Note: Descriptions are shown in the official language in which they were submitted.
CA 02597878 2007-09-13
Capsid Proteins and Uses Therefore
TECHNICAL FIELD
The present invention relates to the development of antigen carriers for
inducing
immune responses, specifically for inducing a cell-mediated immune response
against
the antigen for prophylactic and therapeutic applications. The present
invention also
relates to the development of virus capsid protein based vaccines for
prophylactic and
therapeutic applications.
BACKGROUND OF THE INVENTION
A variety of virus capsid proteins have the intrinsic ability to self-assemble
into highly
organized particles. By using recombinant DNA techniques, capsid proteins can
be
recombinantly produced from different hosts such as mammalian cells, insect
cells,
yeast and E.coli. Often, the produced capsid proteins can self-assemble into
particles
in the hosts that closely resemble virions. The resulted particles are called
virus-like
particles (VLPs). Because of lacking viral genome, VLPs are nonreplicating and
noninfectious (1-14).
There are numerous documented research work and granted patents in the area of
using VLPs prepared from virus capsid proteins as vaccines or using VLPs as
antigen
carriers or antigen delivery systems (or vehicles) to carry desired epitopes
or antigens,
in efforts to enhance the immunogenicity of the carried epitopes or antigens,
and to
prime in vivo class I-restricted cytotoxic responses (1-14, 67).
There is no doubt that VLPs can be expressed abundantly in a variety of
expression
systems by recombinant DNA techniques. There are very little doubts to the
prophylactic or therapeutic potentials of using VLPs as vaccines or using them
as
antigen carriers for eliciting enhanced immune responses, particularly cell-
mediated
immune response against carried antigens or epitopes. Due to their particulate
nature,
VLPs usually can be purified in particles by methods such as salt
precipitation with
ammonium sulfate, density gradient centrifugation, and gel filtration.
However, to use
this technology to produce medicines, in particular for use in humans, there
are still
unsolved problems related to the economically and reproducibly preparing
intact
homogeneous particles from expression host systems with well defined
compositions
able to withstand Iong-term storage (8).
When produced by recombinant DNA technology, VLPs like many other recombinant
proteins will be contaminated with host proteins, lipids, nucleic acids et al.
These
contaminations have to be removed to very low levels to meet the requirements
for
medical application. However, the removal of the contaminations from VLPs is
complicated due to the fact that when VLPs are expressed and assembled in the
CA 02597878 2007-09-13
expression systems, the host proteins and lipids can be incorporated into the
VLPs and
host nucleic acids can be packaged into the VLPs (15-20). Purification of
whole VLPs
wiIl not be able to remove these incorporated or packaged contaminations. More
ever,
VLPs are super-molecular structures with molecular weight normally exceeds
10,00Kd, Possibly due to the poor mass transfer in chromatographic processes
because of the VLPs' massive sizes compare to monomer proteins or other small
molecules, when the separations are conducted by using absorbent resins, the
binding,
elution and fractionation are not as effective and efficient as smaller
molecules.
The importance of being able to purifying totally dissembled capsid proteins
are noted
in US patent No. 6962777 and others (8, 21). VLPs' assembly requires
correctly-folded capsid proteins to start with. Under non-denaturing
conditions, the in
vitro method for the quantitative disassembly and subsequent reassembly of
VLPs is
highly specific for each individual capsid protein and US patent 6962777 might
be the
only published work dealt with this issue with VLPs prepared from human
papillomavirus (HPV) L1 major capsid protein. Many factors significant for
VLPs
formation and stability have not been well elucidated. It is generally known
that
VLPs' disassembly and assembly can be affected by numerous factors. For
example,
pH, ionic strength, post-translational modifications of viral capsid proteins,
disulfide
bonds, and divalent cation bonding. To make this issue even more complicated
is that
the VLPs' disassembly and assembly often require chaperones participation and
for
some VLPs formation, certain specific structure nucleic acids are required (8,
21-36).
Thus, there are numerous interrelated factors which may affect capsid
stability,
assembly and disassembly in vitro, which vary widely even for related viruses.
Further more, the tendency of forming aggregates by partially dissembled or
totally
dissembled capsid proteins is another major obstacles in the process of
producing
homogenous and stable VLPs effectively and efficiently in vitro (8) .
