U.S. patent application number 12/812053 was filed with the patent office on 2010-11-11 for baculovirus-based vaccines.
This patent application is currently assigned to Konkuk University Industrial Cooperation Corp.. Invention is credited to Young-Bong Kim, Hee Jung Lee, Yu-Kyoung Oh, Nuri Park.
Application Number | 20100285056 12/812053 |
Document ID | / |
Family ID | 40853624 |
Filed Date | 2010-11-11 |
United States Patent
Application |
20100285056 |
Kind Code |
A1 |
Kim; Young-Bong ; et
al. |
November 11, 2010 |
Baculovirus-Based Vaccines
Abstract
The present invention relates to a recombinant baculovirus
comprising: (a) a nucleotide sequence encoding a foreign virus
envelope protein; (b) a first promoter operatively linked to the
envelope-encoding nucleotide sequence; (c) a nucleotide sequence
encoding an antigen protein; and (d) a second promoter operatively
linked to the antigen-encoding nucleotide sequence; and a vaccine
composition using the same. The recombinant baculovirus of the
present invention has an excellent efficacy on both humoral and
cellular immune responses against a specific antigen (e.g., HPV
L1), enabling to function as a more efficient DNA vaccine.
Inventors: |
Kim; Young-Bong;
(Gyeonggi-do, KR) ; Lee; Hee Jung; (Seoul, KR)
; Park; Nuri; (Seoul, KR) ; Oh; Yu-Kyoung;
(Seoul, KR) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Assignee: |
Konkuk University Industrial
Cooperation Corp.
Seoul
KR
KR Biotech Co., Ltd.
Seoul
KR
|
Family ID: |
40853624 |
Appl. No.: |
12/812053 |
Filed: |
January 9, 2009 |
PCT Filed: |
January 9, 2009 |
PCT NO: |
PCT/KR09/00136 |
371 Date: |
July 8, 2010 |
Current U.S.
Class: |
424/199.1 ;
435/235.1; 536/23.72 |
Current CPC
Class: |
C12N 2710/20034
20130101; A61P 31/22 20180101; C12N 7/00 20130101; C12N 2710/14171
20130101; C12N 2710/14143 20130101; A61P 31/18 20180101; A61P 31/14
20180101; C12N 2740/10022 20130101; A61P 37/04 20180101; A61K 39/12
20130101; A61P 31/16 20180101; A61P 35/00 20180101; A61P 31/20
20180101; C12N 2710/14145 20130101; C12N 2830/60 20130101; C12N
2830/85 20130101; A61K 2039/5256 20130101; C12N 2800/22 20130101;
C12N 15/86 20130101; C12N 2810/6054 20130101 |
Class at
Publication: |
424/199.1 ;
435/235.1; 536/23.72 |
International
Class: |
A61K 39/12 20060101
A61K039/12; C12N 7/01 20060101 C12N007/01; C07H 21/04 20060101
C07H021/04; A61P 37/04 20060101 A61P037/04; A61P 31/20 20060101
A61P031/20; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 9, 2008 |
KR |
10-2008-0002733 |
Claims
1. A recombinant baculovirus comprising: (a) a nucleotide sequence
encoding a foreign virus envelope protein derived from a
retrovirus; (b) a first promoter operatively linked to the
envelope-encoding nucleotide sequence; (c) a nucleotide sequence
encoding an antigen protein; and (d) a second promoter operatively
linked to the antigen-encoding nucleotide sequence.
2. The recombinant baculovirus according to claim 1, wherein the
antigen comprises a viral antigen, a bacterial antigen, a parasitic
antigen or a cancer antigen.
3. The recombinant baculovirus according to claim 2, wherein the
antigen comprises the viral antigen selected from the group
consisting of HPV (human papillomavirus) antigen, HBV (hepatitis B
virus) antigen, HCV (hepatitis C virus) antigen, HIV (human
immunodeficiency virus) antigen, rotavirus antigen, influenza virus
antigen, HSV (herpes simplex virus) antigen, avian influenza virus
antigen, hog cholera virus antigen, foot-and-mouth disease virus
antigen and Newcastle disease virus antigen.
4. The recombinant baculovirus according to claim 3, wherein the
antigen is HPV antigen.
5. The recombinant baculovirus according to claim 4, wherein the
antigen comprises HPV L1, L2, E6 or E7 protein.
6. The recombinant baculovirus according to claim 4, wherein the
HPV antigen protein is derived from HPV selected from the group
consisting of HPV type 1, 2, 3a, 4, 5, 6b, 7, 8, 9, 10, 11a, 12,
13, 16 and 18.
7-8. (canceled)
9. The recombinant baculovirus according to claim 1, wherein the
virus envelope protein comprises a HERV (human endogenous
retrovirus) envelope protein.
10. The recombinant baculovirus according to claim 9, wherein a
nucleotide sequence encoding the HERV envelope protein comprises a
nucleotide sequence encoding an amino acid sequence of SEQ ID
NO:2.
11. The recombinant baculovirus according to claim 1, wherein the
first promoter is a promoter operable in insect cells.
12. The recombinant baculovirus according to claim 11, wherein the
promoter operable in insect cells comprises IE-1 promoter, IE-2
promoter, p35 promoter, p10 promoter, gp64 promoter or polyhedrin
promoter.
13. The recombinant baculovirus according to claim 1, wherein the
second promoter is a promoter derived from a genome of a mammalian
cell, or a promoter derived from a mammalian virus.
14. The recombinant baculovirus according to claim 13, wherein the
second promoter comprises U6 promoter, H1 promoter, CMV (cytomegalo
virus) promoter, adenovirus late promoter, vaccinia virus 7.5K
promoter, SV40 promoter, HSV tk promoter, RSV promoter, human
elongation factor 1.alpha. (hEF1.alpha.) promoter, methallothionein
promoter, .beta.-actin promoter, human IL-2 gene promoter, human
IFN gene promoter, human IL-4 gene promoter, human lymphotoxin gene
promoter, human GM-CSF gene promoter, TERT promoter, PSA promoter,
PSMA promoter, CEA promoter, E2F promoter, AFP promoter or albumin
promoter.
15-16. (canceled)
17. A method for inducing an immune response against a specific
antigen in a subject, comprising administering a pharmaceutically
effective amount of the vaccine composition of claim 1.
18. The method according to claim 17, wherein the recombinant
baculovirus in the vaccine composition comprises a HPV (human
papillomavirus) antigen gene, and the method is a method for
preventing or treating a HPV infection-induced cancer.
19. A nucleic acid molecule encoding a HERV (human endogenous
retrovirus) envelope protein, comprising the nucleotide sequence of
SEQ ID NO:1.
20. The recombinant baculovirus according to claim 1, wherein the
retrovirus is an endogenous retrovirus.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a recombinant (chimera)
baculovirus and a vaccine composition thereof.
[0003] 2. Description of the Related Art
[0004] HPV (human papillomavirus) is a causative factor in the
development of cervical cancer which occupies approximately 12% of
global women's cancer. Incidence frequency and mortality of
cervical cancer is higher enough to be at a rate of one person per
2 min in the world (Vaccine 24: 5235-5244 (2006)). Presently, about
100 types of HPV have been identified. Of them, HPV type 16 and HPV
type 18 classified as high risk group have been found in the tissue
of cervical cancer at a ratio of not less than 70% (Vaccine 22:
3004-3007 (2004)).
[0005] To modulate cervical cancer, an effective vaccine
development for HPV infection has been attempted. First of all,
there is urgently demanded a vaccine development for preventing
cervical cancer.
[0006] HPV L1 protein as VLPs (virus like particles) has inherent
self-assembly potential, allowing to form external envelopes
without viral genome. It has been reported that VLP contributes to
sufficient induction of immune responses to produce a neutral
antibody having higher titer (Journal of Virology 81 (24):
13927-13931 (2007); Virology 321: 205-216 (2004); Journal of
Medical Virology 80: 841-846 (2008)).
[0007] For efficient gene delivery in vivo, gene delivery systems
have been developed using numerous viral vectors. For the purpose
of gene therapy, viral vectors such as retrovirus and adenovirus
are utilized to deliver HPV16L1 gene to an animal host (Science 260
(5110): 926-932 (1993)). However, utility of these viruses cause
several drawbacks including: (a) virus proliferation in a
replication-dependent manner; (b) cytotoxicity; (c) induction of
early immune responses; and (d) expression of undesired viral
genes.
[0008] By contrast, a baculovirus transfer vector has important
advantages as follows: (a) insertion of a foreign gene having a
relatively large size; and (b) post-translation processing due to
use of insect cells (higher eukaryotic cells). The latter advantage
is very crucial in the senses that the biological and immunological
activity of a recombinant protein expressed by using a baculovirus
transfer vector are almost equivalent to those of original protein
compared with protein produced in prokaryote, E. coli. In addition,
baculovirus has been known to be a biologically safe virus because
its replication is impossible in animal cells and it induces no
cytotoxicity (Virology 125: 107-117 (1983); Hum. Gene Ther. 7:
1937-1945 (1996); Proc. Natl. Acad. Sci. USA 96: 127-132 (1999);
Trends Biotechnol. 20: 173-180 (2002)). It has been known that the
replication of AcNPV (Autographa californica nuclear polyhedrosis
virus) belonging to be an insect virus group is also impossible in
a variety of animal cells, whereas it is possible to deliver a gene
into cells through its infection (Proc. Natl. Acad. Sci. USA 92:
10099-10103 (1995); Proc. Natl. Acad. Sci. USA 93: 2348-2352
(1996)). Previously, it was reported that a specific gene in AcNPV
genome could be highly expressed in animal cells where it is
controlled by an animal promoter (Journal of Virology, 76 (11):
5729-5736 (2002); Vaccine 26 (20): 2451-2456 (2008)).
[0009] Recently, several studies tried to increase a gene transfer
efficiency by introducing Env of other viruses onto the surface of
baculovirus, for example including diverse reports obtaining higher
gene transfer efficiency by introduction of a VSV envelope G
protein onto the surface of baculovirus (Journal of Virology, 78
(16): 8663-8672 (2004); Journal of Urology 250 (2): 276-283 (2006);
Biochemical and Biophysical Research Communications 289 (2):
444-450 (2001); Journal of Virology 75 (6): 2544-2556 (2001)), or
by adding a gp64 protein on virus surface (Human Gene Therapy, 14
(1): 67-77 (2003)). In addition, vaccination using a baculovirus
vector was known to induce immune responses against a hemagglutinin
glycoprotein of influenza virus (Journal of Immunology, 171:
1133-1139 (2003)).
[0010] Throughout this application, various patents and
publications are referenced and citations are provided in
parentheses. The disclosure of these patents and publications in
their entities are hereby incorporated by references into this
application in order to more fully describe this invention and the
state of the art to which this invention pertains.
