KR100663093B1 - Novel Recombinant Poliovirus Vectors and Vaccine Compositions Capable of Inducing Cytotoxic T Lymphocytes - Google Patents

Abstract

The present invention relates to a vaccine composition comprising a novel recombinant poliovirus vector and a recombinant poliovirus derived therefrom, and more particularly, (a) a genome nucleotide sequence of a poliovirus; (b) a nucleotide sequence encoding an additional poliovirus protease cleavage site; (c) an exogenous insertion nucleotide sequence linked to a nucleotide sequence encoding said additional poliovirus protease cleavage site; And (d) a recombinant poliovirus vector comprising a nucleotide sequence encoding a protein transducing peptide domain linked to the foreign insertion nucleotide sequence, and a recombinant poliovirus derived therefrom, wherein the vaccine composition of the present invention has a conventional CTL induction ability. It is significantly increased compared to poliovirus.

Poliovirus, protein permeation domain, cytotoxic T lymphocytes,

Description

Novel Recombinant Poliovirus Vectors and Vaccine Compositions Capable of Inducing Cytotoxic T Lymphocytes             

1 is a genetic map and sequence showing the basic structure of the recombinant poliovirus vector of the present invention.

2 is a view showing the manufacturing process of the recombinant poliovirus vector of the present invention.

3 is a one-step growth curve of the recombinant polioviruses of the present invention.

Figure 4a is a RT-PCR analysis of the genetic stability of the recombinant poliovirus of the present invention: M represents the molecular weight markers and numbers represent the passage number.

Figure 4b is a Western blot analysis of the genetic stability of the recombinant poliovirus of the present invention: the number represents the passage number.

Figure 5 is a photograph showing the results of X-gal staining of HeLa cells treated with protein permeation domain-betagalactosase fusion protein.

6 is a graph showing IgG isotypes in Tg-PVR mouse serum immunized with RPS-Vax / PTD / HCVc recombinant virus in the recombinant poliovirus vector of the present invention.

7 is a graph showing IgG isotypes in Tg-PVR mouse serum immunized with RPS-Vax / PTD / p24 (118 a) recombinant virus in the recombinant poliovirus vector of the present invention.

8 is a graph showing IgG isotypes in Tg-PVR mouse serum immunized with RPS-Vax / PTD / Nef recombinant virus in the recombinant poliovirus vector of the present invention.

9 is a graph showing the proliferation of T cells specifically responding to Nef protein in Tg-PVR mouse splenocytes immunized with RPS-Vax / PTD / Nef recombinant virus.

10 is a graph quantifying IL-4 produced upon proliferation of T cells that specifically respond to Nef protein in Tg-PVR mouse splenocytes immunized with RPS-Vax / PTD / Nef recombinant virus.

11 is a graph quantifying IFN-γ produced upon proliferation of T cells that specifically respond to Nef protein in Tg-PVR mouse splenocytes immunized with RPS-Vax / PTD / Nef recombinant virus.

The present invention relates to a poliovirus vector, and more particularly to a vaccine composition comprising a recombinant poliovirus vector and a recombinant poliovirus derived therefrom.

Poliovirus is a enterovirus belonging to the Picornaviridae , and there are three serotypes (types 1, 2 and 3) as positive-sense single chain RNA viruses (4 and 33). Poliovirus is an infectious virus that infects the digestive system, proliferates in nerve cells, destroys the central nervous system (CNS), and causes polio (poliomyelitis) .Type 1 is the most pathogenic, type 3 and type 2 Toxic (1, 8 and 39).

The poliovirus grows in the cytoplasm of infected cells, and the 7340 base genome RNA covalently binds to VPg, a 22-amino acid viral protein at the 5 'end, and poly (A) tail at the 3' end. Have (2,41). The viral genome acts as mRNA to synthesize a single polyprotein, which is processed by three viral proteases 2A ^pro , 3C ^pro and 3CD (9 and 11). In the transformation step, 2A ^pro acts on a specific tyrosine (Y) -glycine (G) amino acid pair position between P1 and P2 of a single polyprotein to release the P1 capsid precursor protein. The P1 protein is then processed between glutamine (Q) -glycine (G) by 3CD and divided into capsid proteins VP0, VP3 and VP1. The P2 and P3 regions of the genome allow 3C ^pro to act between glutamine (Q) -glycine (G) amino acids to form nonstructural proteins including 3D ^pol involved in viral RNA replication (13, 21 and 38).

As tissue culture of poliovirus became possible in 1949, research on vaccines was actively conducted, and in 1955, inactivated poliovirus was developed as a vaccine, and in 1963, three attenuated oral polio vaccines were used. The development of vaccines (OPV) and Sabin 1, 2 and 3) resulted in a gradual reduction in neurological diseases caused by poliovirus (18).

In the case of the poliovirus oral vaccine, the administration of the poliovirus induced neuropathy is simple due to the advantages of simple administration, excellent mucosal immunity inducing ability (16 and 27), and long-lasting immune response without side effects. Research is underway to use poliovirus as a vector for other vaccine development rather than vaccine research for the prevention of disease (17 and 24).

Several methods have been reported for the development of poliovirus as a vector. First, there is a method of artificially introducing a protein cleavage site and multiple cloning site into a viral capsid protein and inserting an external vaccine gene (3). The 5'-noncoding region of the poliovirus genome RNA overlaps with an internal ribosome entry site (IRES) to create a poliovirus RNA having a dicystron translation enhancement site. Third, poliovirus mini As a replicon method (25,30), there is a method of replacing a structural protein gene with a vaccine gene and co-transforming it with a structural protein expression vector to produce a replication-defective minireplicon (28, 29, 31 and 32). Meanwhile, the present inventors applied the first of the above methods to the Sabin 1 poliovirus to construct a poliovirus vaccine vector, named "RPS- Vax", and filed a patent (see WO 99/07859).

However, the poliovirus vaccine vectors developed to date have problems in inducing cellular immune responses by antigen-specific T cells because the rapid proliferation of the virus does not give the infected cells sufficient time for antigen presentation. There was.

Throughout this specification, numerous patent documents and articles are referred to and their citations are shown in parentheses. The disclosures of cited patents and articles are hereby incorporated by reference in their entirety, so that the level of the technical field to which the present invention belongs and the gist of the present invention are more clearly explained.

The present inventors have diligently researched to develop a new concept poliovirus vaccine vector. As a result, the poliovirus vector produced by introducing a sequence encoding a peptide or polypeptide having cell membrane permeability into a poliovirus vector system was expressed in infected cells. By confirming that antigenic proteins can easily penetrate adjacent cells to facilitate antigen presentation and effectively induce cytotoxic T lymphocyte-response to antigens, the present invention has been completed.

Accordingly, the present invention provides a novel recombinant poliovirus vector.

Another object of the present invention is to provide a vaccine composition comprising a recombinant poliovirus derived from the novel recombinant poliovirus vector and capable of inducing a cellular immune response.