To simply dissemble VLPs, high concentration of Chaotropic agents such as urea
or
guanidine hydrochloride (Gu.HCI) can be used, as these agents will disrupt
non-covalent forces such as hydrogen bonding, Van der Waals interactions, and
the
hydrophobic effect in capsid proteins, and the disulfide bonds in capsid
proteins can
be disrupted simply by reducing agents or oxidative sulfitolysis process. If
just for
producing pure capsid proteins, then to subject VLPs in high concentration of
Gu.HC1
and urea, plus necessary agents to disrupt disulfide bonds in the purification
process
would be advantageous because of the following reasons. (1) Capsid proteins
are
much less likely to form aggregates in high concentration of urea or Gu.HCI,
so the
purification process can be much more efficient and scalable; (2) High
concentration
of urea or Gu.HCI can weak the interactions (hydrogen bonding, Van der Waals
interactions, and the hydrophobic effect) between capsid proteins and
contaminations;
so the purification process can be much more effective in terms at removing
contaminations; (3) VLPs are disintegrated in the high concentration of urea
or
Gu.HCI, so the capsid proteins exhibit more homogenous properties in the
purification process; (4) disintegrated VLPs are much more likely to release
or expose
incorporated or packaged contaminations to the purification forces for
removing them.
CA 02597878 2007-09-13
However, the chaotropic agents such as urea or Gu.HCI are also strong protein
denaturants, proteins are denatured after the treatment with high
concentration of urea
or Gu.HCI, and there is still lack of knowledge on how to correctly refold
denatured
capsid proteins. If denatured capsid proteins are not correctly refolded, they
often
form aggregates instead of self-assemble into VLPs (8, 21).
Therefore, there exists a need in the art for a general method, which would
conduct
the purification of recombinantly expressed capsid proteins in one or more
steps in
high concentration of chaotropic agents (denaturing conditions) plus necessary
agents
to disrupt disulfide bonds, then refold and reassemble of purified homogenous
capsid
proteins.
SUMMARY OF THE INVENTION
The present invention provides compositions for use as antigen or epitope
carriers and
methods to prepare such compositions. In one or more steps of preparation of
the said
compositions, high concentration of chaotropic agents such as urea or Gu.HC1
is (are)
used and the subunits of the compositions are separated and purified in
denatured
forms. The denatured subunits of the said compositions, which contain capsid
proteins
of viruses, are refolded and self-assembled into the compositions, which are
macro-molecules containing multi-subunits, by a process involving gradually
removing out chaotropic agents presented in the denatured subunits by dialysis
or
ultra-filtration. The compositions can be used as antigen or epitope carriers
in
prophylactic or therapeutic applications in the way that authentic VLPs are
used and
may have advantages over authentic VLPs due to their different morphology.
The invention further provides a method for using the compositions as delivery
vehicles for desired moieties. The compositions can also be related to
prophylactic or
therapeutic applications.
DETAILED DESCRIPTION OF THE 1NVENTION
As the refolding and reassembly of capsid proteins are affected by many
factors, most
of them are not well defined and some of the factors are still not unknown, it
will be
difficult to refold and reassemble denatured capsid proteins if some of the
related
factors are unknown or not well defined. To overcome this problem, a fusion
capsid-chaperone protein (FCCP) has been designed based on the following
reasons:
(1) virus capsid proteins have the intrinsic ability to self-assemble into
highly
organized particles, (2) virus capsid proteins can accommodate certain length
of
peptide fused to its N-terminal or C-terminal and still retain the ability to
self-assemble into VLPs (12, 36-37); (3) chaperones can bind and prevent non-
native
or denatured proteins from aggregations and facilitate their folding (38-59),
(4) when
exogenous antigens are particulate in nature, they are presented 1,000 or
10,000-fold
more efficiently than soluble antigen in both class I and class II pathways
(5, 60-68).