DETAILED DESCRIPTION OF THIS INVENTION
[0011] The present inventors have made intensive studies to develop
a baculovirus-based vaccine capable of inducing more enhanced
immune responses against various pathogens. As results, we have
discovered that an expression construct and a recombinant
baculovirus are prepared by combinations of a nucleotide sequence
encoding an antigen gene and a nucleotide sequence encoding a
foreign virus envelope protein, and immunization using the same
leads to induce highly enhanced immune responses, providing a
stable and economic vaccine.
[0012] Accordingly, it is an object of this invention to provide a
recombinant baculovirus.
[0013] It is another object of this invention to provide a vaccine
composition.
[0014] It is still another object to this invention to provide a
method for inducing an immune response against a specific
antigen.
[0015] It is further still another object to this invention to
provide a nucleic acid molecule encoding a HERV (human endogenous
retrovirus) envelope protein.
[0016] It is another object to this invention to provide a
recombinant vector comprising a HERV envelope protein-encoding
nucleic acid molecule.
[0017] It is still another object to this invention to provide a
baculovirus-based gene delivery system comprising a HERV envelope
protein-encoding nucleic acid molecule.
[0018] Other objects and advantages of the present invention will
become apparent from the following detailed description together
with the appended claims and drawings.
[0019] In one aspect of this invention, there is provided a
recombinant baculovirus comprising: (a) a nucleotide sequence
encoding a foreign virus envelope protein; (b) a first promoter
operatively linked to the envelope-encoding nucleotide sequence;
(c) a nucleotide sequence encoding an antigen protein; and (d) a
second promoter operatively linked to the antigen-encoding
nucleotide sequence.
[0020] In another aspect of this invention, there is provided a
vaccine composition comprising the recombinant baculovirus of this
invention as an active ingredient.
[0021] In still another aspect of this invention, there is provided
a method for inducing an immune response against a specific antigen
in a subject, comprising administering the vaccine composition of
this invention.
[0022] The present inventors have made intensive studies to develop
a baculovirus-based vaccine capable of inducing more enhanced
immune responses against various pathogens. As results, we have
discovered that an expression construct and a recombinant
baculovirus are prepared by combinations of a nucleotide sequence
encoding an antigen gene and a nucleotide sequence encoding a
foreign virus envelope protein, and immunization using the same
leads to induce highly enhanced immune responses, providing a
stable and economic vaccine.
[0023] It is the most features of the present invention to utilize
combinations of a nucleotide sequence encoding an antigen gene and
a nucleotide sequence encoding a foreign virus [most preferably,
HERV (human endogenous retrovirus)] envelope protein.
[0024] The recombinant baculovirus of this invention may be useful
for delivery of various antigen genes. The term "antigen gene" or
"nucleotide sequence encoding an antigen protein" used herein
refers to a nucleotide sequence encoding an antigenic protein (for
example, cell or virus envelope protein as an antigen) to be
recognized by an immune system.
[0025] According to a preferable embodiment, the antigen includes a
viral antigen, a bacterial antigen, a parasitic antigen or a cancer
antigen, more preferably a viral antigen or a cancer antigen, and
most preferably a viral antigen.
[0026] Illustrative example of the viral antigen capable of being
used in the present invention includes an antigen derived from
orthomyxoviruses such as influenza virus; retroviruses such as RSV
(respiratory syncytial virus), SIV (simian immunodeficiency virus)
and HIV; herpesviruses such as EBV (Epstein-Barr Virus); CMV
(cytomegalovirus) or HSV (herpes simplex virus); lentiviruses;
rhabdoviruses such as rabies; picomoviruses such as poliovirus;
rotavirus; and parvoviruses. As the viral antigen to be more
concrete, the example of HPV antigen includes HPV L1, L2, E6 or E7
protein; the example of HIV antigen includes a T-cell and B-cell
epitope such as nef, p24, gp120, gp41, tat, rev, pol, env and gp120
(Palker et al., J. Immunol., 142: 3612-3619 (1989)). The example of
HBV envelope antigen is disclosed in Wu et al., Proc. Natl. Acad.
Sci., USA, 86: 4726-4730 (1989). The example of rotavirus antigen
includes VP4 (Mackow et al., Proc. Natl. Acad. Sci., USA, 87:
518-522 (1990)) and VP7 (Green et al., J. Virol., 62: 1819-1823
(1988); influenza virus antigen includes a hemagglutin (HA) and a
nucleoprotein; HSV antigen includes a thymidine kinase (Whitley et
al., In: New Generation Vaccines, pages 825-854); avian influenza
virus antigen includes a hemagglutin; hog cholera virus antigen
includes an envelope protein; foot-and-mouth disease virus antigen
includes an envelope protein; and Newcastle disease virus antigen
includes HN (hemagglutinin-neuraminidase) or F (fusion
protein).
[0027] Exemplary example of the bacterial antigen capable of being
used in the present invention includes an antigen derived from
Mycobacterium spp., Helicobacter pylori, Salmonella spp., Shigella
spp., E. coli, Rickettsia spp., Listeria spp., Legionella
pneumoniae, Pseudomonas spp., Vibrio spp. and Borellia burgdorferi.
More concretely, illustrative example of the bacterial antigen
capable of being used in the present invention includes form-1
antigen of Shigella sonnei (Formal et aZ, Infect. Immun., 34:
746-750 (1981)); an O-antigen of V. cholerae (Forrest et al., J.
Infect. Dis. 159: 145-146 (1989); a FA/I fimbrial antigen of E.
coli (Yamamoto et al., Infect. Immun., 50: 925-928 (1985)) and a
non-toxic B-subunit of thermosensitive toxin (Klipstein et al.,
Infect. Immun., 40: 888-893 (1983)); a pertactin of Bordetella
pertussis (Roberts et al., Vacc., 10: 43-48 (1992)); an adenylate
cyclase-hemolysin of B. pertussis (Guiso et al., Micro. Path., 11:
423-431 (1991)); and a tetanus toxin fragment C of Clostridium
tetani (Fairweather et al., Infect. Immun., 58: 1323-1326
(1990)).
[0028] Exemplified example of the parasitic antigen capable of
being used in the present invention includes an antigen derived
from Plasmodium spp., Trypanosome spp., Giardia spp., Boophilus
spp., Babesia spp., Entamoeba spp., Eimeria spp., Laishmahia spp.,
Schistosome spp., Brugia spp., Fascida spp., Dirofilaria spp.,
Wuchereria spp., and Onchocerea spp. More concretely, the example
of the parasitic antigen capable of being used in the present
invention includes a circumsporozoite antigen of Plasmodium spp.
such as a circumsporozoite antigen of Plasmodium bergerii and a
circumsporozoite antigen of P. falciparum (Sadoff et al., Sci.,
240: 336-337 (1988)); a merozoite surface antigen of Plasmodium
spp. (Spetzler et al., Int. J. Pept. Prot. Res., 43: 351-358
(1994)); a galactose-specific lectin of Entamoeba htstolytica (Mann
et al., Proc. Natl. Acad. Sci., USA, 88: 3248-3252 (1991)); a gp63
of Leishmania spp. (Russell et al., J. Immunol., 140: 1274-1278
(1988)); a paramyosin of Brugia malayi (Li et al., Mol. Biochem.
Parasitol., 49: 315-323 (1991)); and a triose-phosphate isomerase
of Schistosoma mansoni (Shoemaker et al., Proc. Natl. Acad. Sci.
USA, 89: 1842-1846 (1992)).
[0029] Illustrative example of the cancer antigen capable of being
used in the present invention includes a prostate-specific antigen
(Gattuso et al., Human Pathol., 26: 123-126 (1995)), TAG-72 and CEA
(carcinoembryonic antigen) (Guadagni et al., Int. J. Biol. Markers,
9: 53-60 (1994)), MAGE-1 and thyrosinase (Coulie et al., J.
Immunothera., 14: 104-109 (1993)), p53 (WO 94/02167), NY-ESO1
(cancer-testis antigen), AFP (.alpha.-feto protein) and a cancer
antigen 125 (CA-125), or EPCA (Early Prostate Cancer Antigen).
[0030] According to more preferable embodiment, the antigen used in
the present invention includes a virual antigen or cancer antigen,
and most preferably viral antigen.
[0031] Where the antigen gene used in the present invention is a
viral antigen, the antigen preferably includes HPV (human
papillomavirus) antigen, HBV (hepatitis B virus) antigen, HCV
(hepatitis C virus) antigen, HIV (human immunodeficiency virus)
antigen, rotavirus antigen, influenza virus antigen, HSV (herpes
simplex virus) antigen, avian influenza virus antigen, hog cholera
virus antigen, foot-and-mouth disease virus antigen and Newcastle
disease virus antigen.
[0032] More preferably, the antigen includes HPV antigen, much more
preferably HPV L1, L2, E6 or E7 protein, and most preferably HPV L1
protein.
[0033] HPV L1 protein has an original property capable of forming
in vivo or in vitro virus like particles (VLPs) by self-assembly.
L1 protein is the most conserved protein of HPV proteins. According
to a preferable embodiment, the L1 nucleotide sequence used in the
present invention is a nucleotide sequence derived from HPV
selected from the group consisting of HPV type 1, 2, 3a, 4, 5, 6b,
7, 8, 9, 10, 11a, 12, 13, 16 and 18, and more preferably HPV type
16 or 18. For example, the nucleotide sequence encoding a L1
protein is described in GenBank accession Nos. EU118173 (J. Virol.
67 (12): 6929-6936 (1993)), AY383628 and AY383629 (Virology 321
(2): 205-216 (2004)).
[0034] The nucleotide sequence encoding a foreign virus envelope
protein used in this invention may be derived from various viruses
except for baculovirus. Preferably, the envelope protein is derived
from a virus which utilizes a human cell as a host cell, more
preferably a virus which has a target receptor on the surface of a
human cell, and most preferably a virus which is able to induce a
receptor-mediated phagocytosis in a human cell.
[0035] According to a preferable embodiment, the nucleotide
sequence encoding a virus envelope protein used in the present
invention is derived from alphavirus, paramyxovirus, rhabdoviridae,
myxovirus, coronavirus, retrovirus, filovirus or arenavirus, more
preferably retrovirus, and most preferably human endogenous
retrovirus (HERV). HERV is an endogenous virus in human body, most
of which are incorporated in human genome at an inactivated state.
The envelope protein is expressed on the surface of recombinant
virus, inducing phagocytosis by interaction with a receptor of a
human cell.
[0036] According to a preferable embodiment, the nucleotide
sequence encoding a HERV envelope protein is a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO:2, and more
preferably a nucleotide sequence encoding SEQ ID NO:1. The
nucleotide sequence of SEQ ID NO:1 may be optimized to highly
express a HERV envelope protein in insect cells.
[0037] The chimera virus of the present invention is based on
baculovirus. Baculovirus is a rod-shape virus, and its genes are
not expressed in human cells using an insect-specific promoter.
Therefore, baculovirus has been gradually spotlighted as a basic
system of gene therapeutics in the senses that baculovirus induces
no immune responses in human cells by viral gene expression.