According to one aspect of the invention, the invention is (a) the genome nucleotide sequence of the poliovirus; (b) a nucleotide sequence encoding an additional poliovirus protease cleavage site; (c) an exogenous insertion nucleotide sequence linked to a nucleotide sequence encoding said additional poliovirus protease cleavage site; And (d) a nucleotide sequence encoding a protein transduction domain linked to the foreign insertion nucleotide sequence.

Recently, there have been many studies attempting to induce cytotoxic T lymphocytes (hereinafter referred to as "CTL")-response by the MHC class I pathway, with the importance of cellular immunity in various diseases including AIDS (43, 44 and 45).

Accordingly, the inventors have found a surprising fact that the introduction of a peptide or polypeptide having a membrane transduction capacity into a poliovirus vector system and incorporation of an external gene therein leads to a very effective CTL response.

The poliovirus vector of the present invention basically uses an exogenous cleavage site for the poliovirus protease, such a strategy is not only described in the above-mentioned documents but also in the patent applications WO 99/07859 and Korean Patent Application No. 2001-6229, and Also disclosed in US Pat. No. 5,965,124, which is incorporated herein by reference.

According to a preferred embodiment of the present invention, the genome nucleotide sequence of the poliovirus among the sequences included in the vector of the present invention is a poliovirus type 1 (Mahoney), poliovirus type 2 (Lansing) or Genome nucleotide sequence derived from poliovirus type 3 (Leon: Leon). In addition, according to a more preferred embodiment, the genome nucleotide sequence of the poliovirus may be a genome nucleotide sequence derived from attenuated strains Sabin 1, Sabin 2 or Sabin 3, most preferably the probability of being converted to pathogenic Genome nucleotide sequence derived from this lowest Sabine type 1.

According to an embodiment of the invention, said additional poliovirus protease cleavage site is a cleavage site at which the poliovirus 3C, 3CD protease or 2A protease acts, preferably a cleavage site at which the poliovirus 3C protease acts. According to an embodiment of the invention, such additional cleavage sites are formed in specific portions of the poliovirus genome sequence so as not to have a significant adverse effect on the replication and propagation of the poliovirus. For example, between the first and second of the N-terminus of the poliovirus polyprotein, between VP0 and VP3, between VP3 and VP1, between VP1 and 2A ^pro, between 2A ^pro and 2B, between 2B and 2C, 2C and 3A Between 3A and Vpg, between Vpg and 3C ^{pro and} between 3C ^pro and 3D ^pol . Preferably, the additional cleavage site is between the first and second amino acids of the N-terminus of the poliovirus polyprotein, between VP1 and 2A ^pro, between 2A ^pro and 2B, between 2C and 3A or between Vpg and 3C ^pro , Most preferably between the first and second amino acids of the N-terminus.

According to embodiments of the present invention, the exogenous insertion nucleotide sequence may be any sequence, but it is preferable to use a sequence that does not significantly affect the replication and propagation of the poliovirus.

In addition, the exogenous insertion nucleotide sequence is preferably used that is excellent in genetic stability (genetic stability). As used herein, the term "genetic stability" means the continuous maintenance of an exogenous nucleotide sequence as the recombinant poliovirus vector propagates and propagates from one host to another. Such genetic stability is of greater importance when recombinant poliovirus vectors are used as vaccines, and only exogenous sequences exhibiting genetic stability can elicit the same immune response for generations. Therefore, the present inventors have studied to improve the genetic stability of the foreign sequences, and as a result, developed a breakthrough method (Korean Patent Application No. 2001-6229; and Lee et al . J J. Virol 76: 1649-1662 (2002) ). According to the method proposed by the inventors, the genetic stability of the exogenous insertion sequence is large when the sequence size is about 450 bp or less, there is no local A / T rich site, and the G / C distribution is uniform throughout the sequence. Is improved. Sequences having these characteristics can be obtained from naturally-occurring sequences and obtained through mutagenesis. Said mutagenesis can be carried out through site-directed mutagenesis or cassette mutagenesis, which are commonly practiced in the art. In addition, sequences having the above characteristics for genetic stability may be prepared by ligation-free PCR developed by the inventor (see Korean Patent Application No. 2001-6229; Lee et al . J. Virol 76: 1649). -1662 (2002)).

According to an embodiment of the present invention, since the most important of the utility of the poliovirus vector of the present invention is a vaccine, the exogenous insertion sequence in the vector is a polypeptide of a bacterial polypeptide antigen, a viral polypeptide antigen, a fungal polypeptide antigen or a pathogenic eukaryotic cell. A sequence encoding a protein or polypeptide antigen derived from a peptide antigen. In particular, when the vector of the present invention is used as a vaccine, since the most efficient CTL induction is an important use as a defense mechanism against virus invasion, the exogenous insertion nucleotide sequence is HIV (human immunodeficiency virus), SIV (simian immunodeficiency virus), Group consisting of hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), human papilloma virus (HPV), herpes simplex virus (HSV), poliovirus, rotavirus, influenza virus, and pandemic hemorrhagic fever virus A sequence encoding a protein or polypeptide antigen derived from a polypeptide antigen of a pathogenic virus selected from. According to a preferred embodiment of the invention, the sequence encoding a protein or polypeptide antigen is a sequence encoding an amino acid sequence comprising an antigenic determinant region. As such, when a sequence capable of inducing an immune response is used as an exogenous insertion sequence, the vector of the present invention has an increased ability to induce CTL.

In a preferred embodiment of the invention, the sequence encoding the protein transduction domain in the vector comprises at least the amino acid sequence represented by SEQ ID NO: 1. The sequence encoding the protein permeation domain in the vector of the present invention allows efficient CTL induction when the vector of the present invention is used as a vaccine. The protein permeation domain used in the present invention is derived from the Tat protein of HIV-1. Tat protein has the function of a trans-activator in HIV-1 and is known as an essential protein for the replication of the virus. The amino acid sequence of the tart protein is shown in SEQ ID NO: 3 of the attached Sequence Listing, wherein SEQ ID NOs: 49-57 include two lysines and six arginines as basic sites, and SEQ ID NOs: 22-37 contain seven cysteine residues. Containing cysteine-rich sites. The basic site is known as an important site for nuclear localization (Ruben, S. et al., J. Virol. 63: 1-8 (1989); and Hauber, J. et al., J. . Virol 63: 1181-1187 (1989) ), wherein the cysteine-rich region is the metal in the bit - and mediate the formation of the connection dimer (Frankel, AD et al, PNAS USA 85: 6297-6300 (1988..)) , A sequence essential for the activity of trans-activators (Sadaie, MR et al., J. Virol. 63: 1 (1989)).

