WO2014103310A1 - ヒトパラインフルエンザ2型ウイルスベクターを利用したワクチン - Google Patents
ヒトパラインフルエンザ2型ウイルスベクターを利用したワクチン Download PDFInfo
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Definitions
- the present invention relates to an RNA virus vector and an inactivated vaccine that are useful as vaccines for preventing or treating diseases.
- Paramyxoviridae viruses have negative single-stranded RNA as a genome, and a vector capable of high expression of foreign genes by reverse genetics has been prepared, making it an excellent vector for gene therapy or for recombinant vaccines. It is considered to have characteristics. Since the viral life cycle does not include the DNA phase and all transcription and replication occur in the cytoplasm outside the nucleus, the probability of homologous recombination with chromosomal DNA is extremely low, and there is a risk of carcinogenesis due to genetic recombination into the chromosome. small.
- hPIV2 human pareline fenza type 2 virus belonging to the genus Paramyxoviridae Paramyxovirus subfamily Rubravirus, which is a negative single-stranded RNA virus
- an hPIV2 F gene-deficient vector was constructed in which the F gene, which is a viral structural protein, was completely deleted.
- the vector has the characteristics of non-transmissible or single-round-infectious.
- F gene stable expression package cells having high titer vector production ability have been successfully obtained.
- the vector deficient in the F gene can produce an infectious vector that retains the F protein in the viral envelope only in packaged cells in which the F gene is expressed.
- cells / tissues that do not express the F gene cause so-called self-replication, but cannot produce infectious vectors. It can be said that the price is high (Patent Document 1).
- Non-patent Document 1 results showing many efficacy in non-clinical trials, clinical studies, clinical trials, etc. in vaccines or gene therapy using recombinant RNA live virus vectors or attenuated RNA recombinant viruses have been reported (Non-patent Document 1). ⁇ 9).
- live live
- the reason is that when genetically modified live RNA virus is used as a vector, safety measures such as using attenuated virus to reduce pathogenicity or using a non-transmissible vector are being taken.
- the virus and foreign gene products are still produced in large quantities in the infected cells, so that concerns such as effects on homeostasis or mutation of foreign genes cannot be eliminated.
- a vaccine in which the genome is inactivated or decomposed into components so that the virus does not transcribe or replicate is highly safe.
- a formalin treatment method is often used for the production of inactivated vaccines.
- inactivated vaccines that are considered to be highly safe also have safety problems.
- an inactivated RSV vaccine with formalin against RSV belonging to the Paramyxoviridae family was developed, and after the vaccination, severe infection and two fatalities occurred in infants due to natural infection with RSV. Later, the cause was energetically elucidated. It was found that the three-dimensional structure of the F membrane protein was changed by inactivation of the virus with formalin. An antibody having no neutralizing activity against the F membrane protein modified by the vaccination is produced, and an F membrane protein having a normal structure is not present by the formalin treatment, and therefore, an antibody having neutralizing activity against the protein may not be produced. I understood.
- formalin-inactivated RSV is known to be inferior to live RSV in the ability to mature dendritic cells that are antigen-presenting cells, and inactivation by UV treatment or heat treatment without using formalin has been studied. However, none of them have been put into practical use because of their low ability to activate dendritic cells and the induction of an inflammatory reaction.
- a formalin-inactivated vaccine of measles virus belonging to the Paramyxoviridae family has been reported to have eosinophil infiltration and atypical measles.
- inactivated RSV vaccines designed to induce Th1-type immunity in animal studies have been reported to be effective in preventing viral infection, and inactivated vaccines capable of inducing Th1 solve these problems. It is considered possible.
- transgene expression by viral vectors is thought to be high, but there are reports of cases where transgene expression in virally infected cells / tissues is not quantitative or low in expression level and is not effective as a gene therapy vector Has been. In addition, suppression of expression by a pre-existing antibody against the introduced viral vector or suppression of expression by an antibody against the vector following multiple administration of the viral vector has been reported. These reported examples make it difficult to use a virus vector having a high antibody-inducing ability for gene therapy in which a gene expression of a certain amount or more is required even temporarily.