However, the expression of a foreign gene in a baculovirus vector
is sharply induced under the control of a mammalian promoter. In
addition, it is an advantage that the infection by baculovirus
accelerates no replication of human endogenous virus. In contrast
with other viruses for gene therapy, baculovirus may be cultured in
serum-free media, contributing to massive production.
[0038] The construction of recombinant baculovirus and culture of
insect cells are described in Summers and Smith. 1986. A Manual of
Methods for Baculovirus Vectors and Insect Culture Procedures,
Texas Agricultural Experimental Station Bull. No. 7555, College
Station, Tex.; Luckow. 1991. In Prokop et al., Cloning and
Expression of Heterologous Genes in Insect Cells with Baculovirus
Vectors' Recombinant DNA Technology and Applications, 97-152; U.S.
Pat. No. 4,745,051; and EP0340359 in detail, which are herein
incorporated by references.
[0039] For instance, the chimera baculovirus containing a HERV Env
gene and a HPV gene allows a transfer vector delivering a HERV Env
gene and a HPV L1 gene to transfect a cell. The expression
construct containing a HERV Env gene and a HPV L1 gene is flanked
with transposon sequence (e.g., Tn7). The transfer vector is
transfected into a cell (e.g., E. coli) containing a bacmid
(baculovirus shttle vector) with a mini-attTn7 target site and a
helper plasmid with a transposase gene. Transfection of the
transfer vector to E. coli may induce transposition, resulting in a
recombinant bacmid. Subsequently, the recombinant bacmid is
isolated and transfected into suitable insect cells, producing a
chimera baculovirus. The insect cells suitable in the present
invention are not particularly limited, for example including Sf9
(Spodoptera frugiperda), Spodoptera exiaua, Choristoneura
fumiferana, Trichoplusia ni and Spodoptera littoralis, and
Drosophila.
[0040] It is preferable that the nucleotide sequence encoding a
virus envelope protein and HPV L1 in the recombinant baculovirus of
the present invention is involved in a suitable expression
construct. Preferably, the nucleotide sequence encoding a virus
envelope protein and HPV L1 is operatively linked to a promoter in
the expression construct. The term "operatively linked" refers to
functional linkage between a nucleic acid expression control
sequence (such as a promoter, signal sequence, or array of
transcription factor binding sites) and a second nucleic acid
sequence, wherein the expression control sequence affects
transcription and/or translation of the nucleic acid corresponding
to the second sequence. In the present invention, the promoter
linked to the nucleotide sequence encoding a virus envelope protein
and HPV L1 protein may be utilized in various manners.
[0041] According to a preferable embodiment, the first promoter
operatively linked to the envelope-encoding nucleotide sequence is
a promoter operable in insect cells, more preferably baculovirus
IE-1 promoter, IE-2 promoter, p35 promoter, p10 promoter, gp64
promoter or polyhedrin promoter, and most preferably polyhedrin
promoter.
[0042] According to a preferable embodiment, the second promoter
operatively linked to the HPV L1-encoding nucleotide sequence is a
promoter derived from a genome of a mammalian cell, or a promoter
derived from a mammalian virus, more preferably U6 promoter, H1
promoter, CMV (cytomegalo virus) promoter, adenovirus late
promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk
promoter, RSV promoter, human elongation factor 1.alpha.
(hEF1.alpha.) promoter, methallothionein promoter, .beta.-actin
promoter, human IL-2 gene promoter, human IFN gene promoter, human
IL-4 gene promoter, human lymphotoxin gene promoter, human GM-CSF
gene promoter, TERT promoter, PSA promoter, PSMA promoter, CEA
promoter, E2F promoter, AFP promoter or albumin promoter, and most
preferably hEF1.alpha. promoter.
[0043] Preferably, the expression construct used in the present
invention includes a polyadenylation sequence, for example
including hEF1.alpha. polyA, bovine hormone terminator (Gimmi, E.
R., et al., Nucleic Acids Res. 17: 6983-6998 (1989)), SV40-derived
polyadenylation sequence (Schek, N, et al., Mol. Cell Biol. 12:
5386-5393 (1992)), HIV-1 polyA (Klasens, B. I. F., et al., Nucleic
Acids Res. 26: 1870-1876 (1998)), .beta.-globin polyA (Gil, A., et
al., Cell 49: 399-406 (1987)), HSV TK polyA (Cole, C. N. and T. P.
Stacy, Mol. Cell. Biol. 5: 2104-2113 (1985)) or poliomavirus polyA
(Batt, D. B and G. G. Carmichael, Mol. Cell. Biol. 15:4783-4790
(1995)), but not limited to.
[0044] In the recombinant virus of the present invention, each
envelope-encoding nucleotide sequence and HPV L1-encoding
nucleotide sequence may be contained in an expression construct of
a first promoter-envelope-encoding nucleotide sequence-polyA
sequence and a second promoter-HPV L1-encoding nucleotide
sequence-polyA sequence. In addition, envelope- and HPV L1-encoding
nucleotide sequence may be involved as an expression construct of a
first promoter-envelope-encoding nucleotide sequence-second
promoter-HPV L1-encoding nucleotide sequence-polyA sequence.
[0045] According to a preferable embodiment, the recombinant virus
of this invention further includes a gene of interest to be
expressed. The gene of interest to be expressed by the recombinant
virus of this invention is not particularly limited. The gene of
interest in the present invention may be any gene, for example,
including cancer-therapeutic genes encoding proteins having
anti-tumor activity and eventually degenerating tumor cells such as
tumor suppressor genes, immunomodulatory genes [e.g., cytokine
genes, chemokine genes and costimulatory factor genes (for T cell
activity such as B7.1 and B7.2)], suicide genes, cytotoxic genes,
cytostatic genes, pro-apoptotic genes and anti-angiogenic genes,
but not limited to.
[0046] The suicide genes encode proteins capable of conferring to
tumor cells sensitivity to chemotherapeutic agents, or of inducing
toxic conditions in tumor cells. The most well-known suicide gene
is the thymidine kinase (TK) gene (U.S. Pat. Nos. 5,631,236 and
5,601,818). Cells expressing TK are susceptible to selective cell
death by gancyclovir. The tumor suppressor genes encode
polypeptides to inhibit tumorigenesis. The tumor suppressor genes
are inherent in mammalian cells and their deletion or inactivation
is believed to be a prerequisite for tumorigenesis. Examples of the
tumor suppressor genes include members of the tumor suppressor gene
INK4 family, which are exemplified by APC, DPC4, NF-1, NF-2, MTS1,
WT1, BRCA1, BRCA2, VHL, p53, Rb, MMAC-1, MMSC-2, retinoblastoma
gene (Lee et al., Nature, 329: 642 (1987)), gene of adenomatous
polyposis coli protein (U.S. Pat. No. 5,783,666), nasopharyngeal
carcinoma tumor suppressor gene that maps at chromosome 3p21.3
(Cheng et al., Proc. Natl. Acad. Sci., 95: 3042-3047 (1998)),
deleted in colon carcinoma (DCC) gene, MTS1, CDK4, VHL, p100Rb, p16
and p21, and therapeutically effective fragments thereof (e.g.,
p56Rb, p94Rb). It will be understood that other known anti-tumor
genes can be used by those of ordinary skill in the art.
[0047] The term "cytotoxic gene" as used herein, refers to a
nucleotide sequence, the expression of which in a cell elicits a
toxic effect. Examples of the cytotoxic genes include nucleotide
sequences encoding Pseudomonas exotoxin, ricin toxin, diphtheria
toxin, and the like.
[0048] The term "cytostatic gene" as used herein, refers to a
nucleotide sequence, the expression of which in a cell induces an
arrest in the cell cycle. Examples of the cytostatic genes include,
but are not limited to, p21, retinoblastoma gene, E2F-Rb fusion
protein gene, genes encoding cyclin-dependent kinase inhibitors
such as p16, pI5, pI8 and pI9, growth arrest specific homeobox
(GAX) gene (WO 97/16459 and WO 96/30385), and so forth.
[0049] In addition, a variety of therapeutic genes useful in
treating various diseases may be carried in the gene delivery
system of this invention. Non-limiting examples of the therapeutic
genes include genes encoding cytokines (e.g., interferon-.alpha.,
interferon-.beta., interferon-.delta. and interferon-.gamma.),
interleukins (e.g., IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12,
IL-19 and IL-20), colony-stimulating factors (e.g., GM-CSF and
G-CSF), or chemokine genes [monocyte chemotactic protein 1 (MCP-1),
monocyte chemotactic protein 2 (MCP-2), monocyte chemotactic
protein 3 (MCP-3), monocyte chemotactic protein 4 (MCP-4),
macrophage inflammatory protein 1.alpha. (MIP-1.alpha.), macrophage
inflammatory protein 1.beta. (MIP-1.beta.), macrophage inflammatory
protein 1.gamma.(MIP-1.gamma.), macrophage inflammatory protein
3.alpha. (MIP-3.alpha.), macrophage inflammatory protein 3.beta.
(MIP-3.beta.), chemokine (ELC), macrophage inflammatory protein 4
(MIP-4), macrophage inflammatory protein 5 (MIP-5), LD78.beta.,
RANTES, SIS-epsilon (p500), thymus and activation-regulated
chemokine (TARC), eotaxin, 1-309, human protein HCC-1/NCC-2, human
protein HCC-3, and mouse protein C10]. In addition, the therapeutic
genes include genes encoding tissue-type plasminogen activator
(tPA) or urokinase-type plasminogen activator, and LAL-generating
gene to provide sustained thrombolysis for preventing
hypercholesterolemia. Further, polynucleotide sequences available
for treatment of various diseases including cystic fibrosis,
adenosine deaminase deficiency, AIDS and other infectious diseases,
and malignant and inflammatory diseases are known to be useful as
therapeutic genes.
[0050] The term "pro-apoptotic gene" as used herein, refers to a
nucleotide sequence, the expression of which results in the
programmed cell death. Examples of the pro-apoptotic genes include
p53, adenovirus E3-11.6K (derived from Ad2 and Ad5) or adenovirus
E3-10.5K (derived from Ad), adenovirus E4 gene, Fas ligand,
TNF-.alpha., TRAIL, p53 pathway genes and genes encoding a series
of caspases.
[0051] The term "anti-angiogenic gene" as used herein, refers to a
nucleotide sequence, the expression of which results in the
extracellular secretion of anti-angiogenic factors.
Anti-angiogenesis factors include angiostatin, inhibitors of
vascular endothelial growth factor (VEGF) such as Tie 2 (PNAS,
1998, 95, 8795-8800), endostatin, and so on.
[0052] The nucleotide sequences of interest described previously
are available from DNA sequence databases such as GenBank or
EMBL.
[0053] The recombinant virus of the present invention may induce a
receptor-mediated phagocytosis in a human cell by its envelope
protein, and generate immune responses against an antigen protein
in a body through injection of antigen protein to be expressed.
