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.

Claims

1. A method for inducing an immune response against a specific antigen in a mammalian subject in need thereof, comprising: (a) transfecting into an insect cell a recombinant bacmid comprising (i) a nucleotide sequence encoding an envelope protein of an endogenous retrovirus; (ii) a first promoter that is operable in the insect cell and is operatively linked to (i); (iii) a nucleotide sequence encoding a second antigen protein not from any baculovirus and endogenous retrovirus; and (iv) a second promoter that is from a mammalian genome or virus, which is different the first promoter and is operatively linked to (iii); (b) obtaining a recombinant baculovirus vector produced from the insect cell, wherein the recombinant baculovirus vector is an endogenous retrovirus envelope-coated Baculovirus vector to express the second antigen; and (c) administering a pharmaceutically effective amount of the recombinant baculovirus of (b) to the mammalian subject.

2. The method according to claim 1, wherein the second antigen selected from group consisting of a viral antigen, a bacterial antigen, a parasitic antigen or a cancer antigen.

3. The method according to claim 2, wherein the second 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 method according to claim 3, wherein the second antigen is HPV antigen, and the method is a method for preventing or treating a HPV infection-induced cancer.

5. The method according to claim 4, wherein the second antigen selected from group consisting of is HPV L1, L2, E6 or E7 protein.

6. The method according to claim 4, wherein the HPV antigen protein is 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. The method according to claim 1, wherein the endogenous retrovirus envelope protein is a HERV (human endogenous retrovirus) envelope protein.

8. The method according to claim 7, wherein the HERV envelope protein comprises the amino acid sequence of SEQ ID NO: 2.

9. The method according to claim 1, wherein the first promoter operable in the insect cell is selected from the group consisting of IE-1 promoter, IE-2 promoter, p35 promoter, p10 promoter, gp64 promoter and polyhedrin promoter.

10. The method according to claim 1, wherein the second promoter is selected from the group consisting of 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 (hEF1) promoter, methallothionein promoter, -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 and albumin promoter.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 schematically represents a construction procedure of transfer vector, pAc-hEF116L1, used in the present invention. In FIG. 1, black arrow, white arrow, and small black quadrangle indicate polyhedrin promoter, hEF1 promoter, and hEF1 poly(A) signal, respectively.

(2) FIG. 2 schematically represents a construction procedure of transfer vector, pAcHERVenv-hEF116L1, used in the present invention. In FIG. 2, black arrow, white arrow, and small black quadrangle indicate polyhedrin promoter, hEF1 promoter, and hEF1 poly(A) signal, respectively.

(3) FIG. 3 represents an expected scheme of chimera baculovirus transfer vectors and viruses including pAc-hEF116L1 and pAcHERVenv-hEF116L1 construct, respectively. In FIG. 3, black arrow, white arrow, and small black quadrangle indicate polyhedrin promoter, hEF1 promoter, and hEF1 poly(A) signal, respectively.

(4) FIGS. 4-7 are a sequence homology through alignment of nucleotide sequence between a HERV envelope protein synthesized in the present invention (SEQ ID NO:1) and a HERV envelope protein. The nucleotide sequence of HERV envelope protein is shown in GenBank accession No. NM 014590 (SEQ ID NO:3).

(5) FIG. 8 represents RT-PCR to examine expression of HPV 16L1 gene in Huh7 cells infected with Ac-hEF116L1 or AcHERVenv-hEF116L1 construct. NTC indicates a control without template. AcHERVenv-hEF116L1 construct contains an envelope protein of pig endogenous retrovirus and exhibits almost no infectivity to human Huh7 cells.

(6) FIG. 9 is images analyzing HPV 16L1 of normal Huh7 cells and Huh7 cells infected with Ac-hEF116L1 or AcHERVenv-hEF116L1 construct through immunocytochemical staining. AcHERVenv-hEF116L1 construct contains an envelope protein of pig endogenous retrovirus and exhibits almost no infectivity to human Huh7 cells.

(7) 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-hEF116L1 or AcHERVenv-hEF116L1 construct. AcHERVenv-hEF116L1 construct contains an envelope protein of pig endogenous retrovirus and exhibits almost no infectivity to human Huh7 cells.

(8) 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-hEF116L1 construct, HPV 16L1 is positioned at a gene of interest.

(9) 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.

(10) 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.

(11) FIG. 14 represents neutralization response against HPV16 or HPV18 PVs (pseudoviruses) by mouse antiserum immunized with chimera baculovirus of the present invention.

(12) FIG. 15 represents ELISPOT analysis for cell-mediated immune response. To evaluate IFN- 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-hEF116L1 or AcHERVenv-hEF118L1, and (C) serves as a control.

(13) 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

(14) Cell Preparation

(15) 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 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 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 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 g streptomycin/ml.

(16) Synthesis of a Gene Encoding a HERV Envelope Protein

(17) 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.