- the foreign gene product When a foreign gene is introduced and expressed in a viral vector, the foreign gene product does not have a packaging signal, so the foreign gene product is usually not contained in the vector, and the viral vector infects recipient cells after in vivo administration. Then, transcription and replication occur, and foreign gene products are produced for the first time. Therefore, when viral vector infection, transcription / replication, and the like are inhibited by a neutralizing antibody or the like, the expression of a foreign gene is suppressed or reduced.
- the efficiency of virus infection to cells / tissues is low, or transcriptional replication does not occur or the efficiency is very poor even when a virus is infected, and foreign genes are often hardly expressed. It is considered that the in vivo infected cells can suppress the transcriptional replication of the virus by interferon induced by virus infection or the like, and the virus vector cannot reach the target cell due to the extracellular matrix or the like.
- An object of the present invention is a viral vector of the Paramyxoviridae family useful as a vaccine for preventing or treating diseases, and can efficiently introduce an antigen polypeptide (including protein and peptide) into target cells. It is to provide viral vectors and inactivated vectors.
- the inventors of the present invention have made it possible to efficiently and quantitatively carry an antigen protein or peptide to a target cell by fusing and expressing a structural gene of a viral vector and an antigen protein or peptide gene. I found it.
- the inventors also have the following four types of vectors: 1) a type in which an antigen peptide is expressed outside the vector envelope by placing an antigen gene on the 3 ′ end side (hereinafter referred to as a coding chain) of the membrane protein HN gene. 2) A type in which an antigen peptide is expressed inside the vector envelope by placing an antigen gene on the 3 ′ end side of the F gene of the F packaging cell, 3) a vector envelope by combining 1) and 2) above A type that expresses an antigenic peptide at both internal and external positions, and 4) a type that incorporates an antigen onto a vector by fusing the antigen and the membrane / intracellular domain of a membrane protein that functions as a packaging signal to the virus. , Succeeded in building. Thereby, various introduction
- the present invention relates to a viral vector that can efficiently deliver an antigen polypeptide to a target cell, and specifically provides the following inventions: [1] A viral vector in which a gene encoding an antigen polypeptide is incorporated into a virus gene of the Paramyxoviridae family, and the antigen polypeptide is expressed as a fusion protein with a viral structural protein or a part thereof vector: [2] The virus vector according to [1], wherein the fusion protein is a fusion protein with one or more or a part thereof selected from viral HN protein, F protein, and M protein: [3] The gene encoding the antigen polypeptide is 1) Located on the 3 ′ end side of the HN gene, and the antigen polypeptide is expressed outside the vector envelope.
- the viral vector according to any one of [1] to [7], wherein the antigen polypeptide is an influenza virus M2e protein or a fragment thereof: [9]
- the antigen polypeptide is an influenza virus including a virulent influenza virus, parainfluenza type 3 virus, RS virus, Hendra virus, SARS virus, Nipah virus, Lassa virus, dengue virus, West Nile virus, human metapneumovirus, Ebola virus, hantavirus, AIDS virus, hepatitis C virus, Lassa virus, human papilloma virus, rubella virus, rotavirus, norovirus, Crimea-Congo hemorrhagic fever virus, herpes virus, cytomegalovirus, and papilloma virus
- One of the methods [15] Paramyxoviridae virus deficient in the F gene is co-cultured with Vero cells expressing the F gene fused with the F gene or antigen of the Paramyxoviridae virus, and virus particles are extracted from the culture supernatant.
- influenza virus including highly toxic influenza virus, parainfluenza type 3 virus, RS virus, Hendra virus, SARS virus, Nipah virus, Lassa virus, dengue virus, West Nile virus, human metapneumovirus, Ebola virus, hantavirus, AIDS virus, hepatitis C virus, Lassa virus, human papilloma virus, rubella virus, rotavirus, norovirus, Crimea-Congo hemorrhagic fever virus, herpes virus, cytomegalovirus, and papilloma virus
- Antigenic peptide bacterial antigenic peptide selected from group A ⁇ (beta) streptococci, Mycobacterium tuberculosis, Vibrio cholerae, and Mycoplasma, or RSV F
- white white
- the conventional platform-type inactivated virus vector has a problem that the amount of antigen introduced per vector is low, and therefore the antibody production efficiency is low.
- the virus vector of the present invention can contain a large amount of antigenic peptide quantitatively on the virus particle.