Furthermore, the recombinant baculovirus of the present invention
may remarkably induce cellular immune responses as well as humoral
immune responses. Consequently, the recombinant baculovirus of the
present invention may exhibit excellent efficacy on prevention of
various disorders by functions as described above. As demonstrated
in the following examples, the recombinant baculovirus of the
present invention has not only almost similar effect on humoral
immune responses compared with conventional vaccine, gardasil, but
also excellent induction of cellular immunity against HPV, enabling
to function as a HPV vaccine more efficient than gardasil.
[0054] The vaccine composition of the present invention includes:
(a) a therapeutically effective amount of the recombinant
baculovirus; and (b) a pharmaceutically acceptable carrier.
[0055] The recombinant baculovirus contained in the vaccine
composition of the present invention exhibits immunogenicity
against various antigens.
[0056] According to a preferable embodiment, the recombinant
baculovirus contained in the vaccine composition of the present
invention includes HPV antigen genes and the vaccine composition is
a HPV vaccine composition.
[0057] The present composition may be used for prevention or
treatment of various disorders (e.g., cervical cancer, rectal
cancer, vulva cancer, penile cancer or head and neck cancer) caused
by HPV infection, and preferably prevention. Most preferably, the
present composition may be used for prevention or treatment of
cervical cancer, and preferably prevention. The term
"pharmaceutically effective amount" refers to an amount enough to
show and accomplish efficacies and activities of the compound of
this invention for preventing or treating, preferably preventing
the mentioned-above disorders.
[0058] The pharmaceutically acceptable carrier contained in the
vaccine composition of the present invention, which is commonly
used in pharmaceutical formulations, but is not limited to,
includes lactose, dextrose, sucrose, sorbitol, mannitol, starch,
rubber arable, potassium phosphate, arginate, gelatin, potassium
silicate, microcrystalline cellulose, polyvinylpyrrolidone,
cellulose, water, syrups, methylcellulose, methylhydroxy benzoate,
propyl hydroxy benzoate, talc, magnesium stearate, and mineral
oils. The pharmaceutical composition according to the present
invention may further include a lubricant, a humectant, a
sweetener, a flavoring agent, an emulsifier, a suspending agent,
and a preservative.
[0059] Preferably, the vaccine composition according to the present
invention may be administered parenterally, e.g., by intravenous,
intra-abdominal, intramuscular, transdermal or locally.
[0060] A suitable dosage amount of the vaccine composition of the
present invention may vary depending on pharmaceutical formulation
methods, administration methods, the patient's age, body weight,
sex, pathogenic state, diet, administration time, administration
route, an excretion rate and sensitivity for a used pharmaceutical
composition. Generally, a skilled physician may determine and
prescribe an effective dosage for treatment of interest in an easy
manner. Preferably, the vaccine composition of the present
invention may be administered with a daily dose of the recombinant
viruses of 1.times.10.sup.3-1.times.10.sup.15 pfu/ml.
[0061] According to the conventional techniques known to those
skilled in the art, the vaccine composition according to the
present invention may be formulated with pharmaceutically
acceptable carrier and/or vehicle as described above, finally
providing several forms including a unit dose form and a multi-dose
form. Non-limiting examples of the formulations include, but not
limited to, a solution, a suspension or an emulsion in oil or
aqueous medium, an elixir, a powder, a granule, a tablet and a
capsule, and may further comprise a dispersion agent or a
stabilizer.
[0062] In another aspect of this invention, there is provided a
recombinant baculovirus comprising: (a) a nucleotide sequence
encoding a foreign virus envelope protein; (b) a first promoter
operatively linked to the envelope-encoding nucleotide sequence;
(c) a nucleotide sequence encoding a HPV (human papilloma virus) L1
protein; and (d) a second promoter operatively linked to the HPV
L1-encoding nucleotide sequence.
[0063] Since the present recombinant baculovirus comprises the
vaccine composition of this invention as active ingredients
described above, the common descriptions between them are omitted
in order to avoid undue redundancy leading to the complexity of
this specification.
[0064] In still another aspect of this invention, there is provided
a nucleic acid molecule encoding a HERV (human endogenous
retrovirus) envelope protein, comprising a nucleotide sequence of
SEQ ID NO:1.
[0065] The present inventors have made intensive studies to develop
a more efficient gene delivery system based on baculovirus. As
results, we have discovered that where an endogenous retrovirus is
in a cell of interest and has no cytotoxity to the cell, the
introduction of its envelope protein into a gene delivery system
contributes to remarkable improvement of the gene delivery system's
efficiency.
[0066] To develop an improved gene delivery system, we have
optimized baculovirus expression in insect cells by modifying a
nucleic acid molecule encoding a HERV envelope protein which is
introduced into a gene delivery system.
[0067] The envelope gene introduced into the gene delivery system
in the present invention is derived from HERV. HERV is incorporated
into a human genome, and not expressed because it has incomplete
genes as a whole. The present invention modifies a
natural-occurring HERV envelope gene to express a non-expressed
gene of HERV in insect cells in a high-throughput manner.
[0068] The term "nucleic acid" used herein, refers to a DNA
molecule.
[0069] It could be understood that the HERV envelope-encoding
nucleic acid molecule of this invention includes substantially
identical sequences to the sequences set forth in the appended
Sequence Listing. The substantially identical sequences refers to
those showing preferably at least 80%, more preferably at least
85%, still more preferably at least 90%, most preferably at least
95% nucleotide similarity to the sequences of the appended Sequence
Listing, as measured using one of the sequence comparison
algorithms known to those ordinarily skilled in the art, by which
the nucleotide sequence of this invention is maximally aligned
corresponding on random other nucleotide sequences. Methods of
alignment of sequences for comparison are well-known in the art.
Various programs and alignment algorithms are described in: Smith
and Waterman, Adv. Appl. Math. 2: 482 (1981); Needleman and Wunsch,
J. Mol. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol.
Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988);
Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc.
Acids Res. 16: 10881-90 (1988); Huang et al., Comp. Appl. BioSci.
8: 155-65 (1992); and Pearson et al., Meth. Mol. Biol. 24: 307-31
(1994). The NCBI Basic Local Alignment Search Tool (BLAST)
(Altschul et al., J. Mol. Biol. 215: 403-10 (1990)) is available
from several sources, including the National Center for Biological
Information (NBCI, Bethesda, Md.) and on the Internet, for use in
connection with the sequence analysis programs blastp, blasm,
blastx, tblastn and tblastx. It can be accessed at
http://www.ncbi.nlm.nih.gov/BLAST/. A description of how to
determine sequence identity using this program is available at
http://www.ncbi.nlm.nih.gov/BI-AST/blast help.html.
[0070] In another aspect of this invention, there is provided a
recombinant vector comprising the nucleic acid molecule encoding a
HERV envelope protein.
[0071] Since the present recombinant vector comprises the HERV
envelope-encoding sequence described above, the common descriptions
between them are omitted in order to avoid undue redundancy leading
to the complexity of this specification.
[0072] The vector system of this invention may be performed by
various methods known to those skilled in the art and its practical
method is described in Sambrook et al., Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory Press (2001),
which is herein incorporated by reference.
[0073] In each a vector of this invention and an eukaryotic cell
used as an expression vector and the host cell, the promoter
derived from genome of mammalian cells, mammalian viruses or
baculovirus (example: polyhedrin promoter) might be used, and
polyadenylated sequence might be commonly used as the transcription
termination sequence.
[0074] The expression vector of this invention includes an
antibiotics-resistance gene known to those ordinarily skilled in
the art as a selection marker, for example resistant genes against
ampicillin, gentamycin, carbenicillin, chloramphenicol,
streptomycin, kanamycin, geneticin, neomycin and tetracycline.
[0075] According to a preferable embodiment, the vector of the
present invention has a gene map as shown in FIG. 11. The
characteristics of the vector in FIG. 11 are as follows: (a) a HERV
envelope gene expression is controlled by a polyhedrin promoter;
(b) a gene expression of interest is modulated by a hEF1.alpha.
promoter; (c) a hEF1.alpha. polyA signal as a transcription
termination sequence; and (d) two arm of transposon 7 in both side
of expression cassette.
[0076] In still another aspect of this invention, there is provided
a baculovirus-based gene carrier comprising the nucleic acid
molecule encoding a HERV envelope protein.
[0077] Since the present gene carrier is derived from viruses
obtained by infecting the recombinant vector into insect cells
described above, the common descriptions between them are omitted
in order to avoid undue redundancy leading to the complexity of
this specification.
[0078] Recently, the method using virus has been principally
employed as a gene delivery system. Viruses used in the method
include adenovirus, retrovirus, lentivirus, vaccinia and the like.
Most of viruses have limitations for use in a human body because
they has an opportunity to infect or danger a human, whereas
baculovirus is known as a biologically stable virus as it has no
infectivity to a human body and is able to be replicated only in
specific insects. The virus-mediated gene delivery system is
carried out through virus infection which is determined by
interaction between a virus envelope protein and a receptor of a
cell and an animal of interest.
[0079] Focusing on advantages of the baculovirus, the present
invention provides a more improved gene delivery system whereby an
envelope protein of endogenous virus present in an animal of
interest is incorporated on the surface of baculovirus. The
endogenous virus has been known to be widely distributed in all
mammals such as pig, mouse, cat, dog, and so on.
[0080] According to the gene carrier of the present invention, the
envelope protein of human endogenous virus is linked to its
surface, enabling to deliver a gene of interest into a human cell
in a high-throughput and stable manner. Therefore, the gene carrier
of the present invention may be efficiently utilized for
development of gene therapeutics against various disease and
disorders.
[0081] The features and advantages of this invention are summarized
as follows:
[0082] (a) The vaccine of the present invention includes a
recombinant baculovirus containing a nucleotide sequence encoding
an antigen gene and a foreign virus envelope protein.
[0083] (b) The recombinant baculovirus of the present invention may
induce a receptor-mediated phagocytosis in a human cell by the
envelope protein on the surface of baculovirus, and immune
responses in the body injected with the antigen protein (e.g., HPV
L1) to be expressed.
[0084] (c) Furthermore, the recombinant baculovirus of the present
invention may significantly induce cellular immune responses as
well as humoral immune responses.
[0085] (d) Ultimately, the recombinant baculovirus of the present
invention may have an excellent efficacy on prevention of various
diseases (e.g., cervical cancer) induced by a specific antigen as
described above.
[0086] (e) The recombinant baculovirus of the present invention has
not only almost similar effect on humoral immune responses compared
with conventional vaccine, gardasil, but also excellent induction
of cellular immunity against HPV, enabling to function as a HPV
vaccine more efficient than gardasil.
[0087] (f) According to the present invention, a stable and
economic vaccine may be provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0088] FIG. 1 schematically represents a construction procedure of
transfer vector, pAc-hEF1.alpha.16L1, used in the present
invention. In FIG. 1, black arrow, white arrow, and small black
quadrangle indicate polyhedrin promoter, hEF1.alpha. promoter, and
hEF1.alpha. poly(A) signal, respectively.