Since the sequence encoding the protein permeation domain in the vector of the present invention has the amino acid sequence represented by SEQ ID NO: 1 as the minimum sequence, even if other additional sequences are combined in a range that does not show a significant adverse effect on cell membrane permeability, they are included in the sequence of the present invention. do. In addition, since the amino acid sequence itself shown in SEQ ID NO: 1 may be modified in a range that does not show a significant adverse effect on cell membrane permeability according to methods known in the art, such modified sequence should also be interpreted as being included in the sequence of the present invention. do.

As described above, the cysteine-rich region in the protein permeation domain is independent of cell membrane permeability, but rather has a problem of forming disulfide bonds with the material to be transported. Therefore, in a preferred embodiment of the present invention, the protein permeation domain used in the present invention includes a minimum of SEQ ID NOs: 49-57 of the amino acid sequence represented by SEQ ID NO: 3 and lacks SEQ ID NOs: 22-37. In addition, since the protein permeation domain is preferably the smallest possible size, the protein permeation domain used in the present invention contains at least SEQ ID NOs: 49-57 among the amino acid sequences represented by SEQ ID NO: 3, SEQ ID NOs: 22-37 and SEQ ID NO: 73 More preferably, it is lacking -86.

Examples of the sequence that can be used as the protein permeation domain in the vector of the present invention can vary as described above, for example SEQ ID NO: 37-52, SEQ ID NO: 37-58, SEQ ID NO: 38 of the amino acid sequence represented by SEQ ID NO: 1 -58, SEQ ID NOs: 47-58 and SEQ ID NOs: 47-58 are also included. According to a specific embodiment of the present invention described in the following Examples, the sequence of the protein permeation domain is composed of the amino acid sequence represented by SEQ ID NO: 2.

The sequence encoding the above-described protein permeation domain may be directly linked with the exogenous insertion sequence, and may be linked with a suitable linker or spacer therebetween. In addition, the sequence encoding the aforementioned protein permeation domain can be linked to the 5'-end or 3'-end of the exogenous insertion sequence.

According to another aspect of the present invention, the present invention provides a kit comprising: (a) a recombinant poliovirus derived from the above recombinant poliovirus vector; And (b) provides a recombinant poliovirus vaccine composition comprising a pharmaceutically acceptable carrier.

Since the vaccine composition of the present invention basically uses the above-mentioned recombinant poliovirus vector, the matter common to the vaccine composition of the present invention and the recombinant poliovirus vector is omitted in order to avoid excessive redundancy in the specification.

The present invention provides a pharmaceutical vaccine composition comprising a recombinant poliovirus isolated from a host cell transfected with the above-described recombinant poliovirus vector, such as human cervical cancer cells (HeLa cells). The recombinant poliovirus has a sequence encoding a protein or polypeptide antigen comprising an antigenic determining site capable of inducing an immune response.

The vaccine composition of the present invention has the ability to induce humoral, mucosal and cellular immunity, and in particular the CTL inducing ability is greatly increased compared to any conventional poliovirus vaccine vector. In fact, the recombinant poliovirus vaccines developed to date have very low CTL induction ability, and the recombinant virus using a poliovirus vaccine vector shows rapid proliferation and destroys infected cells within a short time after infection. It doesn't happen effectively, so we don't think CTL is effective. However, the recombinant poliovirus used in the vaccine of the present invention overcomes the problems of the conventional poliovirus described above with a protein permeable domain. In other words, the protein or polypeptide antigen expressed by the recombinant poliovirus in the infected cells enters adjacent cells upon destruction of the infected cells, passes through the proteasome, is degraded, and is present on the surface of MHC class I very efficiently, This effectively induces CTL by antigen-specific T cells.

The pharmaceutical composition of the present invention preferably further comprises a pharmaceutically acceptable carrier or diluent, and any carrier or diluent conventionally used for vaccine preparation may be used. For example, chimeric polioviruses prepared according to the present invention can be stabilized in MgCl _2, sucrose and phosphate solutions. The vaccine composition of the present invention can be administered orally or parenterally, most preferably orally.

Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to reaction, Usually a skilled practitioner can easily determine and prescribe a dosage effective for the desired immunity. According to a preferred embodiment of the present invention, when orally administering the vaccine composition of the present invention, a suitable dosage may be administered at a known dosage for sabine vaccines, namely 10 ⁶ TCID ₅₀ or less.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulations here may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of extracts, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

The biggest feature of the vaccine composition of the present invention is the use of recombinant polioviruses with efficient CTL induction ability. For example, given the situation where no effective vaccine is currently developed for HCV, the induction of CTL for HCV by the vaccine composition of the present invention is surprising, which is clearly illustrated in the following examples. .

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.

Example

Experimental material

E. coli  Strains and Plasmids

E. coli used for transformation and plasmid amplification was XL1-Blue [ sup E44 hs R1 rec A1 gyrA 46 thi rel A1 lac ^- F { pro AB ⁺ lacl ^q lacZ Δ M 15 Tn 10 ( tet ^r )}] (Stratagene ) And JM109 { sup EΔ ( lac-pro AB) hsd R17 F ' tra D36 pro AB ⁺ lacl ^q lacZ Δ M 15} (Promega), and the expression of the recombinant plasmid includes BL21 (DE3) pLysS [BF ^- dcm ompT hsdS ( r _B -m _B- ) gal λ (DE3) {pLysS Cam ^r }] (Invitrogen) was used. For expression in bacteria, a prokaryotic expression vector, pTAT (provided by Dr. Dowdy, USA), was used, and the pGEM-T vector (Promega) was used for general cloning and vector modification. As the vector used for poliovirus generation and modification, pTZ-PVS-3m (RPS-vax system, KCTC-0365BP, see Fig. 1) was used.

Cell and virus stock

Cervical cancer cells, HeLa cells (ATCC), were maintained in monolayer culture using DMEM (Dulbecco's modified Eagles medium, GiBcoBRL) containing 10% heat-inactivated fetal bovine serum (FBS, GiBcoBRL). MAGI cells in which the LTR and β-galactosidase genes of HIV-1 were introduced into HeLa cells were selected by adding 100 μg / ml of hygromycin and 200 ug / ml of G418. The cells were cultured in a wet incubator maintained at 5% CO ₂ . The virus of poliovirus sabin type 1 and the modified RPS-Vax system was prepared by in vitro transcription of RNA transcripts by DEAE-dextran method (Van der Welf et al., PNAS 83: 2330-2334 (1986)). Each cell was transfected.

Example 1 Preparation of RPS-Vax / PTD

Position-directed mutagenesis methods were used to add PTD sequences before the MCS of the RPS-Vax system. First of all, since the size of the RPS-Vax system is large (approximately 10.4 kb), it is difficult to perform direct mutagenesis. Therefore, 814 nt (nucleotides) from the T7 promoter including the site of mutagenesis to the poliovirus N-terminal site are generated. Primer T7 primer / sense 5'-TAA TAC GAC TCA CTA TAG-3 '; And fragments obtained by amplification by PCR using polio / anti-sense (814-797) 5′-GGT AGA ACC ACC ATA CGC-3 ′ were introduced into a T-easy vector (Promega). At this time, PCR conditions were confirmed by amplifying DNA fragments in 35 cycles at 94 ° C. 40 seconds, 45 ° C. 30 seconds, and 72 ° C. 40 seconds.