- the structural protein of the fused virus vector particularly the membrane protein itself, has immunogenicity, high immunity can be induced against a low immunogenic antigen polypeptide fused to the protein. That is, the vector itself has an adjuvant activity, and the addition of an adjuvant is unnecessary or less than usual at the time of administration.
- the human parainfluenza virus used in the present invention is a minus single-stranded virus, and the end thereof is modified with triphosphate (5′-PPP).
- This terminal triphosphate followed by a number-based single-stranded RNA moiety serves as a substrate for an intracellular signal called IFIT, induces type 1 interferon having antiviral activity, and contributes to DC cell maturation and the like (Abbas YT., Nature, Vol.
- the vector has a cell adsorbability close to that of a live virus, and can exhibit the ability to induce immunity and DC cell maturation due to the structure described above.
- the virus vector of the present invention can be expected to have a high effect in humans, unlike a virus vector that could not be expected to have a sufficient effect in humans. 7) By introducing the antigen at the gene level, it is not necessary to introduce the antigen after inactivation, and problems associated with the introduction of the antigen after inactivation (low antigen introduction efficiency, destruction of the envelope structure, etc.) can be avoided. 8) The viral vector of the present invention can be constructed so that antigens can be placed outside, inside, or both of the vector envelope, thereby allowing various introductions of a plurality of antigens into the vector.
- an inactivation vector capable of inducing immune immunity comparable to that of a live virus vector against dendritic cells and the like by efficient virus inactivation treatment. It becomes possible to open the way to vaccination of the inactivated vector.
- FIG. 1 shows gene expression efficiency of human parainfluenza type 2 virus in mouse cells (NIH-3T3 cells) and monkey cells (Vero cells). Mouse cells were able to confirm GFP expression up to 10 4 dilution, and monkey cells were able to confirm GFP expression up to 10 7 dilution.
- FIG. 2 shows the copy number of hPIV2 / ⁇ F in human and mouse dendritic cells.
- FIG. 3 shows the infection efficiency of hPIV2 / ⁇ F into mouse dendritic cells.
- FIG. 4 shows maturation marker expression in human dendritic cells after live hPIV2 / ⁇ F / GFP infection.
- FIG. 1 shows gene expression efficiency of human parainfluenza type 2 virus in mouse cells (NIH-3T3 cells) and monkey cells (Vero cells). Mouse cells were able to confirm GFP expression up to 10 4 dilution, and monkey cells were able to confirm GFP expression up to 10 7 dilution.
- FIG. 2 shows the copy number
- FIG. 5 shows MHC and cytokine expression in human dendritic cells after live hPIV2 / ⁇ F / GFP infection.
- FIG. 6 shows the result of virus inactivation treatment with ⁇ -propiolactone.
- A Vector GFP fluorescence on Day 5 and Day 10;
- B Hemagglutination reaction (HA titer).
- C -(E) Ginger live hPIV2 / ⁇ F / GFP, ⁇ -propiolactone-treated hPIV2 / ⁇ F / GFP and formalin-treated antibody values in alveolar lavage fluid after administration of hPIV2 / ⁇ F / GFP (IgG1, IgG2 and IgA ).
- FIG. 7 shows maturation of mouse dendritic cells and induction of MHC expression and cytokines by live and ⁇ -propiolactone (BPL) inactivated hPIV2 / ⁇ F.
- FIG. 8 shows the construction of a vector for producing a fusion protein of hPIV2 HN and antigen.
- A Genomic diagram of hPIV2 ( ⁇ F) in which an antigen is fused with HN (fusion antigen examples: M2e peptide, gp-100 peptide, WT-1 peptide).
- B Conceptual diagram of inactivated hPIV2 ( ⁇ F) in which an antigen is fused with HN.
- FIG. 9 (A) is a plasmid vector diagram for establishing a package cell for producing a fusion protein of hPIV2, a F protein in a package cell and an antigen (example of fusion antigen: M2e peptide, gp-100 peptide, WT-1 peptide) Indicates.
- FIG. 9 (B) shows the result of introducing the antigen of the recovered vector by Western blot.
- FIG. 10 shows (A) an antigen introduction mode into hPIV2 (vector and antigen arrangement diagram), and (B) an antigen introduction result (Western blot) with a vector in which antigens were simultaneously introduced into HN and F.
- FIG. 11 shows the vaccine effect of hPIV2 / ⁇ F / HN-M2e against influenza virus.