The FCCP is composed with a capsid protein and a chaperone protein, the
chaperone
CA 02597878 2007-09-13
protein is linked to capsid protein to its N-terminal or C-terminal via a
peptide bond to
form a single molecule. The objects of this designed FCCP are (1) when the
fusion
protein is recombinantly expressed in a host system, the separation and
purification
process can be conducted in high concentration of chaotropic agents in one or
more
steps, such as up to IOM urea or Gu.HCI can be used in the purification
process,
preferably from 4M-8M for urea and 3-6M for Gu.HCI; (2) the purified
homogenous
FCCP will be refolded with a process involving gradually removing out
chaotropic
agents presented in the purified sample by dialysis or ultra-filtration; (3)
in the
refolding process, FCCP will self-assemble into macro-molecules containing
multi-FCCP subunits, the assembled macro-molecules can be a VLP structure or
another structure with totally different morphology; (4) the macro-molecules
can be
used as antigen or epitope delivery systems, such as protein or peptide based
antigens
or epitopes can be fused to the FCCP by recombinant DNA method to produce
peptide bond linked fusion proteins, or can be chemically linked or conjugated
to the
FCCP; the purified FCCP can be used to incorporate desired moieties, e.g.,
nucleic
acids, proteins, peptides, hormones, anti-cancer agents and antiviral agents
into the
macro-molecules during reassembly. In the FCCP molecules, capsid protein is
the
component mainly responsible for the formation of macro-molecule structures
because of its intrinsic self-assembly ability, chaperone protein is the
component
providing the possibility to process FCCP in denatured form with the
incorporation of
high concentration of chaotropic agents in the purification process and
subsequently
refolding and reassembly. The denatured capsid protein alone often forms
aggregates
in the refolding process, and very often, the denatured capsid protein can not
stay in
non-denaturing solution in soluble forms because of the formation of the
aggregates.
The possible mechanism might be related to the hydrophobic patches in capsid
proteins. Hydrophobic patches in capsid proteins are critical in self-
assembling and
maintaining the VLP structures, and they are buried inside of the capsid
proteins in
the VLP structures (69-74). In the high concentration of denaturant solutions,
such as
urea or Gu.HC1 solutions, capsid proteins are denatured and the buried
hydrophobic
patches are exposed to the solutions. When denaturants are gradually removed
from
the solutions in the refolding process, the gradually increased interaction
among the
exposed hydrophobic patches make the capsid proteins forming aggregates. When
FCCP subjected to the high concentration of urea or Gu.HC1, the hydrophobic
patches
in capsid protein are exposed to the solutions too, but when denaturants are
gradually
removed from the solutions in the refolding process, the exposed hydrophobic
patches
in capsid proteins can be protected by fused chaperone protein, and the FCCP
can stay
in solutions to take refolding and self-assembling process to form soluble
macro-molecules. The FCCP is a different molecule compare to capsid protein,
the
refolding and in vitro assembly process are conducted by gradually removing
out
chaotropic agents presented in the denatured preparation of FCCP, which are
fundamentally different from the nature process of capsid protein's folding
and
self-assembly into VLPs. Because of above reasons, the morphology of the
macro-molecules prepared from this invention may totally differ from authentic
VLPs.
CA 02597878 2007-09-13
One of this invention's findings is that the macro-molecules formed by the
self-assembling of FCCP without the morphology of authentic VLPs can be very
immunogenic, and may have the following advantages over authentic VLPs: (1)
pre-existing immunity to authentic VLPs might be circumvented by the use of
macro-molecules with different morphology; (2) existing immune tolerance to
authentic capsid proteins might be circumvented by the use of the macro-
molecules
with different morphology; (3) the use of macro-molecules with different
morphology
might circumvent the problem of interference with commercial anti-capsid
protein
assays; (4) macro-molecules are much more stable in the solutions. The strong
immunogenicity of the macro-molecules could be due to (1) when exogenous
antigens
are particulate in nature, they are presented 1,000 or 10,000-fold more
efficiently than
soluble antigen in both class I and class II pathways, and macro-molecule
structures
might have features of particle antigens, (2) innate immunity might be able to
recognize some conserved sequences in capsid protein in FCCP, which can work
synergistically to generate strong, lasting immunological responses.
Furthermore the
capsid protein in the FCCP can be utilized to package nucleic acids. Some
nucleic
acids such as double strand RNA and unmethylated CpG-DNA are well known for
their ability to greatly enhance the immune responses (75-83).