Subsequently, JM109 was transformed to select positive clones, followed by insertional mutagenesis PCR with mutagenesis primers. Among the primers used at this time, m / PTD / Sense is 5'-TAC GGG CGC AAG AAG CGC CGC CAG CGC CGC CGC CCG CGG GTT AAC CGG -3 'and m / PTD / Anti-sense is 5'- CAT TAT GAT ACA ATT GTC -3 '. Mutagenesis used Pwo polymerase (Roche) to obtain blunt-ended PCR products, synthesized DNA fragments at 94 ℃ 15 seconds, 41 ℃ 30 seconds and 72 ℃ 2 minutes 10 cycles, then 94 ℃ 15 seconds as a template And 72 ° C., 2 min 25 cycles to amplify the complete DNA fragment. Mutagenic positive clones were selected and confirmed by sequencing. The modified RPS-Vax / T-vector clone thus obtained was digested with EcoR I and Sst II restriction enzymes to obtain an insertion sequence, which was then cleaved alone with Sst II and then introduced into the RPS-Vax system partially digested with EcoR I. .

Using the RPS-Vax / PTD vector constructed according to the experimental method described above, RNA transcripts were synthesized through in vitro transcription according to the method described in the following Examples, and then transfected into HeLa cells to confirm that viruses were produced. In addition, the introduced gene was confirmed to be genetically stable during the 12th passage culture.

Example 2: Cloning of Various Epitopes in an RPS-Vax / PTD System

To introduce some sequences of p24 of HIV-1, nef2 (sequence containing hot CTL region of nef), p17 (full length sequence) and some sequences of Tat72 gene and core gene of HCV into RPS-Vax system The following primers were used to PCR the genes: First, HXBc2 cDNA was used by the National Institutes of Health NIH AIDS Reference and Reagent Program for HIV-1 p24, nef2, p17, and Tat72 PCR. Primers for the Tat72 gene of -1 are Tat72 / SacII / sense 5'-AG CCT CCG CGG ATG GAG CCA GTA GAT CCT-3 'and Tat72 / EagI / anti-sense 5'-AC GTT CGG CCG CTT TGA TAG AGA AGC TTG-3 '; Primers for p24 of HIV-1 include p24-100 & 169aa / SacII / sense (1185-1203) 5'-AGG CCT CCG CGG CCT ATA GTG CAG AAC ATC-3 ', p24-100aa / EagI / anti-sense (1470- 1487) 5'-GG CCT CGG CCG TCC CCT TGG TTC TCT CAT-3 ', p24-118aa / SacII / sense (1282-1299) 5'-ATT AAT CCG CGG AGC CCA GAA GTG ATA CCC-3', p24- 118aa / EagI / anti-sense (1618-1635) 5'-ATT AAT CGG CCG AAT GCT GGT AGG GCT ATA-3 'and p24-169aa / EagI / anti-sense (1675-1691) 5'-AGG CCT CGG CCG ATA GAA CCG GTC TAC ATA-3 'was used; HCV core-E1-E2 gene was used as a target gene for the synthesis of HCV core from Professor Sung Young-Chul of the Faculty of Life Sciences, Pohang University of Science and Technology. Among these primers, HCV-c / HpaI / sense ( 1-18) 5'-CAG GTC GTT AAC ATG AGC ACA AAT CCT AAA-3 'and HCV-c / EagI / anti-sense (286-300) 5'-AGG CCT CGG CCG GGG TGA CGA GAG CCA-3' Was used. The primers for the nef2 gene of HIV-1 were nef-2 / sense 5'-TTATTACCGCGGGATGGGGTGGGAGCAGTA-3 'and nef-2 / antisense 5'-TTAATTCGGCCGGTGTAGCAAGCTGTTGTCC-3'; p17 / sense 5'-TATTAACCGCGGATGGGTGCGAGAGCGTCA-3 'and p17 / antisense 5'-TATATACGGCCGGTAATTTTGGCTGACCTGG-3' were used as primers for the p17 gene.

p24 (100, 118 and 169 aa), nef2, p17 and Tat72 genes were used as primers designed to have Sst II and Eag I, and HCV-core (100 aa) Hpa I and Eag I restriction enzyme sites. PCR conditions were carried out 25 cycles of 94 ℃ 30 seconds, 45 ℃ 30 seconds and 72 ℃ 40 seconds to produce the gene to be inserted.

After the PCR product was treated with each restriction enzyme, each recombinant plasmid was synthesized by introducing into the prepared RPS-Vax / PTD system. Positive clones were identified by PCR using restriction enzyme experiments and the following primers: polio / sense (680-697) 5'-CAT TGA GTG TGT TTA CTC-3 'and polio / anti-sense (814-797) 5 '-GGT AGA ACC ACC ATA CGC-3'. At this time, the PCR conditions were performed 25 cycles of 94 ℃ 30 seconds, 45 ℃ 30 seconds and 72 ℃ 40 seconds.

Example 3: in vitro  Warrior

The plasmid DNA of each of the recombinant polioviruses was opened using restriction enzymes ( Sac I or Sal I, NEB), and then purified using phenol / chloroform extraction (3 times repeated) and ethanol precipitation. The ring-opened plasmid DNA was reacted for 1 hour at 37 ° C using T7 RNA polymerase (NEB) to perform in vitro transcription, and residual DNA was removed by treating RNase-free DNase I at 37 ° C for 30 minutes. RNA transcripts were purified by phenol / chloroform extraction (3 times) and ethanol precipitation and used for experiments.

Example 4 Transfection of Recombinant RNA Transcripts into Mammalian Cells

Recombinant RNA transcripts from in vitro transcription were transfected into HeLa cells (adenocarcinoma; cervix) using the DEAE-dextran method (Van der Welf et al., PNAS 83: 2330-2334 (1986)). First, 60-mm dish, grown in HeLa cells (3 x 10 ⁵⁾ a single-layer phosphate-buffered saline (PBS, NaCl 9 g, NaHPO 4 0.359g, Na 2 HPO 4 1.094 g / ℓ) to the washed two times, and 1 Μg RNA was dissolved in RNase-free DDW and mixed with 0.1 mg of DEAE-dextran and stored on ice. Then, after standing for 15 minutes, the RNA mixture was overlaid on the cells and incubated at 37 ° C. for 15 minutes, shaking every 5 minutes to prevent the cells from drying. After 15 min incubation, washed twice with PBS, 2 ml of low glucose DMEM was added, and incubated in a wet incubator maintained at 5% CO ₂ until total cytopathic effect was seen. Viral titers were measured by TCID ₅₀ assay in HeLa cells (BWJ Mahy, Virology a practical approach , IRL press, p. 13 (1985)). As a result, a chimeric poliovirus was generated in HeLa cell monolayer, and the recombinant of the present invention produced a progeny virus having replication ability.