- FIG. 12 shows the vaccine effect of hPIV2 / ⁇ F / HN-gp-100 on melanoma cells.
- FIG. 14 (A) shows the membrane / intracellular domain of F membrane protein that functions as an extracellular region of M2e antigen or RSV F (including modifications) and a packaging signal for viruses in the NotI region of hPIV2 / ⁇ F plasmid. The figure which introduce
- FIG. 14 (B) shows confirmation of M2e expression in the infected cells of the collected vector.
- the present invention provides a viral vector in which a gene encoding an antigen polypeptide is incorporated into a viral structural gene of the Paramyxoviridae family, and the antigen polypeptide is a fusion protein with a viral structural protein. And an inactivated vector capable of inducing immunity similar to a live (live) virus.
- the HN gene of the F gene-deficient hPIV2 vector and / or the F gene contains the M2e peptide gene of universal influenza virus, the gp100 peptide gene of melanoma antigen, or F gene-deficient hPIV2 vectors in which the WT-1 cancer antigen gene was expressed in a fused form were constructed.
- an F gene-deficient hPIV2 vector was constructed by fusing the M2e antigen and the membrane / intracellular domain of the F membrane protein containing the packaging signal to the foreign gene introduction part of the F gene-deficient hPIV2 vector.
- the inactivated or live vector thus obtained was found to have a very high vaccine effect against influenza virus and a melanoma growth inhibitory effect.
- a particularly preferred paramyxoviridae virus used in the present invention is a human parainfluenza type 2 virus, and its genome is a monocistronic single-stranded minus RNA of about 15,000 bases.
- viral structural gene products NP protein, P (phospho) protein, M (matrix) protein, F (fusion) protein, HN (hemagglutinin neuraminidase) protein and L (large) protein are encoded on the genome in that order. .
- V (buoy) protein is produced by RNA editing.
- Nucleocapsid protein (NP) binds to the RNA genome to form a helically symmetric ribonucleoside protein complex (nucleocapsid, RNP).
- NP protein Of the proteins encoded by the viral genome, NP protein, P (phospho) protein, and L (large) protein are necessary for the formation of RNP.
- F (fusion) protein and HN (hemagglutinin neuraminidase) protein exist on the virus envelope, and are responsible for adsorption and fusion to the receptor.
- the M (matrix) protein interacts with the cytoplasmic domains of the F and HN proteins, the envelope lipid bilayer and RNP, and is important for budding of virus particles.
- human parainfluenza type 2 virus is an RNA virus that grows in the cytoplasm, no gene is integrated into the chromosome of the host cell.
- this virus is known to infect human airway mucosa and induce mucosal immunity centered on IgA, humoral immunity by IgG, and cellular epidemic immunity.
- IgA human airway mucosa
- IgG humoral immunity by IgG
- cellular epidemic immunity since there have been no serious reports of human (adult) infections so far, it is considered extremely useful as a viral vector for treatment.
- Virus vector means a virus particle in which a gene to be expressed in an infected cell is packaged together with a virus genome gene, and a vector not containing a virus genome capable of producing an infectious virus. In the present specification, the latter is particularly referred to as an “inactivation vector” and can be obtained by inactivating a nucleic acid by treatment with a drug or the like.
- an “antigen polypeptide” is a polypeptide capable of inducing an immune response in a subject administered with the viral vector of the present invention.
- examples thereof include a cancer antigen or a part thereof, a protein derived from bacteria or viruses, or one of the proteins.
- useful antigen polypeptides for use as influenza vaccines include influenza virus HA protein, NA protein, M2 protein or M2e protein, or fragments thereof.
- infectious disease-related transducing antigen polypeptides include influenza virus (including highly toxic influenza virus), parainfluenza type 3 virus, RS virus, Hendra virus, SARS virus, Nipah virus, Lassa virus, dengue virus, West Nile virus, Human metapneumovirus, Ebola virus, Hantavirus, AIDS virus, hepatitis C virus, Lassa virus, human papillomavirus, rubella virus, rotavirus, norovirus, Crimea-Congo hemorrhagic fever virus, herpes virus, cytomegalovirus, papillomavirus, etc.
- antigenic peptides of bacteria such as group A ⁇ (beta) -type streptococci, Mycobacterium tuberculosis, Vibrio cholerae, Mycoplasma and the like.