Those skilled in the art will recognize and appreciate that in the FCCP
molecule,
capsid protein can be a whole protein, part of the whole protein, science
mutated or
variant of capsid proteins which still retain the ability of self-assembly;
chaperone
protein can be a full length protein, a functional equivalent, such as, a
fragment of
whole chaperone protein, a science mutated or a variant of chaperone protein.
Many
chaperones are heat shock proteins, that is, proteins expressed in response to
elevated
temperatures or other cellular stresses (38-59). The reason for this behaviour
is that
protein folding is severely affected by heat and, therefore, some chaperones
act to
repair the potential damage caused by misfolding. Other chaperones are
involved in
folding newly made proteins as they are extruded from the ribosome.
There are many different families of chaperones; each family acts to aid
protein
folding in a different way. In bacteria like E. coli, many of these proteins
are highly
expressed under conditions of high stress, for example, when placed in high
temperatures. For this reason, the term "heat shock protein" has historicaIly
been used to
name these chaperones. The prefix "Hsp" designates that the protein is a heat
shock
protein.
Some of the common chaperone familys are Hsp60, Hsp70, Hsp90, HsplOO and small
moleculae weight family of Hsp proteins (38-59). Chaperones are not limited to
Hsp
proteins and those skilled in the art will recognize that present unknown
chaperones
can be used to produce FCCP in the method provideed in this invention when
they are
discovered. In this invention, the idea is to have a peptide or a protein
joined to a
capsid protein via a peptide bond, then the fused protein can be processed in
one or
more steps in high concentration of chaotrapic agents such as urea or Gu.HCI
solution,
the purified fusion protein then can be subjected to refold and self assemble
process
by gradually removing chaotrapoic agents out from the samples. After the
purification,
CA 02597878 2007-09-13
refolding and assembling process, the chaperone protein might be cliped off
from
FCCP with a chemical method, or by an enzyemtic method, such as, a specific
enzyme cleavage site can be designed at the joint of the capsid protein and
chaperone
protein . For example, asp asp asp asp lys can be recognized by enterokinase,
and this
sequence can be introduced into the joint of the capsid protein and chaperone
protein,
after refolding and reassembling, the enterokinase can be used to clip off the
chaperone protein.
Thus, the objects of the invention are to solve the problems of the prior art
and
provide a novel method for utilizing viral capsid proteins.
More specifically, it is an object of the invention to provide a novel method
for
utilizing capsid proteins to produce macro-molecules, which are capsid protein
based
vaccines. The macro-molecules are self-assembled by FCCP, which is a capsid
protein
fused to a chaperone protein or their functional fragments fused together.
Still more specifically, it is an object of the invention to use the macro-
molecules as
antigen carriers for eliciting enhanced immune responses, particularly cell-
mediated
immune response against carried antigens or epitopes. Such as, protein or
peptide
based antigens or epitopes can be fused to the FCCP by recombinant DNA method,
or
desired antigens or epitopes can be chemically linked or conjugated to the
FCCP.
It is also an object of the invention to provide a method which enables the
purification
of FCCP in one or more steps in denatured form using high concentration of
chaotropic agents such as urea or Gu.HCI, the purified homogenous FCCP
subsequently refolded and reassembled into macro-molecules by gradually
removing
chaotropic agents out from the denatured FCCP sample.
It is another object of the invention to provide a method for packaging or
encapsulating desired moieties in macro-molecules, e.g., therapeutic or
diagnostic
agents.
It is still another object of the invention to provide a novel delivery system
to
incorporate desired moieties, e.g., nucleic acids, proteins, peptides,
hormones,
anti-cancer agents and antiviral agents into the macro-molecules during
reassembly.
It is still another object of the invention to provide a novel method with the
ability to
prepare homogenous and well defined capsid protein based vaccines in a
scalable
process.
It is still another object of the invention to provide a novel method to
prepare capsid
protein based vaccines with improved quality, e.g., improved homogeneity,
immunogenicity, and stability.
It is still another object of the invention to provide a novel method to
prepare capsid
CA 02597878 2007-09-13
protein based vaccines with the potential to circumvent pre-existing inununity
to
authentic VLPs.
It is still another object of the invention to provide a novel method to
prepare capsid
protein based vaccines with the potential to circumvent existing immune
tolerance to
authentic capsid proteins.