Example 5: One-Step Growth Curve

To determine the proliferative capacity of each chimeric poliovirus, Sabin 1 and chimeric poliovirus were infected with MOI = 10 after monolayer culture of HeLa cells of 5 × 10 ⁵ cells in a 60 mm plate. After incubation for 1 hour at room temperature to allow the virus to adsorb to the cells, washed twice with PBS to remove the residual virus and incubated in a 5% CO ₂ wet incubator with 3 ml of complete DMEM. 100 μl of the supernatant was taken at 3 hour intervals after infection, and virus titers were determined by TCID ₅₀ assay, and a one-step growth curve was prepared.

As can be seen in Figure 3, the growth rate difference between the Sabin 1 and chimeric poliovirus showed a maximum value of about 2-3, but when the harvest time is delayed by 3-6 hours, the titer of the chimeric poliovirus is Sabin 1 It appeared similar to

Example 6: Genetic Stability of Insertion Sequences in Chimeric Poliovirus

Example 6-1: Analysis by RT-PCR

In order to confirm the genetic stability of the insertion sequence in the chimeric poliovice prepared according to the present invention, the chimeric poliovirus produced in the above example was transfected into HeLa cells and incubated at 37 ° C. for 18 hours, respectively. Each passage of was transfected with MOI = 10, and poliovirus was harvested at each passage to obtain total RNA by phenol-chloroform extraction and ethanol precipitation. Subsequently, 1 μg of viral RNA and 0.5 μg of primer (5′-CAT TGA GTG TGT TTA CTC-3 ′) were denatured at 70 ° C. for 10 minutes, immediately transferred to ice and added with a reverse transcriptase reaction solution (50 mM Tris). ㆍ HCl pH 8.3, 65 mM KCl, 3 mM MgCl ₂ , 10 mM DTT, 1 mM dNTP mixture, 20 U RNAsin) and 200 U of MMLV reverse transcriptase (Moloney Murine Luekemia virus RTase, Promega) were added at 42 ° C. 60 After reacting for a minute, cDNA was synthesized. After the reaction was completed, the enzyme was inactivated by incubating at 100 ° C. for 3 minutes. The gene region introduced in the synthesized cDNA was prepared using Sabin 1 primer (polio / sense (680-697) 5'-CAT TGA GTG TGT TTA CTC-3 'and 5'-GGT AGA ACC ACC ATA CGC-3'). Amplification by PCR. PCR was performed using Taq. A total of 35 cycles of 94 ° C. 40 seconds, 45 ° C. 30 seconds, 72 ° C. 40 seconds were performed using the polymerase. The PCR product was electrophoresed on agarose gel to observe the band pattern. The results are summarized in the following Table 1. The genetic stability in Table 1 indicates the number of passages in which the sequence inserted into the RPS-Vax / PTD remains stable during the proliferation in the host cell.

RPS-Vax / PTD Type of Insertion Sequence Size of insertion sequence (bp) Genetic Stability of Insertion Sequences Tat72 249 - p24 (100aa) 333 12 p24 (118aa) 381 12 p24 (169aa) 540 6 HCV / c (100aa) 333 12 nef2 (131aa) 393 12 p17 (132aa) 396 12

As can be seen in Table 1, the gene encoding the foreign antigen inserted in the chimeric RPS-Vax / PTD of the present invention remained stable up to 12 passages. On the other hand, p24 (169 aa) is estimated to have reduced genetic stability compared to other foreign genes due to the size of the insertion sequence.

As can be seen in Figure 4a, HCV core, HIV-1 nef, HIV-1 p17, HIV-1 p24 (100aa, 118aa) gene fragment inserted RPS-Vax / PTD / HCVc, RPS-Vax / PTD / nef2 and RPS-Vax / PTD / p17, RPS-Vax / PTD / p24-123, RPS-Vax / PTD / p24-118 showed genetic stability of the insertion sequence up to 12 passages.

Example 6-2: Analysis by Western Blot

HeLa cells were infected with Sabin type 1 and recombinant poliovirus (MOI = 10) and then incubated at 37 ° C. for 8 hours. Infected cells were collected and washed twice with PBS. The cells were then resuspended in PBS followed by 0.01 mg of 2 × sample buffer (62.5 mM Tris-Cl pH 6.8, 10% glycerol, 2% SDS, 1% β-mercaptoethanol, bromophenol blue and xylene cyanol) / ML) was added and boiled for 10 minutes at 100 ℃, the nuclei were removed by centrifugation. Then, some of the protein in the cell debris was electrophoresed on a 12% SDS-PAGE gel and then transferred to an NC (nitrocellulose) membrane to perform western hybridization as follows: First, the protein was run on the gel and then transferred to a transfer buffer ( Gels were soaked for 15 min in 48 mM Tris-Cl, 39 mM glycine, 20% methanol and 1.3 mM SDS). Subsequently, 6 sheets of 3M paper and NC membrane were soaked in the same transfer buffer, and then superimposed on a semi-dry transfer blot in the order of 3M paper, NC membrane, gel and 3M paper and transferred to 20 volts for 30 minutes. The extent of metastasis was confirmed with pre-stain markers. The membrane was blocked for 30 minutes in blocking solution (5% non-fat dry milk w / v, 0.02% NaN ₃ in PBS), and then prepared primary Ab (anti-HCV core mAb; Biogenesis, and recombinant PTD produced in our laboratory). 50 μg of -Nef protein was mixed with Freud's adjuvant to give 2 weeks of booster vaccination at 6 weeks of age after 2 weeks of immunization to 6-week-old Balb / c, and diluted 1/500 of PBS with antiserum). Primary reaction was carried out for 3 hours. Thereafter, washing with PBS three times to remove residual Ab, and 2nd Ab for 1st Ab was diluted with PBS (1 / 10,000) and reacted for 1 hour. After the second reaction, washed three times with PBS again, and then mixed with 10 ml of AP buffer (100 mM NaCl, 5 mM MgCl ₂ and 100 mM Tris-Cl, pH 9.5) by adding 66 μl of NBT and 33 μl BCIP to develop a band. Or the band was identified using an ECL kit (Amersham pharmacia biotech).

As can be seen in Figure 4b, it can be seen that the genes inserted up to 12 passages are stably expressed in both RPS-Vax / PTD / HCVc inserted with the core gene fragment of HCV and RPS-Vax / PTD / nef inserted with the Nef gene. have.

