positive for PERT assay. Cells lysates were
prepared
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similarly for the following samples: mock transfection with empty V lJns
vector; no
vector control; transfection with V lJns-tpa-pol (codon optimized); and
transfection
with VlJns-IApol (codon optimized). Samples were serially diluted to 1:100,000
in
PERT buffer and 24 replicates for each sample at this dilution were assayed
for RT
activity.
Rodent imm.unizatiofz with optimized afzd virus-derived pol plasznids - To
compare the immunogenic properties of wt-pol (codon optimized) and virus-
derived
pol gene, cohorts of BALB/c mice (N=10) were vaccinated with 1 ~,g, 10 ~,g,
and 100
~,g doses of V 1R-wt-pol (codon optimized) and V lJns-wt-pol plasmid (virus
derived).
At 5 weeks post dose l, 5 of 10 mice per cohort were boosted with the same
dose of
plasmid they initially received. In all cases, the vaccines were suspended or
diluted in
6 mM sodium phosphate, 150 mM sodium chloride, pH 7.2, and the total dose was
injected to both quadricep muscles in 50 ~.L aliquots using a 0.3-mL insulin
syringe
with 28-1/2G needles (Becton-Dickinson, Franklin Lakes, NJ).
Anti-RT ELISA - Anti-RT titers were obtained following standard secondary.
antibody-based ELISA. Maxisorp plates (NLTNC, Rochester, NY) were coated by
overnight incubation with 100 ~,L of 1 ~.g /mL HIV-1 RT protein (Advanced
Biotechnologies, Columbia, MD) in PBS. The plates were washed with PBS/0.05%
Tween 20 using Titertek MAP instrument (Hunstville, AL) and incubated for
approximately 2h with 200 ~,Llwell of blocking solution (PBS/0.05% tween/1%
BSA). The blocking solution was decanted; 100 ~.L aliquot of serially diluted
serum
samples were added per well and incubated for 2 h at room temperature. An
initial
dilution of 100-fold is performed followed by 4-fold serial dilution. The
plates were
washed and 100 ~,L of 1/1000-diluted HRP-rabbit anti-mouse IgG (ZYMED, San
Francisco, CA) were added with 1 h incubation. The plates were washed
thoroughly
and soaked with 100 ~,L 1,2-phenylenediamine dihydrochloride/hydrogen peroxide
(DAKO, Norway) solution for 15 min. The reaction was quenched by adding 100
~,L
of 0.5M HZS04 per well. OD4~2 readings were recorded using Titertek Multiskan
MCC/340 with S20 stacker. Endpoint titers were defined as the highest serum
dilution that resulted in an absorbance value of greater than or equal to O.1
OD492 (2.5
times the background value).
ELlspot assay - Antigen-specific INFy-secreting cells from mouse spleens
were detected using the ELIspot assay (Miyahira, et al., 1995, Quantification
of
antigen specific CD8+ T cells using an ELISPOT assay. J. lynmunol. Methods
1995,
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181, 45-54). Typically, spleens were collected from 3-5 mice/cohort and pooled
into
a tube of 8-mL complete RPMI media (RPMI1640, 10% FBS, 2mM L-glutamine,
100U/mL Penicillin, 100 u/mL streptomycin, 10 mM Hepes, 50 uM (3-ME).
Multiscreen opaque plates (Millipore, France) were coated with 100 ~,L/well of
5
~.g/mL purified rat anti-mouse IFN-~y IgGl, clone R4-6A2 (Pharmingen, San
Diego,
CA), in PBS at 4°C overnight. The plates were washed with
PBS/penicillin/streptomycin in hood and blocked with 200 ~,L/well of complete
RPMI media for 37 °C for at least 2 h. The mouse spleens were ground
on steel
mesh, collected into 15m1 tubes and centrifuged at 1200rpm for 10 min. The
pellet
was treated with 4 mL ACK buffer (Gibco/BRL) for 5 min at room temperature to
lyse red blood cells. The cell pellet was centrifuged as before, resuspended
in
complete RPMI media (5 ml per mouse spleen), filtered through a cell strainer.
and
counted using a hemacytometer. Block media was decanted from the plates and to
each well, 100 ~,L of cell samples (5x105 cells per well) and 100 ~.L of the
antigen
, solution were added. To the control well, 100 p.L of the media were added;
for
specific responses, peptide pools containing either CD4+ or CD8+ epitopes were
added. In all cases, a final concentration of 4 ~,g/mL per peptide was used.