It is still another object of the invention to provide a novel method to
prepare capsid
protein based vaccines with the potential to circumvent the problem associated
with
authentic VLPs of interference with commercial anti-capsid protein based
assays.
The following examples are provided in order to demonstrate and further
illustrate the
present invention, and are not to be construed as limiting the scope thereof.
SEQUENCE LISTS
Sequence 1: The amino acid sequence of the fusion protein of E7-Core-Hsp65 in
example 1.
Sequence 2: The DNA sequence of Ankegens 2479bp for E7-Core-Hsp65 in example
1.
EXAMPLES
Example 1
FCCP molecule carrying an HPV antigen
A chaperone protein-Hsp65 derived from Mycobacterium bovis BCG hsp65 gene (84)
is fused to the C-terminal of the nucleocapsid protein (core antigen) of
Hepatitis B virus
(HBV) subtype ADW2 (85-86) to form a FCCP molecule. An E7 protein from human
papillomavirus type 16 (87) is fused to the N-terminal of the FCCP molecule.
The
amino acid sequence of the fusion protein of E7-Core-Hsp65 is listed in
attached
Sequence 1. According to the amino acid sequence, the DNA sequence was
designed
and synthesized.
The synthesized DNA sequence was named Ankegens 2479bp and cloned into Smal
digested pBluescript II SK (+/-) from Stratagene (88) to produce
pBSK-Ankegens-2479bp. The DNA sequence of Ankegens 2479bp for
E7-Core-Hsp65 is listed in attached Sequence 2.
Example 2
Expression and purification of E7-Core-Hsp65 fusion protein
CA 02597878 2007-09-13
E7-Core-BCG65 DNA fragment was cut from pBSK-Ankegens-2479bp by Ndel and
EcoRl then subcloned into pET-23a (89) corresponding sites to produce
pET-23a-2479. The pET-23a-2479 was transformed into Rosetta-gami(DE3) from
Novagen. E7-Core-BCG65 fusion protein was expressed in E. coil cells by
fermentation and induction of transformed Rosetta-gami(DE3) cells with 0.5mM
isopropyl-thio-galatopyranoside according to Novagen's pET System Manual.
After
fermentation, cells were harvested by centrifugation. Cells were washed once
by
suspending 100g cell paste in 1000m1 of buffer A (100mM Tris-Hcl pH 9.0; 5mM
EDTA) then centrifuging at 8500rpm for 30 minutes. Discarded the supernatant
then
re-suspended the pelleted cells with 1000m1 of buffer B(50mM sodium acetate;
2mM
EDTA). The suspended cells were ruptured by homogenization process with
pressure
at 760bar, and then centrifuged at 8500rpm for 30 minutes. The supernatant was
collected and the volume was measured. Urea was added to the supernatant
according
to 0.7g urea for lmi supernatant, and then sodium chloride was added to final
concentration at 100 mM, L-Cysteine was added to final concentration at 20mM.
The
solution was stirred at room temperature to have all the urea dissolved then
stirred at 4
`C for overnight. After overnight stirring, the sample was applied to an XK-50
column (GE Health) containing 300ml of SP-Sepharose resin (GE Health), which
was
previously washed with 1 M sodium chloride and equilibrated with buffer C(50mM
sodium acetate; 100mM NaCI; 2mM EDTA; 8M urea; 10mM L-Cysteine). After
sample loading, the column was washed with 10 column-volumes of buffer D(50mM
sodium acetate; 100mM NaCI; 2mM EDTA; 8M urea; 10mM L-Cysteine; 2.5%
Triton-X-100) overnight to remove endotoxin. After overnight washing with
buffer D,
the column was washed with 5 column-volumes of buffer C to remove Triton-X-
100,
and then the column was washed with 3 column-volumes of buffer E(50mM
sodium acetate; 300mM NaCl; 2mM EDTA; 8M urea; 10mM L-Cysteine) to remove
contaminations. E7-Core-BCG65 fusion protein was eluted from the column with
buffer D (50mM sodium acetate; 800mM NaCI; 2mM EDTA; 8M urea; 10mM
L-Cysteine). Pooled eluted protein was dialyzed against 4X 40 volumes of
buffer F
(50mM sodium acetate, 6Murea) to remove NaCl and L-Cysteine. After dialysis,
Oxidative sulfitolysis was performed by adding sodium sulfite and sodium
tetrathionate to final concentrations of 200mM and 50mM respectively and
incubating
for overnight at room temperature. The sulfitolyzed sample was diluted 5
volumes
with buffer F then applied to an XK-50 column with 150m1 of Q-Sepharose resin
(GE
Health), which was previously washed with 1M NaCI and equilibrated with buffer
F.