Example 7: Staining of Cells with PTD-β-galactosidase Fusion Protein

Example 7-1: Expression and Purification of PTD-β-galactosidase

PTD- by bonding the pTAT vector β- galactosidase or β- galactosidase coding gene was introduced into the transformed a recombinant vector in E.Coli {BL21 (DE3) pLysS} , protein expression and purification was carried out. First, E.Coli transformed with the recombinant plasmid was incubated in 5 ml of 2x YTA broth containing 50 µg / ml of ampicillin, and then 500 µl of the cultured strain was inoculated into 500 ml culture broth. After culturing for another day by adding 0.4 mM IPTG to OD = 0.8, the bacteria were harvested by centrifugation at 8000 rpm for 4 minutes. Subsequently, grinding buffer A (50 mM glycine, 50 mM Tris-HCl, 10 mM imidazole) containing 1 mM PMSF (phenyl methyl sulfonyl fluoride) was added to the harvested bacteria, left at 4 ° C. for 30 minutes and sonicated. Cells were disrupted by 1 ml of 50% slurry Ni-NTA resin (Qiagen) was added to the bacterial extract supernatant obtained by centrifugation at 1,5000 rpm for 15 minutes at 4 ° C, and the mixture was bounded at 4 ° C for 8 hours. The 5 ml column was then loaded with the protein bound resin and then washed with wash buffer (50 mM glycine, 50 mM Tris-HCl and 20 mM imidazole) at 10 times the bed volume, followed by elution buffer (250 mM imitate). Dozol) was used to purify undenatured protein. The amount and purity of the recovered protein was confirmed by 12% SDS-PAGE gel. Each protein isolated in this way was used for X-gal staining of HeLa cells.

Example 7-2: X-gal staining of HeLa cells

HeLa cells of 5 × 10 ⁵ cells were incubated in a single layer in a 60 mm plate and washed twice with PBS. After addition of 1.5 ml of serum-free medium Opti-MEM, each protein of PTD / β-galactosidase fusion protein or β-galactosidase (without PTD tagging) was added (30 μg / ml in Opti-). MEM) and incubated at 37 ° C. for 30 minutes. After washing twice with PBS again, 1.5 ml of Opti-MEM was added and incubated at 37 ° C. for a predetermined time. X-gal staining was washed twice with PBS according to Kimpton and Emerman method (42), followed by 2 ml of fixative (1% formaldehyde, 0.2% glutaraldehyde in PBS), and left at room temperature for 5 minutes. It was. Then, after washing twice with PBS, 2 ml of dye solution (4 mM potassium ferrocyanide, 4 mM potassium ferricyanide, 2 mM MgCl ₂ , 0.4 mg / ml X-gal in PBS) was added, followed by room temperature. It was observed after reacting for 1 hour.

As can be seen in FIG. 5, PTD-tagging effectively transfers β-galactosidase into HeLa cells, and after treatment with PTD-conjugated β-galactosidase, is decomposed or processed after about 12 hours to effect staining. It can be seen that is reduced. These results indicate that when a recombinant virus is introduced by introducing an external gene into the RPS-Vax / PTD system, the virus is expressed in the form of a complex protein bound to PTD when the foreign protein introduced as the virus proliferates. It is easy to enter into the antigen presentation effectively suggests that it is very effective in inducing CTL immunity.

Example 8: Determination of antigen-specific IgG isotypes in immunized mice

Example 8-1: Immunization of Tg-PVR Mice by Virus and Protein

RNA transcripts for each of RPS-Vax, PTD-HCV / core (p), RPS-Vax / HCVc and RPS-Vax / PTD / HCVc constructed in Example 2 above were obtained from HeLa cells as in Example 3 above. Then, the virus was obtained by transfection with Example 4 and DEAE-dextran method.

On the other hand, PTD-HCV / core protein was obtained by native purification by cloning into the pET43.1a (+) vector as follows. HCV-EcoRI / sense 5'- CAT GAA TTC TAC GGC CGC AAG AAA CGC CGC CAG CGC CGC CGC ATG AGC ACA AAT CCT AAA -3 'and HCV-XhoI / antisense 5'- CCG CTC GAG TTA DNA fragments were synthesized by PCR using AGC AGA AAC TGG GGT-3 'in 94 ° C 30 sec, 53 ° C 30 sec and 72 ° C 2 min 35 cycles. DNA fragments synthesized with Xho I and Eco R1 restriction enzyme and pET43.1a (+) were cut and inserted. After insertion was confirmed by PCR, JM109 E. coli competent cells were transformed. The expressed protein was purified using a Ni-NTA column in the same manner as in Example 7-1 after dissolving E. coli.

Using the virus and protein obtained in the above process, 5-6-week-old Tg-PVR mice (produced by Dr. Nomoto, Tokyo University, Japan) were grown and proliferated at the Animal Resources Center of Biotechnology Research Institute). Immunization: 1st vaccination (im) virus 1 × 10 ⁷ , 1 week after 2nd vaccination (im) virus 5 × 10 ⁶ , 1 week after 3rd vaccination (ip) virus 5 × 10 ⁶ , and 3 weeks after 4 Primary boosting inoculation (ip) 30 μg PTD-HCV core protein.

Blood was collected three days after the last boosting procedure from the immunized mice.

In immunization with PTD-HCV / core protein only, 30 μg of PTD-HCV / core protein was used in all four steps, and the inoculation site was identical to the inoculation with virus.

Example 8-2: HCV Core Protein-Specific IgG Isotype Determination by ELISA

The PTD-HCV / core protein (2 μg / ml) obtained in Example 8-1 was coated on a 96-well plate for ELISA for 1 hour at 37 ° C. and washed three times with PBS-T. The plate was then blocked with 1% skim milk at 37 ° C. for 1 hour. Then, the primary antibody (diluted with 1/500 of the serum of each mouse obtained in Example 8-1) was added and reacted at 37 ° C. for 2 hours, and then washed three times with PBS-T buffer. Secondary antibodies (diluted alkaline phosphatase-conjugated mouse IgG1, G2a, G2b and G3 each at 1/1000, Pharmingen) were added and reacted at 37 ° C for 1 hour, followed by PBS-T. Wash three times. Subsequently, the substrate of alkaline phosphatase (p-Nitrophenyl Phosphate tablet 5 mg, Sigma Diagnostics: tablet / 5 ml) was added to the substrate solution (10% Diethanolamine buffer: 10% Diethanolamine, 0.2 g / LNaN ₃ , 0.1 g / ℓ MgCl). ₂ ) was added to 100 μl per well and absorbance at OD ₄₀₅ with a microplate spectrometer (Model 550, BioRad) at 30 minutes and 1 hour. Measured.

As can be seen in Figure 6, only the mouse serum immunized with RPS-Vax / PTD / HCVc was observed to express a high concentration of IgG2a antibody that specifically responds to HCV core protein. These results indicate that RPS-Vax / PTD / HCVc effectively induces Th1-type T cell immunity directly involved in the cytotoxic T lymphocyte (CTL) immune response ( Vaccine , 20: 1308-15 (2002). The results show that the chimeric poliovirus of the present invention can efficiently induce not only humoral and mucosal immunity but also cellular immunity against the introduced foreign antigen.