(18) Cloning of Transfer Vector

(19) 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 baculovirus expression system. To express HPV 16L1 protein in animal cells using the recombinant baculovirus system, a human elongation factor 1 (hEF1) 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-HPV 16L1-hEF1 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 (SEQ ID NO:4); and antisense primer, 5-TTAATTAACCCACGTTTCAACATG-3 (SEQ ID NO:5).

(20) 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 1 (Invitrogen) transfer vector, generating a pAc-hEF116L1 vector (See, FIG. 1). A HERV envelope protein gene was cut with Sail from pUC57-HERVenv vector and then inserted into pFastBac 1 vector. After cutting pGEM-Teasy/hEF116L1 with NotI, hEF116L1 was inserted into pFastBac 1-HERVenv transfer vector, constructing pAcHERVenv-hEF116L1 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).

(21) Construction of a Recombinant Baculovirus

(22) 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-hEF116L1 and Ac-hEF116L1, respectively (See, FIG. 3). Finally, titers of recombinant baculoviruses were determined in Sf9 cells using a plaque assay. Meanwhile, recombinant baculoviruses (AcHERVenv-hEF118L1) were prepared according to the mentioned-above method for preparing recombinant baculoviruses (AcHERVenv-hEF116L1) except for using a HPV 18L1 gene (GenBank accession No. AY383629).

(23) Transfection of a Gene into Huh7 Cells Using a Recombinant Baculovirus

(24) Huh7 cells were seeded into a 24-well plate at a concentration of 110.sup.5 cells/well and cultured at 37 C. After incubation for 12 hrs, the cells were washed with PBS, and then infected with Ac-hEF116L1 and AcHERVenv-hEF116L1 virus of 100 MOI (multiplicity of infectivity), respectively. Then, the cells were cultured at 37 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.

(25) Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis

(26) 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 l of PCR reaction mixture was mixed with 2.5 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 C. for 3 min; and 30-cycle step of denaturing at 94 C. for 30 sec, annealing at 62 C. for 20 sec and elongating at 72 C. for 20 sec. The primers used were: sense primer, 5-CAGGGCCACAACAACGGCATCTGCTGGG-3 (SEQ ID NO:6); and antisense primer, 5-GGCTGCAGGCCGAAGTTCCAGTCCTCCA-3 (SEQ ID NO:7). 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.

(27) Quantitative Analysis Using Real-Time PCR (Q-PCR)

(28) 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 l of DyNAmo HS SYBR Green qPCR kit reaction solution and 5 l of sample buffer containing primers and templates. The primers used were: 16L1 sense primer, 5-CAGCGAGACCACCTACAAGA-3 (SEQ ID NO:8); and antisense primer, 5-GCTGTTCATGCTGTGGATGT-3 (SEQ ID NO:9). The resulting PCR products were expected as about 138 bp. PCR products were obtained by pre-denaturing step at 95 C. for 5 min, and 45-cycle step of denaturing at 94 C. for 10 sec, annealing at 62 C. for 20 sec and elongating at 72 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).

(29) Immunocytochemistry

(30) Huh7 cells were divided into a glass slide, and then transfected with Ac-hEF116L1 and AcHERVenv-hEF116L1 virus of 100 MOI (multiplicity of infectivity), respectively. After transfection for 48 hrs, the cells were fixed with 4% formaldehyde at 4 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 C. for 10 min. Next, the cells were washed with PBS and blocked with PBS containing goat serum at 37 C. for 30 min, followed by incubating with HPV 16L1 monoclonal antibody (Camvir-1) at 4 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.

(31) Gardasil

(32) Gardasil (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.

(33) Mouse

(34) 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.

(35) Mouse Immunization

(36) Recombinant baculoviruses were diluted with sterile PBS at a total volume of 100 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.

(37) Table 1.

(38) TABLE-US-00001 Experimental Immunization (interval of 2-week) group First Second Third Group 1 Gardasil Gardasil Gardasil Group 2 AcHERVenv-hEF116L1 AcHERVenv-hEF116L1 AcHERVenv-hEF116L1 or or or AcHERVenv-hEF118L1 AcHERVenv-hEF118L1 AcHERVenv-hEF118L1 Group 3 AcHERVenv-hEF116L1 AcHERVenv-hEF116L1 Gardasil or or AcHERVenv-hEF118L1 AcHERVenv-hEF118L1 Group 4 AcHERVenv-hEF116L1 Gardasil Gardasil or AcHERVenv-hEF118L1 Group 5 AcHERV Gardasil Gardasil Group 6 AcHERV AcHERV Gardasil Group 7 AcHERV AcHERV AcHERV Group 8 PBS PBS PBS
ELISA

(39) Sixty 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 g/ml, and incubated at 4 C. for 14-16 hrs. Each well was blocked at 37 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.

(40) Pseudoviruses (PVs) Preparation

(41) 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 g of each pfwB and p16L1/L2. In addition, cells were transfected with 9 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 C. until next experiment.

(42) Neutralization Analysis

(43) 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 110.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 level of sample treated with normal mouse serum.