After sample loading, the column was washed with 2 column-volumes of 95%
buffer
F and 5% buffer G (50mM sodium acetate; 1M NaCI; 6Murea), and then
E7-Core-BCG65 fusion protein was eluted with a lineal gradient from 95% buffer
F
and 5% buffer G to 50% buffer F and 50% buffer G over 8 column-volumes. Eluted
E7-Core-BCG65 fusion protein was pooled, and then dialyzed against 1X40
volumes
of Tris.HCl pH9.0, 1X40 volumes of Tris.HCl pH7.5 with 100mM NaCI to remove
urea and refold E7-Core-BCG65 fusion protein. The endotoxin levels in the
final
preparations (E7-Core-BCG65 in Tris.HCl pH7.5 with 100mM NaCI) were below
CA 02597878 2007-09-13
5EU/mg protein.
Example 3
Therapeutic and prophylactic effects of E7-Core-BCG65 treatment in mice
The E7-Core-BCG65 is an FCCP carrying an E7 antigen from HPV type 16, and the
E7 expressing TC-1 tumor cells were used to evaluate the therapeutic and
prophylactic applications of E7-Core-BCG65 on mice bearing TC-1 tumor or being
challenged with TC-1 tumor.
Female C57BL/6 mice, six to eight weeks old (20.0 2.0g) were purchased from
SHANGHAI SLAC LABORATORY ANIMAL CO. LTD. Quality Control No.:
SCXK(Shanghai)2003-0003 0
TC-1 cell line expressing HPV16 E7 protein was derived from primary lung cells
of
C57BU6 mice by immortalization and transformation with HPV16 E7 gene and an
activated human C-Ha-ras gene as described in Lin et al. (90). TC-1 cells were
grown
in RPMI1640 medium supplemented with 10% fetal calf serum, 2mM nonessential
amino acids, 2mM L-glutamine, 1mM pyruvate, Penicillin/Streptomycin, and the
cells
were harvested by trypsinization, the cells were washed three times with PBS
then
re-suspended in PBS. 1X105 TC-1 cells were inoculated subcutaneously into the
mice and the mice were treated with E7-Core-BCG65 or saline subcutaneously
according to their experiment groups.
Animal experiment groups:
Groups Mice Dose Time of E7-Core-BCG65 Treatment
Treatment
Therapeutic 8 500ug 48h and 16 days after inoculation of TC-1
Application E7-Core-BCG6
8 100ug 48h and 16 days after inoculation of TC-1
E7-Core-BCG6
5
8 20ug 48h and 16 days after inoculation of TC-1
E7-Core-BCG6
5
Prophylactic 8 100ug Two treatments with 14 days in between
Application E7-Core-BCG6 Inoculation of TC-1 14 days after second
5 treatment
8 20ug Two treatments with 14 days in between
E7-Core-BCG6 Inoculation of TC-1 14 days after second
5 treatment
Control 6 Saline 48h and 16 days after inoculation of TC-1
CA 02597878 2007-09-13
The mice were monitored for the presence or absence of tumor by palpation and
the
volume of the tumor was measured with Vernier Caliber by 2 orthogonal
dimensions
twice a week; these measurements were extrapolated to mm3 and are presented as
average tumor volume standard error of the mean. The life span of the mice
was
recorded.
In control group, the presence of the tumor was observed 4 days after TC-1
inoculation; the average volume of the tumor was grown to 40mm3 10 day after
inoculation and 7499.84mm3 36 days after inoculation. All mice in the control
group
died within 60 days after inoculation.
In therapeutic group, mice were treated with E7-Core-BCG65 48h and 16 days
after
TC-1 inoculation; the average volume of the tumor was grown to 181.89mm3
(500ug),
671.34mm3 (IOOug) and 2148.57mm3 (20ug) 36 days after inoculation. All mice
were alive 60 days after inoculation.