- Antigen polypeptides useful for vaccines for cancer treatment include gp100, MUC1, NY-ESO-1, MelanA / MART1, TRP2, MAGE, CEA, CA125, HER2 / neu, WT1, and PSA. It is done.
- a fragment refers to a polypeptide having a partial sequence of a protein to be an antigen and capable of inducing an immune response when administered to a subject.
- the immune reaction includes both humoral immunity and cellular immunity.
- the antigen polypeptide is expressed as a fusion protein with a viral structural protein or a part of the structural protein.
- the antigen polypeptide expressed in the virus is taken into the virus particle and delivered to cells infected with the virus. That is, in the present invention, not only is the antigen polypeptide gene incorporated on the viral gene transcribed and translated in the infected cells, but the viral particles themselves have the antigen polypeptide, which Can be reliably delivered to recipient cells.
- a gene encoding the antigenic polypeptide is incorporated into a viral gene so as to be linked to a gene encoding a structural protein of the virus or a part of the structural protein.
- genes encoding viral structural proteins include hPIV2 HN gene, F gene, M gene, NP gene, P gene and L gene.
- One type of antigen gene may be used, but two or more types of antigen genes may be linked. This can induce an immune response against two or more types of antigens. It is also possible to introduce a plurality of antigens simultaneously into a plurality of genes among the HN gene, F gene, M gene, NP gene, P gene, and L gene.
- Such a vector can be constructed by a conventional method using a conventional recombinant DNA technique.
- the antigen polypeptide is expressed as a fusion protein with one or more selected from HN protein, F protein, and M protein.
- the antigen polypeptide can be expressed on the viral envelope.
- the antigen polypeptide can be expressed inside the virus particle when incorporated in the form of fusion to the N-terminal side of the HN protein, fusion to the N- or C-terminal side of the F protein, or fusion with the M protein.
- an F protein in which an antigen peptide is fused to the C-terminal side of the F protein is constructed and combined with a packaging cell that supplies the protein to a trans, a large amount of antigen is expressed inside the envelope of F-deficient hPIV2. be able to.
- the antigen peptide can be fused with the membrane / intracellular region of the viral HN protein or F protein, and the antigen can be included on the vector envelope.
- HN protein and F protein have antigenicity per se, and even higher immunogenicity can be imparted to antigen polypeptides having low immunogenicity by fusing with HN protein and F protein. It becomes possible.
- an F gene deficient (referred to as ⁇ F) virus is used as the human parainfluenza type 2 virus.
- the F protein of the human parainfluenza type 2 virus is a protein that is required when the viral nucleocapsid is introduced into the host by fusing the viral envelope and the cell membrane in the viral replication / transcription process.
- the F gene-deficient human parainfluenza type 2 virus produces infectious hPIV2 carrying the F gene in packaging cells, but does not produce infectious virus in cells that do not express the F protein. Therefore, virus particles having self-propagating ability cannot be constructed after infecting a subject's cells, and other cells are not infected, so that the virus is highly safe.
- Vero cells are used as packaging cells that can be infected with F gene-deficient virus and produce virus particles.
- Vero cells are particularly permissive for human pareline fenza type 2 virus F protein, have no interferon expression, and can stably proliferate even when the F gene of human parainfluenza type 2 virus is constantly expressed. And virus particles can be produced efficiently.
- a defective virus particle having an infectivity for supplying F gene in trans by using a Vero cell holding an F gene fused with an antigen polypeptide as a packaging cell instead of a Vero packaging cell that expresses the F gene. Can be manufactured.
- the present invention provides a method for producing a human parainfluenza type 2 virus vector that expresses an antigen polypeptide as a fusion protein with a viral structural protein.
- This method includes the steps of co-culturing human parainfluenza type 2 virus in which a gene encoding a fusion protein is incorporated into a viral gene, with Vero cells, and isolating virus particles from the culture supernatant.
- the viral vector of the present invention has been subjected to nucleic acid inactivation treatment.
- the nucleic acid inactivation treatment refers to inactivating only the viral genome while maintaining the three-dimensional structure of the envelope proteins such as F protein and HN protein and having the functions of these proteins.
- the nucleic acid inactivation treatment can be performed by, for example, nucleic acid alkylating agent treatment, hydrogen peroxide treatment, UV irradiation, radiation irradiation, or heat treatment.