[0089] FIG. 2 schematically represents a construction procedure of
transfer vector, pAcHERVenv-hEF1.alpha.16L1, used in the present
invention. In FIG. 2, black arrow, white arrow, and small black
quadrangle indicate polyhedrin promoter, hEF1.alpha. promoter, and
hEF1.alpha. poly(A) signal, respectively.
[0090] FIG. 3 represents an expected scheme of chimera baculovirus
transfer vectors and viruses including pAc-hEF1.alpha.16L1 and
pAcHERVenv-hEF1.alpha.16L1 construct, respectively. In FIG. 3,
black arrow, white arrow, and small black quadrangle indicate
polyhedrin promoter, hEF1.alpha. promoter, and hEF1.alpha. poly(A)
signal, respectively.
[0091] FIGS. 4-7 are a sequence homology through alignment of
nucleotide sequence between a HERV envelope protein synthesized in
the present invention and a HERV envelope protein. The nucleotide
sequence of HERV envelope protein is shown in GenBank accession No.
NM 014590.
[0092] FIG. 8 represents RT-PCR to examine expression of HPV 16L1
gene in Huh7 cells infected with Ac-hEF1.alpha.16L1 or
AcHERVenv-hEF1.alpha.16L1 construct. "NTC" indicates a control
without template. AcHERVenv-hEF1.alpha.16L1 construct contains an
envelope protein of pig endogenous retrovirus and exhibits almost
no infectivity to human Huh7 cells.
[0093] FIG. 9 is images analyzing HPV 16L1 of normal Huh7 cells and
Huh7 cells infected with Ac-hEF1.alpha.16L1 or
AcHERVenv-hEF1.alpha.16L1 construct through immunocytochemical
staining. AcHERVenv-hEF1.alpha.16L1 construct contains an envelope
protein of pig endogenous retrovirus and exhibits almost no
infectivity to human Huh7 cells.
[0094] FIG. 10 is a bar graph showing quantitative analysis by
real-time PCR using a Delta-Delta CT method to determine expression
level of HPV 16L1 mRNA in Huh7 cells infected with
Ac-hEF1.alpha.16L1 or AcHERVenv-hEF1.alpha.16L1 construct.
AcHERVenv-hEF1.alpha.16L1 construct contains an envelope protein of
pig endogenous retrovirus and exhibits almost no infectivity to
human Huh7 cells.
[0095] FIG. 11 represents a gene map of vector pFB HERV-hEF
constructed in an embodiment of the present invention.
Abbreviation: Polh promoter, polyhedrin promoter; HERVenv, envelope
gene of HERV; Tn7R, right arm; and Tn7L, left arm. In
AcHERVenv-hEF1.alpha.16L1 construct, HPV 16L1 is positioned at a
gene of interest.
[0096] FIG. 12 represents ELISA for IgG antibody response in serum
immunized with chimera baculovirus of the present invention. Sample
and anti-mouse IgG were used at a dilution ratio of 1:100 and
1:2,000, respectively. The bars from left to right correspond to
1-week, 3-week, 5-week, 9-week and 14-week in each group.
[0097] FIG. 13 is to measure IgG antibody response in vaginal
washing solution immunized with chimera baculovirus of the present
invention. Sample and anti-mouse IgG were used at a dilution ratio
of 1:50 and 1:1,000, respectively. The y axis indicates absorbance
at 405 nm. The bars from left to right correspond to 1-week,
3-week, 5-week, 9-week and 14-week in each group.
[0098] FIG. 14 represents neutralization response against HPV16 or
HPV18 PVs (pseudoviruses) by mouse antiserum immunized with chimera
baculovirus of the present invention.
[0099] FIG. 15 represents ELISPOT analysis for cell-mediated immune
response. To evaluate IFN-.gamma. expression, ELISPOT was carried
out in spleen cells. CD8.sup.+ T cells were stimulated with HPV 16
PVs or HPV18 PVs. (A) indicates an experimental group immunized
with gardasil, (B) indicates an experimental group immunized with
AcHERVenv-hEF1.alpha.16L1 or AcHERVenv-hEF1.alpha.18L1, and (C)
serves as a control.
[0100] The present invention will now be described in further
detail by examples. It would be obvious to those skilled in the art
that these examples are intended to be more concretely illustrative
and the scope of the present invention as set forth in the appended
claims is not limited to or by the examples.
EXAMPLES
Materials and Methods
Cell Preparation
[0101] Insect cells, Sf9 (ATCC CRL-1711), were cultured in TC-100
media supplemented with 10% FBS (fetal bovine serum, Gibco BRL) and
1% penicillin/streptomycin (Gibco BRL) at 27.degree. C. 293TT cells
(Schiller Lab, USA NCI) were incubated in DMEM (Dulbecco's modified
minimal essential medium) supplemented with 10% FBS and hygromycin
B (400 .mu.g/ml; Invitrogen Corp.). Human liver cell line, Huh7
cells (JCRB0403) were incubated in DMEM supplemented with 10% FBS
(Gibco BRL) and 1% penicillin/streptomycin (Gibco BRL) at
37.degree. C. under the atmosphere of 5% CO.sub.2. HeLa cells
(ATCC) were cultured in DMEM supplemented with 10% FBS, 100 U
penicillin/ml, and 100 .mu.g streptomycin/ml.
Synthesis of a Gene Encoding a HERV Envelope Protein
[0102] HERV (human endogenous retrovirus) is an endogenous virus in
a human body, most of which are incorporated in human genome at an
inactivated state. To obtain a HERV envelope protein, a gene
encoding the HERV envelope protein was directly synthesized to
optimize its nucleotide sequence suitable for expression in insect
cells (GeneScript). The nucleotide sequence encoding the
synthesized HERV envelope protein was inserted into EcoRV site of
pUC57 vector (GeneScript), constructing pUC57-HERVenv.
Cloning of Transfer Vector
[0103] Construction of a recombinant baculovirus containing a
procedure of transfer vector cloning were carried out according to
Invitrogen's protocol using a Bac-to-Bac.TM. baculovirus expression
system. To express HPV 16L1 protein in animal cells using the
recombinant baculovirus system, a human elongation factor 1.alpha.
(hEF1.alpha.) promoter and a HPV 16L1 gene were inserted into an
AcMNPV (autographa californica multiple nuclear polyhedrosis virus)
transfer vector. In PCR amplification, a plasmid DNA (p16L1L2)
containing a `hEF1.alpha.-HPV 16L1-hEF1.alpha. poly(A) signal`
construct was used as a template (Schiller Lab, USA NCI;
Christopher B. Buck et al., J. Virol. 82 (11): 5190-5197 (2008)).
The primer sequence used was as follows: sense primer,
5'-GGCTCCGGTGCCCGTCAGTGGGCA-3'; and antisense primer,
5'-TTAATTAACCCACGTTTCAACATG-3'.
[0104] The PCR-amplified products were cloned into pGET-Teasy
vector (Promega). The vector was restricted with EcoRI, and
subsequently the fragments were inserted into EcoRI site of
pFastBac.TM.1 (Invitrogen) transfer vector, generating a
pAc-hEF1.alpha.16L1 vector (See, FIG. 1). A HERV envelope protein
gene was cut with Sail from pUC57-HERVenv vector and then inserted
into pFastBac.TM.1 vector. After cutting pGEM-Teasy/hEF1.alpha.16L1
with NotI, hEF1.alpha.16L1 was inserted into pFastBac.TM.1-HERVenv
transfer vector, constructing pAcHERVenv-hEF1.alpha.16L1 vector
(See, FIG. 2). To confirm OFR (open reading frame) of the transfer
vectors cloned, gene sequences were analyzed using ABI gene
sequence analyzer (ABI).
Construction of a Recombinant Baculovirus
[0105] Each recombinant transfer vectors cloned were transfected
into DH10Bac (Invitrogen), producing recombinant bacmids
(baculovirus shuttle vector). Selection of recombinant bacmids was
carried out by PCR using M13 primer (Invitrogen). Three types of
bacmids were transfected into Sf9 cells using lipofectamine
(Invitrogen) for construction of recombinant baculoviruses. At 4
days post-infection, produced viruses were collected and infected
repeatedly into new Sf9 cells to produce viruses with high titer.
Afterwards, selected recombinant viruses were designated as
AcHERVenv-hEF1.alpha.16L1 and Ac-hEF1.alpha.16L1, respectively
(See, FIG. 3). Finally, titers of recombinant baculoviruses were
determined in Sf9 cells using a plaque assay. Meanwhile,
recombinant baculoviruses (AcHERVenv-hEF1.alpha.18L1) were prepared
according to the mentioned-above method for preparing recombinant
baculoviruses (AcHERVenv-hEF1.alpha.16L1) except for using a HPV
18L1 gene (GenBank accession No. AY383629).
Transfection of a Gene into Huh7 cells Using a Recombinant
Baculovirus
[0106] Huh7 cells were seeded into a 24-well plate at a
concentration of 1.times.10.sup.5 cells/well and cultured at
37.degree. C. After incubation for 12 hrs, the cells were washed
with PBS, and then infected with Ac-hEF1.alpha.16L1 and
AcHERVenv-hEF1.alpha.16L1 virus of 100 MOI (multiplicity of
infectivity), respectively. Then, the cells were cultured at
37.degree. C. for 10 hrs, and transferred to fresh DMEM
supplemented with 10% FBS and 1% penicillin/streptomycin, followed
by further incubation for 48 hrs. The extent of expression of HPV
16L1 was examined in each virus as follows.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Analysis
[0107] Using RNeasy mini kit (Qiagen, Valencia, Calif.), total RNA
was isolated from Huh7 cells transfected and DNA was removed by
treatment of deoxyribonuclease I (DNaseI, Promega, Madison, Wis.).
Purified RNA was reverse transcribed with M-MuLV reverse
transcriptase (Bioneer, USA) to synthesize cDNA. 7.5 .mu.l of PCR
reaction mixture was mixed with 2.5 .mu.l of cDNA and PCR was
carried out using Thermal Cycler PCR (GeneAmp PCR system 9700,
Perkin-Elmer Cetus, USA). PCR condition was as follows: hot-start
step at 94.degree. C. for 3 min; and 30-cycle step of denaturing at
94.degree. C. for 30 sec, annealing at 62.degree. C. for 20 sec and
elongating at 72.degree. C. for 20 sec. The primers used were:
sense primer, 5'-CAGGGCCACAACAACGGCATCTGCTGGG-3'; and antisense
primer, 5'-GGCTGCAGGCCGAAGTTCCAGTCCTCCA-3'. The resulting PCR
products were expected as about 275 bp. To normalize PCR efficiency
between samples, 18S rRNA (ribosomal RNA) housekeeping gene was
used. The amplified PCR products were detected on a 1.5% agarose
gel.