Each
sample/antigen mixture were performed in triplicate wells. Plates were
incubated at
37°C, 5% C02, 90% humidity for 20-24 h. The plates were washed with
PBS/0.05%
Tween 20 and incubated with 100 ~,L/well of 1.25 ~.g/mL biotin-conjugated rat
anti-
mouse IF'N-Y mAb, clone XMG1.2 (Pharmingen) at 4°C overnight. The
plates were
washed and incubated with 100 p,L/well 1/2500 dilution of strepavidin-alkaline
phosphatase conjugate (Pharmingen) in PBS/0.005% Tween/5% FBS for 30 min at
37 °C. Following a wash, spots were developed by incubating with 100
~1/well 1-step
NBTIBCIP (Pierce Chemicals) for 6-10 min. The plates were washed with water
and
allowed to air dry. The number of spots in each well was determined using a
dissecting microscope and the data normalized to 10~ cell input.
Results - Ifi vitro expressioyz of Pol in mammalian cells - Heterologous
expression of the optimized wt or IA pol genes (V1R-wt-pol (codon optimized),
VlJns-IApol (codon optimized), VlJns-tpa-IApol (codon optimized)) in 293 cells
(Figure 8) yielded a single polypeptide of correct approximate molecular size
(90-kDa) for the RT-1N fusion product. In contrast, no expression could be
detected
by transfecting cells with 1 and 10 ~,g of the VlJns-wt-pol, which bears the
virus-
derived pol.
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Ultrasefisitive RT assay of cells tra~zsfected with Pol constructs - Table 4
summarizes the levels of polymerase activity from mock (vector only) control,
IApol
(codon optimized)and wt-pol plasmids (codon optimized). Results indicate that
the
wild-type POL transfected cells contained RT activity approximately 4-5 logs
higher
than the 293 cell only baseline values. Mock transfected cells contained
activity no
higher than baseline values: The RT activity from opt-IApol-transfected cells
was
also found to be no different than baseline values; no individual reaction
tube resulted
in RT activity higher than the established cut-off value of 56.
Table 4
Sam 1e Av . RT/tubeStandard deviationMinimum Maximum
Vector 16.25 18.52 0.0 42.99
only
IApol (codon2.99 8.01 0.0 35.20
optimized)
Wt-pol 126147 21338 68973 152007
(codon
o timized)
Comparative immunogenicity of optimized and virus-derived pol plasmid - To.
compare the in vivo potencies of both constructs, BALB/c mice (N=10 per group)
were vaccinated with escalating doses (l, 10, 100 ~,g) of either VlJns-wt-pol
(virus
derived) or V1R-wt-pol (codon optimized). At 5 wks post dose 1, 5 of 10
animals
were randomly boosted with the same vaccine and dose they received initially.
Figure 9 shows the geometric mean titers of the BALB/c cohorts determined at 2
wks
past boost. No significant anti-RT titers can be observed from animals
immunized
with one or two doses of the wt-pol plasmid (virus derived). In contrast,
animals
vaccinated with the humanized gene construct gave cohort anti-RT titers
(>1000)
significantly above background levels at doses above 10 ug. The responses seen
at 10
and 100 ug dose of V1R-wt-pol (codon optimized) were boosted approximately
10-fold with a second immunization, reaching titers as high as 10~.
Spleens from all mice in each of the cohorts were collected to be analyzed for
IFN-'y
secretion following stimulation with mixtures of either CD4+ peptide epitopes
or
CD8+ peptide epitopes. The results are shown in Figure 10. All wt-pol
vaccinees did
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not show any significant cellular response above the background controls. In
contrast,
strong antigen-stimulated IFN-'y secretion were observed in a dose-responsive
manner
from animals vaccinated with one or two doses of 10 or more p,g of the wt-pol
(codon
optimized) construct.
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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