In the prophylactic group, mice were treated with E7-Core-BCG65 twice in 14
days,
and after second treatment, mice were inoculated with TC-1; the average volume
of
the tumor was grown to22.43mm3 (100ug) and 89.08mm3 (20ug) 36 days after
inoculation. All mice were alive 60 days after inoculation.
Table 1 The average tumor volume in different experiment groups (mm) (z s)
'Iherapeutic Group Prophylactic Group Control
Date
(day) SOOug 100ug 20ug 100ug 20ug Group
(n=8) (n=8) (n=8) (n=8) (n=8) (n=6)
8.65 5.40 16.33 8.83 42.54 24.55* 2.32 1.06 5.56 2.91 39.00 19.28
13 41.70 20.90 51.01 20.37 84.72-+36.72* 1.97 2.44 10.58 25.56 133.57 69.64
16 31.91 12.26 49.96 20.62 189.07 91.07* 1.97 1.42 5.24 2.08 320.20 149.14
19 35.69 10.52 156.28 46.49 208.49 85.46 2.85 1.49 25.65 10.43 782.65 257.69
22 43.89 21.13 224.71 107.46 357.47 159.47 2.44 1.98 35.24 80.41 1033.81
594.12
25 109.04 47.41 257.01 107.19 756.40 258.40 4.52 2.78 17.38 6.76 2414.19
1201.87
28 127.68 56.24 395.56 128.72 892.54 364.47 18.81 5.42 69.63 24.46 4432.67
1824.46
31 156.124-49.46 525.81 152.94 1527.21 510.46 20.57 10.46 85.57 25.85 6024.54
2465.46
34 181.89 75.53 671.34 301.28 2048.57 1050.57 22.42 18.60 89.08 43.09 7499.84
3722.56
37 250.52 120.59 785.69 268.85 3051.65 1253.32 56.83 25.56 173.59 89.41
9483.58 4565.74
40 396.88+~08.12 921.87 368.03 3887.08 1889.08 59.81 26.79 173.92 86.39
13141.43 5077.39
Those skilled in the art will recognize, or be able to ascertain that the
basic
construction in this invention can be altered to provide other embodiments
which
utilize the process of this invention. Therefore, it will be appreciated that
the scope of
this invention is to be defmed by the claims appended hereto rather than the
specific
embodiments which have been presented hereinbefore by way of example.
CA 02597878 2007-09-13
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CA 02597878 2007-09-13
87. HPV 16-E7 (GenBank accession #K02718)
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CA 02597878 2007-09-13
HMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVT
FCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPMDIDPYKEFGATVEL
LSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWV
GNNLEDPASRDLWNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTP
PAYRPPNAPILSTLPETTWRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQCM
AKTIAYDEEARRGLERGLNALADAVKVTLGPKGRNWLEKKWGAPTITNDGVSIAKE
IELEDPYEKIGAELVKEVAKKTDDVAGDGTTTATVLAQALVREGLRNVAAGANPLGL
KRGIEKAVEKVTETLLKGAKEVETKEQIAATAAISAGDQSIGDLIAEAMDKVGNEGV
ITVEESNTFGLQLELTEGMRFDKGYISGYFVTDPERQEAVLEDPYILLVSSKVSTVK
DLLPLLEKVIGAGKPLLIIAEDVEGEALSTLWNKIRGTFKSVAVKAPGFGDRRKAM
LQDMAILTGGQVISEEVGLTLENADLSLLGKARKVVVTKDETTIVEGAGDTDAIAGR
VAQIRQEIENSDSDYDREKLQERLAKLAGGVAVIKAGAATEVELKERKHRIEDAVRN
AKAAVEEGIVAGGGVTLLQAAPTLDELKLEGDEATGANIVKVALEAPLKQIAFNSGL