Example 9 HIV-1 p24 Protein-Specific IgG Isotype Determination in Immunized Mice

In the same manner as the immunization process for the HCV core protein, the immunization against the HIV-1 p24 protein was performed. In this case, the recombinant virus used for mouse immunization was RPS-Vax, RPS-Vax / p24 (118 aa) and RPS-Vax / PTD / p24 (118 aa) constructed in Example 2, and the PTD-p24 full-length protein as a control. Was used together. PTD-p24 full-length protein was obtained by cloning in a dissolved state by cloning in a pTAT vector as follows. p24 is p24 / XhoI / sense (1186-1203) 5'-TGC CCT CTC GAG CCT ATA GTG CAG AAC ATC-3 'and p24 / EcoRI / antisense (1878-1860) 5'-TAC AGA ATT CGC CAA AAC TCT TGC DNA fragments were synthesized by PCR with HXBc2 cDNA as a template at 94 ° C 30 seconds, 50 ° C 30 seconds and 72 ° C 1 min 35 cycles using CTT AT-3 '. DNA fragments synthesized with Xho I and Eco R1 restriction enzymes and pTAT vectors were cleaved and then inserted by ligation. After insertion was confirmed by PCR, JM109 E. coli competent cells were transformed. The expressed protein was purified using a Ni-NTA column in the same manner as in Example 7-1 after dissolving E. coli.

Immunization procedures in Tg-PVR mice were performed in the same manner as in Example 8-1. ELISA analysis was also performed in the same manner as in Example 8-2.

As can be seen in Figure 7, the production of IgG2a antibody that specifically responds to the p24 antigen was observed significantly higher only in the mouse serum immunized with the RPS-Vax / PTD / p24 (118 aa) virus among the immunized mice. These results suggest that RPS-Vax / PTD / p24 (118 aa) effectively induces Th1 type cellular immunity against HIV-1 p24 antigen, and furthermore can effectively induce CTL against HIV-1. It means that there is. Taken together, these results indicate that the use of the chimeric poliovirus vector of the present invention can efficiently induce not only humoral and mucosal immunity but also antigen-specific cellular immunity against the introduced antigen.

Example 10 Determination of HIV-1 Nef Protein-Specific IgG Isotypes in Mouse Serum Immunized with RPS-Vax / PTD / Nef Recombinant Virus

Example 10-1: Expression of PTD-nef Protein

Primers of sense / GGG CTC GAG ATG GGA GGC AAG TGG TCA AAA and antisense / GGG GAA TTC TCA GTT CTT GAA GTA CTC CGG so that the Nef gene can be cleaved with Xho I and Eco R1 restriction enzymes after amplification, the respective cut ends with Xho I and Eco R1, and is also inserted through the connection in response to the pTAT-HA vector digested with Xho I and Eco R1. After insertion was confirmed by PCR, JM109 E. coli competent cells were transformed. The expressed protein was purified using a Ni-NTA column in the same manner as in Example 7-1 after dissolving E. coli.

Example 10-2: PTD-Nef Protein-Specific IgG Isotype Determination by ELISA

The PTD-nef protein (2 μg / ml) obtained in Example 10-1 was coated on a 96-well plate for ELISA for 1 hour at 37 ° C. and washed three times with PBS-T. The plates were then blocked with 3% Scheme Milk at 37 ° C. for 1 hour. Then, the primary antibody (diluted with 1/500 of the serum of each mouse obtained in Example 8-1) was added and reacted at 37 ° C. for 2 hours, and then washed three times with PBS-T. , Secondary antibody (dilution of alkaline phosphatase-conjugated mouse IgG1, G2a, G2b and G3 to 1/1000 each, pharmingen) was added and reacted at 37 ° C for 1 hour, followed by 3 with PBS-T. Washed twice. Substrate of alkaline phosphatase (p-Nitrophenyl Phosphate tablet 5 mg, Sigma Diagnostics: tablet / 5 ml) in buffer (10% Diethanolamine buffer: 10% Diethanolamine, 0.2 g / LNaN ₃ , 0.1 g / L MgCl ₂ ) After dissolving and adding 100 μl per well and reacting for 30 minutes, OD ₄₀₅ was measured using a microplate spectrometer (Model 550, BioRad). As a result, as shown in Figure 8, in the mice immunized with the RPS-Vax / PTD / nef virus it was confirmed that both of the two IgG2a appear relatively higher than the other control. This indirectly demonstrates that the virus effectively induced Th1-type T cell immune responses to the Nef protein. In addition, when immunized with PTD-Nef protein in mice immunized with RPS-Vax / PTD / nef virus, the level of IgG2a was lowered and increased when immunized virus again, which is also RPS-Vax / PTD / Once again, nef recombinant virus effectively induces Nef-specific Th1 type cellular immune response.

These results strongly suggest that the presentation of antigen by RPS-Vax / PTD / nef virus can induce a cellular immune response much more effectively than when immunized with PTD-Nef protein alone. These facts suggest that the PTD-Nef protein expressed during virus propagation effectively penetrates adjacent cells using PTD-domain, and the proteins presented and produced in MHC class I through proteasomes in the cytoplasm can effectively amplify cellular immune responses. It is because it is possible.

Example 11 T Cell Proliferation Assay in Spleen Cells

Mice immunized with RPS-Vax / PTD / nef recombinant virus were sacrificed by cervical dislocation and spleens were extracted. The spleens were finely chopped and the spleen cells were extracted by passing through the mesh. Subsequently, 10 ml of erythrocyte lysis solution was added and reacted at room temperature for 10 minutes. After washing twice with PBS, the cells were resuspended in 10 ml RPMI-10 medium to adjust the concentration of cells to 5 × 10 ⁶ / ml. Some of the extracted splenocytes were washed with PBS, and then, mytomycin was reacted for 30 minutes at 37 ° C. so that the final concentration was 50 μg / ml. After washing twice with PBS and suspended in 10 ml RPMI-10, PTD-nef was added to the final 100 ㎍ / ㎖, and then treated for 12 hours to use as a feeder (feeder cell). The splenocytes cultured for one day with the final 20 units / ml of IL-2 were used as the effector cells. The cultured cells treated with PTD-nef and the reaction cells (5 X 10 ⁶ / ml) were 1 1: 1 (100 μl: 100 μl) were mixed and incubated. After incubating the cultured cells and the reaction cells for 3 days, ³ H-thymidine 1 μCi was added to the culture solution and incubated for 16 hours. After harvesting the cells in the filter by using a cell harvester (Skatron Inc), dry the cells and place them in a container, add 2 ml of the radioactive solution (Scintillation Cocktail, Amersham Co.) per container, and then use the liquid radiometer. (liquid-scintillation counter: LS-650, Beckman) was used to measure the radioactivity introduced into the cells.

As a result of immunization and testing of two mice, as shown in FIG. 9 , the RPS-Vax / PTD / nef2-immunized mice showed a relatively higher T cell proliferation capacity than the mice immunized with RPS-Vax. These results show that T cells reacting with nef2 in splenocytes effectively proliferate after immunization, demonstrating that cellular immunity that specifically responds to nef is effectively induced by the RPS-Vax / PTD system.