Quantitative Analysis Using Real-Time PCR (Q-PCR)
[0108] To evaluate expression level of HPV 16L1 mRNA in cells
infected, quantitative analysis using real-time PCR (Q-PCR) was
performed as described previously (Dhar et al., 2001). The
expression level of total HPV 16L1 mRNA was analyzed four-times
using real-time PCR machine (Roter Gene 3000, Corbett Research,
Australia). PCR reaction mixture was added with 5 .mu.l of
DyNAmo.TM. HS SYBR.TM. Green qPCR kit reaction solution and 5 .mu.l
of sample buffer containing primers and templates. The primers used
were: 16L1 sense primer, 5'-CAGCGAGACCACCTACAAGA-3'; and antisense
primer, 5'-GCTGTTCATGCTGTGGATGT-3'. The resulting PCR products were
expected as about 138 bp. PCR products were obtained by
pre-denaturing step at 95.degree. C. for 5 min, and 45-cycle step
of denaturing at 94.degree. C. for 10 sec, annealing at 62.degree.
C. for 20 sec and elongating at 72.degree. C. for 20 sec. After PCR
reaction, the copy number and melting curve analysis of target
molecules were performed using Roter-Gene ver. 6.0 program (Roter
Gene 3000, Corbett Research, Australia).
Immunocytochemistry
[0109] Huh7 cells were divided into a glass slide, and then
transfected with Ac-hEF1.alpha.16L1 and AcHERVenv-hEF1.alpha.16L1
virus of 100 MOI (multiplicity of infectivity), respectively. After
transfection for 48 hrs, the cells were fixed with 4% formaldehyde
at 4.degree. C. for 12 hrs, and washed with PBS (phosphate buffered
saline), followed by further incubating with PBS containing 0.5%
Triton X-100 at 37.degree. C. for 10 min. Next, the cells were
washed with PBS and blocked with PBS containing goat serum at
37.degree. C. for 30 min, followed by incubating with HPV 16L1
monoclonal antibody (Camvir-1) at 4.degree. C. overnight. The cells
were washed with PBS for 30 min, and then incubated with a mouse
IgG-horseradish peroxidase antibody for 1 hr. After washing with
PBS, the cells were observed under a confocal laser scanning
microscope (FV-1000 spectral, Olympus, Japan) to detect HPV 16L1
protein.
Gardasil
[0110] Gardasil.TM. (MERCK & CO, USA, MSD, Korea) as a HPV
quadrivalent vaccine (type 6, 11, 16 and 18) served as a positive
control of immune responses in this experiment.
Mouse
[0111] Four-week old female BALB/c mice were purchased from
Orient-Bio Inc. (Korea), and housed under filter-tip conditions
accessible in water and feed.
Mouse Immunization
[0112] Recombinant baculoviruses were diluted with sterile PBS at a
total volume of 100 .mu.l, and mice were immunized by intramuscular
injection at the base of the bottom leg with viruses at a
concentration of 10.sup.7 PFU (plaque forming unit). Twenty-four
BALB/c mice were classified into eight groups (Table 1). Each mouse
group was injected according to selected prime/boost regime.
Immunization was carried out three-times at an interval of 2-week,
and blood and vaginal washes were harvested at 1-week after each
immunization. Before analysis, anti-serum was heat-denatured.
TABLE-US-00001 TABLE 1 Experimental Immunization (interval of
2-week) group First Second Third Group 1 Gardasil Gardasil Gardasil
Group 2 AcHERVenv-hEF1.alpha.16L1 AcHERVenv-hEF1.alpha.16L1
AcHERVenv-hEF1.alpha.16L1 or or or AcHERVenv-hEF1.alpha.18L1
AcHERVenv-hEF1.alpha.18L1 AcHERVenv-hEF1.alpha.18L1 Group 3
AcHERVenv-hEF1.alpha.16L1 AcHERVenv-hEF1.alpha.16L1 Gardasil or or
AcHERVenv-hEF1.alpha.18L1 AcHERVenv-hEF1.alpha.18L1 Group 4
AcHERVenv-hEF1.alpha.16L1 Gardasil Gardasil or
AcHERVenv-hEF1.alpha.18L1 Group 5 AcHERV Gardasil Gardasil Group 6
AcHERV AcHERV Gardasil Group 7 AcHERV AcHERV AcHERV Group 8 PBS PBS
PBS
ELISA
[0113] Sixty .mu.l of MBP-L1 (Bioprogen Co., Ltd., Korea) that
HPV16 L1 is linked to maltose binding protein (MBP) was added to
each well of a ELISA plate at a concentration of 1 .mu.g/ml, and
incubated at 4.degree. C. for 14-16 hrs. Each well was blocked at
37.degree. C. for 2 hrs with a blocking buffer (5% skim milk in PBS
containing 0.1% Tween-20). After washing with PBS containing 0.05%
Tween-20 and 0.05% NP-40, serum samples diluted in blocking buffer
(1:100) were added to each well, and incubated at room temperature
for 1 hr. For IgG detection, anti-mouse IgG-HRP (SC-2030, Santa
Cruz Biotechnology, Inc., Santa Cruz, Calif.) diluted in blocking
buffer (1:2,000) was added to each well. To detect IgA, anti-mouse
IgA-HRP (SC-3791, Santa Cruz Biotechnology, Inc., Santa Cruz,
Calif.) diluted in blocking buffer (1:1,000) was added to each
well. OPD (o-phenylenediamine) substrate in 0.1 M citrate buffer
(pH 4.7) was added to each well, and then the absorbance was
measured at 450 nm.
Pseudoviruses (PVs) Preparation
[0114] According the method proposed by Schiller (J. Virol. 78 (2):
751-757 (2004)), cotransfection of 293T cells was carried out to
prepare PVs. 293T cells were seeded in 25 T flask 16 hrs before
transfection, and transfected with the mixture of L1/L2-plasmid and
pfwB plasmid expressing enhanced green fluorescent protein (GFP)
using Lipofectin (Invitrogen). The nucleotide map of plasmids used
is described in
http://ccr.cancer.gov/Staff/links.asp?profileid=5637. To prepare
HPV16 PVs, cells were transfected with 9 .mu.g of each pfwB and
p16L1/L2. In addition, cells were transfected with 9 .mu.g of each
pfwB and p18L1/L2 to prepare HPV18 PVs. After 4-6 hrs, the media of
transfected cells were exchanged. The cells were harvested 48 hrs
post-transfection. The supernatant was aliquoted and stored at
-80.degree. C. until next experiment.
Neutralization Analysis
[0115] The mixture of diluted serum of immunized mouse and PVs were
incubated at room temperature for 1 hr., the mixture was inoculated
into HeLa cells seeded at a concentration of 1.times.10.sup.4 for
16 hrs before inoculation. After incubation for 2 days, GFP
expression was observed under a fluorescence microscope.
Neutralizing titer was indicated as a reciprocal of maximal
dilution rate of serum which reduces GFP expression level to 1/2
level of sample treated with normal mouse serum.
IFN-.gamma. Enzyme-Linked Immunospot (ELISPOT)
[0116] A 96-well plate was coated with 200 ng of anti-mouse
IFN-.gamma. capturing antibody (BD Bioscience) in 100 .mu.l PBS at
4.degree. C. overnight. The plate was blocked in 100 .mu.l RPMI
1640 with 10% FBS at 37.degree. C. for 2 hrs, and spleen cells with
a density of 1.times.10.sup.6 were seeded into the plate duplicate.
PVs of 2.times.10.sup.6 IFU (infectious unit) were inoculated into
the plate, followed by incubating at 37.degree. C. for 24 hrs. The
plate was washed with PBS containing 0.05% Tween 20 three times to
remove the cells. Each well was added with 20 ng of
sterile-filtered anti-mouse IFN-.gamma. detecting antibody in PBS
with 10% FBS, and then incubated at room temperature for 2 hrs.
After the plate was washed with PBS containing 0.05% Tween 20 three
times, 100 .mu.l dilution solution of streptavidin-alkaline
phosphatase (1:1,000) was added. The plate was incubated at room
temperature for 1 hr, and washed with PBS containing 0.05% Tween 20
three times, followed by washing with PBS three times. The plate
was added with 100 .mu.l of AEC substrate reagent (BD Biosciences,
CA, USA) and incubated for 10 min. The plate was washed with
distilled water to stop reaction. The spot was quantitated using an
ELISPOT reader (AID Elispot Reader ver. 4, Germany). The well
containing media without treatment of spleen cells served as a
negative control. The count of background well was depreciated from
samples.
Results
Gene Synthesis of a HERV Envelope Protein
[0117] For construction of a transfer vector, a HERV envelope
protein gene (Env) was prepared through gene synthesis, and
optimized for codon usage of insect to be effectively expressed in
insect cells. Likewise, the amino acid sequence of synthetic HERV
envelope protein was partially modified in a state maintaining the
amino acid sequence of HERV envelope protein as described
previously. The nucleotide sequence and amino acid sequence of HERV
envelope protein (1,617 by in length) used in the present invention
are described in SEQ ID NO:1 and SEQ ID NO:2, respectively. As
shown in FIGS. 4-7, the nucleotide sequence of synthetic HERV
envelope protein was compared with that of conventional HER
envelope protein, suggesting a homology of 73.5% in the level of
nucleotide sequence.
Construction of a Recombinant Baculovirus
[0118] To construct recombinant baculoviruses, two types of
transfer vectors, pAc-hEF1.alpha.16L1 and
pAcHERVenv-hEF1.alpha.16L1, were planned, and expected forms of
baculoviruses were indicated (FIG. 3). To insert an envelope
protein of baculovirus, a polyhedrin promoter was followed by
inserting a gene of HERV envelope protein (Env), and a HPV 16L1
gene was controlled by hEF1.alpha.. HERV Env may induce a
receptor-mediated phagocytosis in human cells. FIG. 1 schematically
represents a cloning method of pAc-hEF1.alpha.16L1, and FIG. 2
briefly represents a cloning method of pAcHERVenv-hEF1.alpha.16L1.
The cloning of pAcHERVenv-hEF1.alpha.18L1 was performed according
to the same method.
[0119] Under regulation of an insect virus promoter, HERV envelope
protein has characteristics of being highly expressed in insect
cells but being hardly expressed in animal cells. On the contrary,
HPV 16L1 protein is possible to be highly efficiently expressed in
animal cells but being hardly or very lowly expressed in insect
cells due to utilization of human elongation factor 10 promoter
(hEF1.alpha.). Recombinant bacmids were prepared using each plasmid
cloned, and transfected into Sf9 cells, producing viruses with
higher titer.
Efficiency Measurement for Transfection of HPV 16L1 Gene to Huh7
Cells
[0120] To check transfection efficiency of HPV 16L1 gene according
to modification of baculovirus envelope, Huh7 cells were infected
with Ac-hEF1.alpha.16L1 and AcHERVenv-hEF1.alpha.16L1 virus at MOI
of 100, respectively. Expression level of HPV 16L1 mRNA was
examined using RT-PCR. As shown in FIG. 8, HPV 16L1 products of
about 275 by in length were detected in cells infected with
Ac-hEF1.alpha.16L1 and AcHERVenv-hEF1.alpha.16L1 virus using
electrophoresis. However, there was a difference to what extent HPV
16L1 gene was amplified. The amplified amount of HPV 16L1 gene in
AcHERVenv-hEF1.alpha.16L1 baculovirus having HERV envelope protein
in its envelope was higher than that in baculovirus having no
modification in its envelope.