(44) IFN- Enzyme-Linked Immunospot (ELISPOT)

(45) A 96-well plate was coated with 200 ng of anti-mouse IFN- capturing antibody (BD Bioscience) in 100 l PBS at 4 C. overnight. The plate was blocked in 100 l RPM 1640 with 10% FBS at 37 C. for 2 hrs, and spleen cells with a density of 110.sup.6 were seeded into the plate duplicate. PVs of 210.sup.6 IFU (infectious unit) were inoculated into the plate, followed by incubating at 37 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- 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 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 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.

(46) Results

(47) Gene Synthesis of a HERV Envelope Protein

(48) 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 bp 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.

(49) Construction of a Recombinant Baculovirus

(50) To construct recombinant baculoviruses, two types of transfer vectors, pAc-hEF116L1 and pAcHERVenv, hEF116L1, 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. HERV Env may induce a receptor-mediated phagocytosis in human cells. FIG. 1 schematically represents a cloning method of pAc-hEF116L1, and FIG. 2 briefly represents a cloning method of pAcHERVenv-hEF116L1. The cloning of pAcHERVenv-hEF118L1 was performed according to the same method.

(51) 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). Recombinant bacmids were prepared using each plasmid cloned, and transfected into Sf9 cells, producing viruses with higher titer.

(52) Efficiency Measurement for Transfection of HPV 16L1 Gene to Huh7 Cells

(53) To check transfection efficiency of HPV 16L1 gene according to modification of baculovirus envelope, Huh7 cells were infected with Ac-hEF116L1 and AcHERVenv-hEF116L1 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 bp in length were detected in cells infected with Ac-hEF116L1 and AcHERVenv-hEF116L1 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-hEF116L1 baculovirus having HERV envelope protein in its envelope was higher than that in baculovirus having no modification in its envelope.

(54) Immunocytochemistry analysis was carried out to observe under a microscope in Huh7 cells infected with Ac-hEF116L1 and AcHERVenv-hEF116L1 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-hEF116L1 and Ac-hEF116L1 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.

(55) 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-hEF116L1 virus is considered as 1, the gene copy number in cells infected with AcHERVenv-hEF116L1 virus is evaluated as 4.17-fold.

(56) TABLE-US-00002 TABLE 2 GOI GOI Norm. - Relative Virus name CT count CT CT CT concentration Normalization AcHERVenv- 22.89 2 19.45 3.44 2.06 4.17 hEF116L1 AcPERVenv- 25.85 2 18.24 7.61 2.11 0.23 hEF116L1 AchEF116L1 23.93 2 18.44 5.5 0 1 Yes
Immune Response in Mouse

(57) Mouse was intramuscularly injected with AcHERVenv, AcHERVenv-hEF116L1, or AcHERVenv-hEF118L1 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-hEF116L1 or AcHERVenv-hEF118L1 (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-hEF116L1 or AcHERVenv-hEF118L1 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-hEF116L1 or AcHERVenv-hEF118L1 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-hEF116L1 or AcHERVenv-hEF118L1 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.

(58) 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-hEF116L1 or AcHERVenv-hEF118L1 (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-hEF116L1 or AcHERVenv-hEF118L1 may induce mucosal immune response in mouse.

(59) Neutralization of HPV Type 16, HPV Type 18, and BPV PVs by Mouse Anti-Serum

(60) 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-hEF116L1 or AcHERVenv-hEF118L1 developed in the present invention. Considered standard as 50% of neutralizing activity, in Group 3 and 4 boosted with gardasil after priming of AcHERVenv-hEF116L1 or AcHERVenv-hEF118L1, 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-hEF116L1, neutralizing antibody titer was measured at the highest titer of 312,500. As results, it is expected that the priming of AcHERVenv-hEF116L1 may improve boosting effect of gardasil.

(61) Cellular Immune Response Analysis

(62) To assess T-cell immune responses in immunized mouse, ELISPOT analysis was carried out. About 500 spots were observed in spleen cells (110.sup.6) of mouse in Group 2 immunized with AcHERVenv-hEF16L1 or AcHERVenv-hEF118L1 three-times, whereas no spot was observed in Group 1 immunized with gardasil or a negative control due to secretion of IFN-. Of mice injected with gardasil, AcHERVenv-hEF16L1 or AcHERVenv-hEF118L1, and PBS, strong HPV16-specific T-cell response (secretion of IFN-) was generated in mice immunized with AcHERVenv-hEF16L1 or AcHERVenv-hEF118L1, and no cellular immune responses were detected in the experimental group immunized with gardasil (FIG. 15).

(63) In conclusion, AcHERVenv-hEF16L1 or AcHERVenv-hEF118L1 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-hEF16L1 or AcHERVenv-hEF118L1 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-hEF16L1 or AcHERVenv-hEF118L1 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-hEF16L1 or AcHERVenv-hEF118L1 chimera baculovirus vaccine of the present invention is more stable and economic than gardasil in respect of vaccine efficacy.

(64) 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.

REFERENCES

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