Example 12 Cytokine Analysis

In the same manner as in Example 11, the supernatant was harvested after 7 days of incubation after splenocyte extraction, pulsing and antigen provision of mice immunized with RPS-Vax / PTD / nef recombinant virus. Anti-mouse IL-4 Ab (MM-450C, Pharmingen Inc.) diluted in PBS at 2 μg / ml, anti-mouse IFN-γAb (MM-700 diluted in sodium carbonate-bicarbonate buffer at 1 μg / ml , Pharmingen Inc.) was coated on a Maxisorb ELISA plate (Costar) for 8 hours at 4 ℃. After blocking for 2 hours at room temperature with 4% BSA-PBS, 50 μl of the harvested supernatant was added and reacted at room temperature for 1 hour. Biotin-labeled antibody (MM-450D, MM-700B; Pharmingen Inc.) was diluted to 0.1 µg / ml, added 50 µl and left at room temperature for 30 minutes, washed solution (50 mM Tris-0.2% Tween20, pH). 7.9-8.1) three times. 100 μl of HRP-conjugated streptoavidine (N-100, Pharmingen Inc.) diluted 1: 4000 was added thereto, left at room temperature for 30 minutes, and washed three times with a washing solution. Subsequently, 100 μl of TMB substrate solution (N-300, Pharmingen Inc.) was added thereto, and the mixture was left at room temperature for 30 minutes, and then 100 μl of stop solution (0.18 MH ₂ SO ₄ ) was added thereto. Absorbance at 450 nm was then measured with an ELISA meter (Bio-Rad).

As shown in FIG. 10, IL-4 was hardly detected during T cell proliferation of mice immunized with RPS-Vax / PTD / nef recombinant virus, but IFN-γ was expressed in RPS-Vax / PTD. T cell proliferation of two mice immunized with / nef recombinant virus was measured at a relatively higher concentration at least two-fold higher than the control (see FIG. 11). These results indicate that most T cells proliferated during Nef-specific T cell proliferation analyzed in splenocytes of mice immunized with RPS-Vax / PTD / nef virus shown in FIG. 9 are cells involved in Th1 type cellular immune response. These results also demonstrate that viruses recombined with the RPS-Vax / PTD system effectively induce cellular immune responses associated with CTL induction.

The present invention provides novel recombinant poliovirus vectors. In addition, the present invention provides a vaccine composition comprising a recombinant poliovirus derived from a novel recombinant poliovirus vector and greatly improved the CTL inducibility of the introduced gene. Vaccine candidates produced with the poliovirus vector of the present invention can significantly improve the cellular immunity specific to the introduced antigen, in particular, CTL induction ability, greatly improving the utility and value of the poliovirus vector in the development of a vaccine for CTL induction .

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<110> CreaGene Inc. <120> Novel Recombinant Poliovirus Vectors and Vaccine Compositions          Capable of Inducing Cytotoxic T Lymphocytes <130> Crea-2 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Protein transduction peptide <400> 1 Arg Lys Lys Arg Arg Gln Arg Arg Arg   1 5 <210> 2 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Protein transduction peptide <400> 2 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg   1 5 10 <210> 3 <211> 86 <212> PRT <213> Human immunodeficiency virus type 1 <400> 3 Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser   1 5 10 15 Gln Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe              20 25 30 His Cys Gln Val Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly          35 40 45 Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Ser Gln Thr      50 55 60 His Gln Val Ser Leu Ser Lys Gln Pro Thr Ser Gln Ser Arg Gly Asp  65 70 75 80 Pro Thr Gly Pro Lys Glu                  85

Claims (13)

Recombinant poliovirus vaccine vector for inducing cytotoxic T lymphocyte (CTL) -response, comprising: (a) the genome nucleotide sequence of a poliovirus; (b) a nucleotide sequence encoding an additional poliovirus protease cleavage site; (c) a nucleotide sequence encoding a foreign antigen linked to a nucleotide sequence encoding said additional poliovirus protease cleavage site; And (d) a nucleotide sequence encoding a protein permeable domain linked to the nucleotide sequence encoding said foreign antigen. The method of claim 1, wherein the nucleotide sequence encoding the exogenous antigen is a sequence encoding a protein or polypeptide antigen derived from a bacterial polypeptide antigen, a viral polypeptide antigen, a fungal polypeptide antigen or a polypeptide antigen of a pathogenic eukaryotic cell. Recombinant poliovirus vaccine vector, characterized in that. The nucleotide sequence encoding the exogenous antigen is human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), Sequence encoding a protein or polypeptide antigen derived from a polypeptide antigen of a pathogenic virus selected from the group consisting of human papilloma virus (HPV), herpes simplex virus (HSV), poliovirus, rotavirus, influenza virus and pandemic hemorrhagic fever virus Recombinant poliovirus vaccine vector, characterized in that. 4. The recombinant poliovirus vaccine vector according to claim 3, wherein the sequence encoding the protein or polypeptide antigen is a sequence encoding an amino acid sequence comprising an antigenic determining site. 2. The recombinant poliovirus vaccine vector according to claim 1, wherein the genome nucleotide sequence of the poliovirus is derived from the genome nucleotide sequence of savin 1, sabin 2 or savin 3 poliovirus. 6. The recombinant poliovirus vaccine vector according to claim 5, wherein the genome nucleotide sequence of the poliovirus is derived from the genome nucleotide sequence of a Sabine type 1 poliovirus. The recombinant poliovirus vaccine vector of claim 1, wherein the additional poliovirus protease cleavage site is a cleavage site to which the poliovirus 3C protease or 2A protease acts. 8. The recombinant poliovirus vaccine vector according to claim 7, wherein the additional poliovirus protease cleavage site is a cleavage site on which the poliovirus 3C protease acts. The recombinant poliovirus vaccine vector according to claim 1, wherein the protein permeation domain essentially comprises an amino acid sequence represented by SEQ ID NO: 1. 10. The recombinant poliovirus vaccine vector of claim 9, wherein the protein permeation domain essentially comprises SEQ ID NOs: 49-57 of the amino acid sequence represented by SEQ ID NO: 3 and lacks SEQ ID NOs: 22-37. 11. The recombinant poliovirus vaccine vector according to claim 10, wherein the protein permeation domain essentially comprises SEQ ID NOs: 49-57 of the amino acid sequence represented by SEQ ID NO: 3 and lacks SEQ ID NOs: 22-37 and SEQ ID NOs: 73-86. . 12. The recombinant poliovirus vaccine vector according to claim 11, wherein the protein permeation domain comprises an amino acid sequence represented by SEQ ID NO: 2. Recombinant poliovirus vaccine composition comprising: (a) a recombinant poliovirus vaccine derived from the recombinant poliovirus vaccine vector of any one of claims 1 to 12; And (b) a pharmaceutically acceptable carrier.

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