[0121] Immunocytochemistry analysis was carried out to observe
under a microscope in Huh7 cells infected with Ac-hEF1.alpha.16L1
and AcHERVenv-hEF1.alpha.16L1 virus. At 48 hrs after infection, the
cells were stained with a HPV 16L1 monoclonal antibody (Camvir-1)
and a mouse IgG-horseradish peroxidase antibody, and observed under
a confocal laser scanning microscope to determine whether HPV 16L1
protein is or not. As shown in FIG. 9, it could be demonstrated
that the fluorescence was overall detected in the cells infected
with HERVenv-hEF1.alpha.16L1 and Ac-hEF1.alpha.16L1 virus compared
to Huh7 cells having no virus infection. However, the following
experiments were further performed to significantly differentiate
the extent of fluorescence between two samples.
[0122] To determine a transfer efficiency of HPV 16L1 gene using
infection, quantitative analysis by real-time PCR (Q-PCR) was
carried out. The accuracy of Q-PCR analysis was normalized by a
standard curve. The experiments were repeated four times, and
relative quantitation was obtained from a Delta-Delta CT method
using Roter-Gene ver. 6.0 as shown in FIG. 10. As described in the
following table 2, it could be appreciated that where the gene copy
number in cells infected with Ac-hEF1.alpha.16L1 virus is
considered as 1, the gene copy number in cells infected with
AcHERVenv-hEF1.alpha.16L1 virus is evaluated as 4.17-fold.
TABLE-US-00002 TABLE 2 GOI GOI .DELTA. - Relative Virus name CT
count Norm.CT .DELTA.CT .DELTA.CT concentration Normalization
AcHERVenv- 22.89 2 19.45 3.44 -2.06 4.17 -- hEF1.alpha.16L1
AcPERVenv- 25.85 2 18.24 7.61 2.11 0.23 -- hEF1.alpha.16L1
AchEF1.alpha.16L1 23.93 2 18.44 5.5 0 1 Yes
Immune Response in Mouse
[0123] Mouse was intramuscularly injected with AcHERVenv,
AcHERVenv-hEF1.alpha.16L1, or AcHERVenv-hEF1.alpha.18L1 at a
concentration of 10.sup.7 PFU. Gardasil-injected group was used as
a positive control, and AcHERVenv- or PBS-injected group served as
a negative control. Immune responses of each group were compared.
HPV16L1-specific IgG antibody or HPV18L1-specific IgG antibody were
detected from mouse serum immunized using ELISA. Prior to
immunization, noticeably low level of IgG antibody was detected in
the serum from AcHERVenv- or PBS-injected group as expected. As
shown in FIG. 12, IgG antibody response was detected in only serum
of gardasil-injected group (Group 1) after first immunization,
whereas not significantly in serum of the group injected with
AcHERVenv-hEF1.alpha.16L1 or AcHERVenv-hEF1.alpha.18L1 (Group 2).
IgG antibody response against HPV16 and HPV18 in serum of mouse
immunized with gardasil two-times, was enhanced about 2.7-fold and
2-fold higher than that in serum of mouse after first immunization,
respectively. In serum of mouse immunized with gardasil
three-times, IgG antibody response against HPV16 and HPV18 was
enhanced about 1.3-fold and 1.3-fold higher than that in serum of
mouse after second immunization, and 3.5-fold and 2.5-fold higher
than that in serum of mouse after second immunization, respectively
(See, Group 1 in FIG. 12). IgG antibody response against HPV16 and
HPV18 in serum of mouse immunized with AcHERVenv-hEF1.alpha.16L1 or
AcHERVenv-hEF1.alpha.18L1 two-times, was enhanced about 3-fold and
2-fold higher than that in serum of mouse after first immunization,
respectively. In serum of mouse immunized with
AcHERVenv-hEF1.alpha.16L1 or AcHERVenv-hEF1.alpha.18L1 three-times,
IgG antibody response against HPV16 and HPV18 was enhanced about
1.1-fold and 1.1-fold higher than that in serum of mouse after
second immunization, and 3.3-fold and 2.4-fold higher than that in
serum of mouse after second immunization, respectively (See, Group
2 in FIG. 12). Therefore, it could be appreciated that IgG antibody
response against HPV16 and HPV18 in serum of mouse immunized with
AcHERVenv-hEF1.alpha.16L1 or AcHERVenv-hEF1.alpha.18L1 is similar
to that immunized with gardasil. Given that IgG antibody response
was also observed in 9-week and 14-week after first immunization,
it was evident that the immunity was continuously maintained.
[0124] Secretory IgA response was determined by ELISA using vaginal
washes of immunized mouse. It was demonstrated that IgA antibody is
secreted not only in the gardasil-injected experimental group but
also in the experimental group injected with
AcHERVenv-hEF1.alpha.16L1 or AcHERVenv-hEF1.alpha.18L1 (FIG. 13).
It was evident that in addition to first immunization, IgA antibody
secretion was also increased in second and third immunization, and
further the immunity was persisted because IgA antibody response
was observed in 9-week and 14-week. Hence, it could be appreciated
that the immunization with AcHERVenv-hEF1.alpha.16L1 or
AcHERVenv-hEF1.alpha.18L1 may induce mucosal immune response in
mouse.
Neutralization of HPV Type 16, HPV Type 18, and BPV PVs by Mouse
Anti-Serum
[0125] Neutralizing activity of anti-serum was determined depending
on the extent of inhibiting infectivity of HPV16 or HPV18 PVs
against GFP-expressing plasmid in HeLa cells. Titer of neutralizing
antibody was indicated as a reciprocal of serum amount under
conditions that serum is maximally diluted (i.e., serum diluted at
a multiple of 5) and GFP expression level of samples with serum
treatment is reduced to 50% or 90% compared to that of samples
without serum treatment. Neutralizing activity of diluted serum
against HPV16 or HPV18 PVs in each experimental group is shown in
FIG. 14. FIG. 14 represents a neutralization titer that HPV16 or
HPV18 PVs were reduced to 50%, and neutralizing antibody titers
after second and third immunization than first immunization were
highly enhanced in all experimental groups. After third
immunization, neutralizing antibody titer in Group 1 and 2 was
156,250, and observed in higher level without significant
difference in B cell humoral immune responses between
gardasil-injected group and group injected with
AcHERVenv-hEF1.alpha.16L1 or AcHERVenv-hEF1.alpha.18L1 developed in
the present invention. Considered standard as 50% of neutralizing
activity, in Group 3 and 4 boosted with gardasil after priming of
AcHERVenv-hEF1.alpha.16L1 or AcHERVenv-hEF1.alpha.18L1,
neutralizing antibody titer was further increased from 234,375 to
312,500. Interestingly, in Group 4 boosted with gardasil two-times
after priming of AcHERVenv-hEF1.alpha.16L1, neutralizing antibody
titer was measured at the highest titer of 312,500. As results, it
is expected that the priming of AcHERVenv-hEF1.alpha.16L1 may
improve boosting effect of gardasil.
Cellular Immune Response Analysis
[0126] To assess T-cell immune responses in immunized mouse,
ELISPOT analysis was carried out. About 500 spots were observed in
spleen cells (1.times.10.sup.6) of mouse in Group 2 immunized with
AcHERVenv-hEF.alpha.16L1 or AcHERVenv-hEF1.alpha.18L1 three-times,
whereas no spot was observed in Group 1 immunized with gardasil or
a negative control due to secretion of IFN-.gamma.. Of mice
injected with gardasil, AcHERVenv-hEF.alpha.16L1 or
AcHERVenv-hEF1.alpha.18L1, and PBS, strong HPV16-specific T-cell
response (secretion of IFN-.gamma.) was generated in mice immunized
with AcHERVenv-hEF.alpha.16L1 or AcHERVenv-hEF1.alpha.18L1, and no
cellular immune responses were detected in the experimental group
immunized with gardasil (FIG. 15).
[0127] In conclusion, AcHERVenv-hEF.alpha.16L1 or
AcHERVenv-hEF1.alpha.18L1 chimera baculovirus effectively
transferred a DNA vaccine into an animal body in a stable manner,
leading to almost similar effect on humoral immune responses
compared with conventional vaccine, gardasil. Inoculation of both
AcHERVenv-hEF.alpha.16L1 or AcHERVenv-hEF1.alpha.18L1 chimera
baculovirus and gardasil resulted in much higher neutralizing
antibody titer than that of gardasil alone. As expected, gardasil
generated no cellular immunity, whereas AcHERVenv-hEF.alpha.16L1 or
AcHERVenv-hEF1.alpha.18L1 chimera baculovirus permits to express L1
gene in APC (antigen presentation cell) as a DNA vaccine, inducing
very strong cellular immunity. Taken together, a novel
AcHERVenv-hEF.alpha.16L1 or AcHERVenv-hEF1.alpha.18L1 chimera
baculovirus vaccine of the present invention is more stable and
economic than gardasil in respect of vaccine efficacy.
[0128] Having described a preferred embodiment of the present
invention, it is to be understood that variants and modifications
thereof falling within the spirit of the invention may become
apparent to those skilled in this art, and the scope of this
invention is to be determined by appended claims and their
equivalents.