EPGWAEKVRNLPAGHGLNAQTGVYEDLLAAGVADPVKVTRSALQNAASIAGLFLTT
EAVVADKPEKEKASVPGGGDMGGMDF
Sequence 1: The amino acid sequence of the fusion protein of E7-Core-Hsp65
V
CA 0259787:8 2007-09-13
catatgcatggagatacacctacattgcatgaatatatgttagatttgcaaccagagaca
actgatctctactgttatgagcaattaaatgacagctcagaggaggaggatgaaatagat
ggtccagctggacaagcagaaccggacagagcccattacaatattgtaaccttttgttgc
aagtgtgactctacgcttcggttgtgcgtacaaagcacacacgtagacattcgtactttg
gaagacctgttaatgggcacactaggaattgtgtgccccatctgttctcagaaaccaatg
gacattgacccttataaagaatttggagctactgtggagttactctcgtttttgccttct
gacttctttccttccgtcagagatctcctagacaccgcctcagctctgtatcgggaagcc
ttagagtctcctgagcattgctcacctcaccatactgcactcaggcaagccattctctgc
tggggggaattgatgactctagctacctgggtgggtaataatttggaagatccagcatcc
agggatctagtagtcaattatgttaatactaacatgggtttaaagatcaggcaactattg
tggtttcacatatcttgccttacttttggaagagagactgtacttgaatatttggtatct
ttcggagtgtggattcgcactcctccagcctatagaccaccaaatgcccctatcttatca
acacttccggaaactactgttgttagacgacgggaccgaggcaggtcccctagaagaaga
actccctcgcctcgcagacgcagatctcaatcgccgcgtcgcagaagatctcaatctcgg
gaatctcaatgtatggccaagacaattgcgtacgacgaagaggcccgtcgcggcctcgag
cggggcttgaacgccctcgccgatgcggtaaaggtgacattgggccccaagggccgcaac
gtcgtcctggaaaagaagtggggtgcccccacgatcaccaacgatggtgtgtccatcgcc
aaggagatcgagctggaggatccgtacgagaagatcggcgccgagctggtcaaagaggta
gccaagaagaccgatgacgtcgccggtgacggcaccacgacggccaccgtgctggcccag
gcgttggttcgcgagggcctgcgcaacgtcgcggccggcgccaacccgctcggtctcaaa
cgcggcatcgaaaaggccgtggagaaggtcaccgagaccctgctcaagggcgccaaggag
gtcgagaccaaggagcagattgcggccaccgcagcgatttcggcgggtgaccagtccatc
ggtgacctgatcgccgaggcgatggacaaggtgggcaacgagggcgtcatcaccgtcgag
gagtccaacacctttgggctgcagctcgagctcaccgagggtatgcggttcgacaagggc
itacatctcggggtacttcgtgaccgacccggagcgtcaggaggcggtcctggaggacccc
tacatcctgctggtcagctccaaggtgtccactgtcaaggatctgctgccgctgctcgag
aaggtcatcggagccggtaagccgctgctgatcatcgccgaggacgtcgagggcgaggcg
ctgtccaccctggtcgtcaacaagatccgcggcaccttcaagtcggtggcggtcaaggct
cccggcttcggcgaccgccgcaaggcgatgctgcaggatatggccattctcaccggtggt
caggtgatcagcgaagaggtcggcctgacgctggagaacgccgacctgtcgctgctaggc
aaggcccgcaaggtcgtggtcaccaaggacga.gaccaccatcgtcgagggcgccggtgac
accgacgccatcgccggacgagtggcccagatccgccaggagatcgagaacagcgactcc
gactacgaccgtgagaagctgcaggagcggctggccaagctggccggtggtgtcgcggtg
atcaaggccggtgccgccaccgaggtcgaact.caaggagcgcaagcaccgcatcgaggat
gcggttcgcaatgccaaggccgccgtcgagga.gggcatcgtcgccggtgggggtgtgacg
ctgttgcaagcggccccgaccctggacgagctgaagctcgaaggcgacgaggcgaccggc
gccaacatcgtgaaggtggcgctggaggcccc:gctgaagcagatcgccttcaactccggg
ctggagccgggcgtggtggccgagaaggtgcgcaacctgccggctggccacggactgaac
gctcagaccggtgtctacgaggatctgctcgc:tgccggcgttgctgacccggtcaaggtg
acccgttcggcgctgcagaatgcggcgtccat.cgcggggctgttcctgaccaccgaggcc
gtcgttgccgacaagccggaaaaggagaaggc:ttccgttcccggtggcggcgacatgggt
ggcatggatttctgaattc
Sequence 2: DNA Sequence of Ankegens 2479bp for E7-Core-Hsp65 Fusion Protein