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Sequence CWU 1
1
211617DNAArtificial SequenceSynthesized sequence of HERV envelope
1atg gcc ctg ccc tac cac att ttt ctg ttc acc gtg ctg ctg cct tcc
48Met Ala Leu Pro Tyr His Ile Phe Leu Phe Thr Val Leu Leu Pro Ser1
5 10 15ttc acc ctg aca gcc cct cca cca tgc agg tgt atg aca tcc tcc
tct 96Phe Thr Leu Thr Ala Pro Pro Pro Cys Arg Cys Met Thr Ser Ser
Ser 20 25 30ccc tac cag gag ttt ctg tgg cgg atg cag aga ccc ggc aac
atc gat 144Pro Tyr Gln Glu Phe Leu Trp Arg Met Gln Arg Pro Gly Asn
Ile Asp 35 40 45gcc cca agc tac cgg tcc ctg agc aaa ggc acc ccc acc
ttc aca gcc 192Ala Pro Ser Tyr Arg Ser Leu Ser Lys Gly Thr Pro Thr
Phe Thr Ala 50 55 60cac aca cac atg ccc aga aac tgc tat cac tcc gcc
acc ctg tgc atg 240His Thr His Met Pro Arg Asn Cys Tyr His Ser Ala
Thr Leu Cys Met65 70 75 80cac gcc aat acc cac tat tgg acc gga aaa
atg att aat cct tct tgc 288His Ala Asn Thr His Tyr Trp Thr Gly Lys
Met Ile Asn Pro Ser Cys 85 90 95cct ggc ggc ctg ggc gtg acc gtg tgc
tgg aca tac ttt aca cag acc 336Pro Gly Gly Leu Gly Val Thr Val Cys
Trp Thr Tyr Phe Thr Gln Thr 100 105 110ggg atg agc gac ggc ggg ggc
gtg cag gac cag gcc cgg gaa aag cac 384Gly Met Ser Asp Gly Gly Gly
Val Gln Asp Gln Ala Arg Glu Lys His 115 120 125gtg aaa gaa gtg atc
agc cag ctg aca agg gtg cac gga aca agc tcc 432Val Lys Glu Val Ile
Ser Gln Leu Thr Arg Val His Gly Thr Ser Ser 130 135 140cct tat aag
gga ctg gac ctg tct aag ctg cac gag aca ctg cgg aca 480Pro Tyr Lys
Gly Leu Asp Leu Ser Lys Leu His Glu Thr Leu Arg Thr145 150 155
160cac acc agg ctg gtg agc ctg ttc aac aca acc ctg aca ggc ctg cac
528His Thr Arg Leu Val Ser Leu Phe Asn Thr Thr Leu Thr Gly Leu His
165 170 175gaa gtg tcc gcc cag aat cct acc aat tgt tgg atc tgc ctg
cca ctg 576Glu Val Ser Ala Gln Asn Pro Thr Asn Cys Trp Ile Cys Leu
Pro Leu 180 185 190aat ttc cgg cct tac gtg tcc atc cct gtg ccc gag
cag tgg aat aat 624Asn Phe Arg Pro Tyr Val Ser Ile Pro Val Pro Glu
Gln Trp Asn Asn 195 200 205ttc tct acc gaa atc aat acc acc tcc gtg
ctg gtg ggc cca ctg gtg 672Phe Ser Thr Glu Ile Asn Thr Thr Ser Val
Leu Val Gly Pro Leu Val 210 215 220tcc aac ctg gag att aca cac acc
agc aat ctg acc tgt gtg aag ttt 720Ser Asn Leu Glu Ile Thr His Thr
Ser Asn Leu Thr Cys Val Lys Phe225 230 235 240tcc aac acc aca tat
acc acc aac agc cag tgc att agg tgg gtg acc 768Ser Asn Thr Thr Tyr
Thr Thr Asn Ser Gln Cys Ile Arg Trp Val Thr 245 250 255ccc ccc acc
cag att gtg tgc ctg cca tct ggg atc ttc ttt gtg tgc 816Pro Pro Thr
Gln Ile Val Cys Leu Pro Ser Gly Ile Phe Phe Val Cys 260 265 270ggc
aca agc gcc tac cgc tgt ctg aac ggg agc tcc gag agc atg tgc 864Gly
Thr Ser Ala Tyr Arg Cys Leu Asn Gly Ser Ser Glu Ser Met Cys 275 280
285ttt ctg agc ttc ctg gtg ccc cca atg acc atc tat aca gag cag gac
912Phe Leu Ser Phe Leu Val Pro Pro Met Thr Ile Tyr Thr Glu Gln Asp
290 295 300ctg tat tct tac gtg atc tct aaa cca cgc aac aag cgg gtg
cca att 960Leu Tyr Ser Tyr Val Ile Ser Lys Pro Arg Asn Lys Arg Val
Pro Ile305 310 315 320ctg cca ttc gtg atc ggg gcc ggg gtg ctg ggc
gcc ctg ggc acc ggg 1008Leu Pro Phe Val Ile Gly Ala Gly Val Leu Gly
Ala Leu Gly Thr Gly 325 330 335atc gga ggc atc aca act agt aca cag
ttc tac tac aaa ctg tct cag 1056Ile Gly Gly Ile Thr Thr Ser Thr Gln
Phe Tyr Tyr Lys Leu Ser Gln 340 345 350gaa ctg aac ggc gac atg gag
agg gtg gcc gat tct ctg gtg acc ctg 1104Glu Leu Asn Gly Asp Met Glu
Arg Val Ala Asp Ser Leu Val Thr Leu 355 360 365cag gac cag ctg aac
tcc ctg gcc gcc gtg gtg ctg cag aat cgg agg 1152Gln Asp Gln Leu Asn
Ser Leu Ala Ala Val Val Leu Gln Asn Arg Arg 370 375 380gcc ctg gat
ctg ctg acc gcc gaa cgg ggc ggc acc tgt ctg ttt ctg 1200Ala Leu Asp
Leu Leu Thr Ala Glu Arg Gly Gly Thr Cys Leu Phe Leu385 390 395
400ggg gag gaa tgc tgc tat tat gtg aac cag tcc gga atc gtg acc gag
1248Gly Glu Glu Cys Cys Tyr Tyr Val Asn Gln Ser Gly Ile Val Thr Glu
405 410 415aag gtg aag gag atc cgc gac agg atc cag agg cgg gcc gaa
gag ctg 1296Lys Val Lys Glu Ile Arg Asp Arg Ile Gln Arg Arg Ala Glu
Glu Leu 420 425 430aga aat acc ggc cca tgg ggc ctg ctg tct cag tgg
atg ccc tgg att 1344Arg Asn Thr Gly Pro Trp Gly Leu Leu Ser Gln Trp
Met Pro Trp Ile 435 440 445ctg cca ttc ctg ggc ccc ctg gcc gcc att
atc ctg ctg ctg ctg ttt 1392Leu Pro Phe Leu Gly Pro Leu Ala Ala Ile
Ile Leu Leu Leu Leu Phe 450 455 460ggc ccc tgt atc ttc aac ctg ctg
gtg aat ttc gtg tct agc aga atc 1440Gly Pro Cys Ile Phe Asn Leu Leu
Val Asn Phe Val Ser Ser Arg Ile465 470 475 480gag gcc gtg aag ctg
cag atg gag cct aag atg cag tcc aag aca aaa 1488Glu Ala Val Lys Leu
Gln Met Glu Pro Lys Met Gln Ser Lys Thr Lys 485 490 495atc tat cgc
cgc cct ctg gac aga ccc gcc agc cct aga tct gac gtg 1536Ile Tyr Arg
Arg Pro Leu Asp Arg Pro Ala Ser Pro Arg Ser Asp Val 500 505 510aat
gac att aag ggc acc cca cca gag gag atc tcc gcc gcc cag ccc 1584Asn
Asp Ile Lys Gly Thr Pro Pro Glu Glu Ile Ser Ala Ala Gln Pro 515 520
525ctg ctg agg ccc aac tct gcc ggg agc agc tga 1617Leu Leu Arg Pro
Asn Ser Ala Gly Ser Ser 530 5352538PRTArtificial SequenceSynthetic
Construct 2Met Ala Leu Pro Tyr His Ile Phe Leu Phe Thr Val Leu Leu
Pro Ser1 5 10 15Phe Thr Leu Thr Ala Pro Pro Pro Cys Arg Cys Met Thr
Ser Ser Ser 20 25 30Pro Tyr Gln Glu Phe Leu Trp Arg Met Gln Arg Pro
Gly Asn Ile Asp 35 40 45Ala Pro Ser Tyr Arg Ser Leu Ser Lys Gly Thr
Pro Thr Phe Thr Ala 50 55 60His Thr His Met Pro Arg Asn Cys Tyr His
Ser Ala Thr Leu Cys Met65 70 75 80His Ala Asn Thr His Tyr Trp Thr
Gly Lys Met Ile Asn Pro Ser Cys 85 90 95Pro Gly Gly Leu Gly Val Thr
Val Cys Trp Thr Tyr Phe Thr Gln Thr 100 105 110Gly Met Ser Asp Gly
Gly Gly Val Gln Asp Gln Ala Arg Glu Lys His 115 120 125Val Lys Glu
Val Ile Ser Gln Leu Thr Arg Val His Gly Thr Ser Ser 130 135 140Pro
Tyr Lys Gly Leu Asp Leu Ser Lys Leu His Glu Thr Leu Arg Thr145 150
155 160His Thr Arg Leu Val Ser Leu Phe Asn Thr Thr Leu Thr Gly Leu
His 165 170 175Glu Val Ser Ala Gln Asn Pro Thr Asn Cys Trp Ile Cys
Leu Pro Leu 180 185 190Asn Phe Arg Pro Tyr Val Ser Ile Pro Val Pro
Glu Gln Trp Asn Asn 195 200 205Phe Ser Thr Glu Ile Asn Thr Thr Ser
Val Leu Val Gly Pro Leu Val 210 215 220Ser Asn Leu Glu Ile Thr His
Thr Ser Asn Leu Thr Cys Val Lys Phe225 230 235 240Ser Asn Thr Thr
Tyr Thr Thr Asn Ser Gln Cys Ile Arg Trp Val Thr 245 250 255Pro Pro
Thr Gln Ile Val Cys Leu Pro Ser Gly Ile Phe Phe Val Cys 260 265
270Gly Thr Ser Ala Tyr Arg Cys Leu Asn Gly Ser Ser Glu Ser Met Cys
275 280 285Phe Leu Ser Phe Leu Val Pro Pro Met Thr Ile Tyr Thr Glu
Gln Asp 290 295 300Leu Tyr Ser Tyr Val Ile Ser Lys Pro Arg Asn Lys
Arg Val Pro Ile305 310 315 320Leu Pro Phe Val Ile Gly Ala Gly Val
Leu Gly Ala Leu Gly Thr Gly 325 330 335Ile Gly Gly Ile Thr Thr Ser
Thr Gln Phe Tyr Tyr Lys Leu Ser Gln 340 345 350Glu Leu Asn Gly Asp
Met Glu Arg Val Ala Asp Ser Leu Val Thr Leu 355 360 365Gln Asp Gln
Leu Asn Ser Leu Ala Ala Val Val Leu Gln Asn Arg Arg 370 375 380Ala
Leu Asp Leu Leu Thr Ala Glu Arg Gly Gly Thr Cys Leu Phe Leu385 390
395 400Gly Glu Glu Cys Cys Tyr Tyr Val Asn Gln Ser Gly Ile Val Thr
Glu 405 410 415Lys Val Lys Glu Ile Arg Asp Arg Ile Gln Arg Arg Ala
Glu Glu Leu 420 425 430Arg Asn Thr Gly Pro Trp Gly Leu Leu Ser Gln
Trp Met Pro Trp Ile 435 440 445Leu Pro Phe Leu Gly Pro Leu Ala Ala
Ile Ile Leu Leu Leu Leu Phe 450 455 460Gly Pro Cys Ile Phe Asn Leu
Leu Val Asn Phe Val Ser Ser Arg Ile465 470 475 480Glu Ala Val Lys
Leu Gln Met Glu Pro Lys Met Gln Ser Lys Thr Lys 485 490 495Ile Tyr
Arg Arg Pro Leu Asp Arg Pro Ala Ser Pro Arg Ser Asp Val 500 505
510Asn Asp Ile Lys Gly Thr Pro Pro Glu Glu Ile Ser Ala Ala Gln Pro
515 520 525Leu Leu Arg Pro Asn Ser Ala Gly Ser Ser 530 535
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References