- the viral vector is treated with ⁇ -propiolactone, which is a nucleic acid alkylating agent.
- ⁇ -propiolactone may be added to the virus culture and incubated at 4 ° C. for about 24 hours.
- the preferred concentration of ⁇ -propiolactone is 0.004% to 0.05%, more preferably 0.004% to 0.01% in terms of final concentration.
- the virus By using an F gene-deficient virus, even if a live virus remains due to drug treatment, the virus lacks the ability to grow, and there is no risk of growth within the recipient, and the high safety of the inactivated vaccine is maintained. Is possible.
- the genome can be inactivated while retaining the hemagglutination activity of the viral vector and the ability to mature dendritic cells.
- the virus particle itself has an adjuvant activity, there is an advantage that it is not necessary to use it together with an adjuvant that induces immunity to an antigen, or the adjuvant concentration can be reduced.
- the virus vector of the present invention infects cells via sialic acid receptors. Since sialic acid is present in many cells / tissues, vector administration routes include nasal spray, pulmonary, oral, sublingual, intradermal, subcutaneous, and direct administration to veins, Ex vivo administration to immune-inducing cells such as dendritic cells can be considered.
- the virus vector of the present invention can be typically administered to mammalian cells including humans as a spray.
- the propellant can be prepared by a conventional method.
- the culture supernatant containing the virus vector is concentrated if necessary, suspended in a buffer solution such as PBS, a virus vector stable solution or physiological saline together with an appropriate carrier or excipient, and then filtered as necessary.
- Etc. and can be prepared by sterilizing by filtration, and then filling an aseptic container. You may add a stabilizer, a preservative, etc. to a propellant as needed.
- the expression vector thus obtained can be administered by inhalation to a subject.
- the culture supernatant containing the virus vector is concentrated if necessary, suspended in a buffer solution such as PBS or physiological saline together with an appropriate carrier or excipient, and then sterilized by filtration with a filter or the like as necessary. It can then be prepared by filling aseptic containers. A stabilizer, a preservative and the like may be added to the injection as necessary.
- the expression vector thus obtained can be administered to a subject as an injection.
- Example 1 Confirmation of transgene expression in African green monkey cells (Vero cells) and mouse cells (NIH3T3 cells) by hPIV2 / ⁇ F
- Vero cells and NIH3T3 cells were cultured in a single layer upon vector infection.
- HPIV2 / ⁇ F into which the GFP gene was introduced was diluted to 10 8 from the stock solution. Since the vector does not generate an infectious vector, it exhibits GFP fluorescence only in the primary infected cells. The diluted virus was added for 1/10 of the cell culture solution and cultured for 3 days.
- Example 2 Confirmation of proliferation vector copy number in human and mouse dendritic cells by hPIV2 / ⁇ F (Recovery of human dendritic cells) Based on ethical rules, CD14 positive cells were recovered from peripheral blood of healthy individuals and cultured for 1 day. On the 4th and 7th days, GM-CSF was added to the culture solution at 50 ng / mL and IL-4 at 25 ng / mL, and the cells were cultured. GFP gene-introduced cells were infected with hPIV2 / ⁇ F so that the multiple infectivity (MOI) was 25, 50, 100, and the N gene region portion of hPIV2 in the cells 3 days after infection was quantitatively analyzed. PCR was performed and the copy number of the genome was measured.
- MOI multiple infectivity
- Bone marrow was recovered from the femur of B57BL / 6 and BALB / c mice based on the ethical regulations, and the residue was removed and cultured (RPM-1640 medium, 10% FBS). The medium was changed to the culture medium every 2 days of culture, and GM-CSF (20 ng / mL) and IL-4 (20 ng / mL) were added. GFP gene-introduced cells were infected with hPIV2 / ⁇ F so that the multiple infectivity (MOI) was 25, 50, 100, and the N gene region portion of hPIV2 in the cells 3 days after infection was quantitatively analyzed. PCR was performed and the copy number of the genome was measured. As a result of quantitative PCR using the N region part, it was found that the number of copies per cell of the N region part was about 5 times higher in human dendritic cells than in mouse dendritic cells (FIG. 2).
- Example 3 Confirmation of infection efficiency in dendritic cells by hPIV2 / ⁇ F Based on the ethical rules, CD14 positive cells were collected from peripheral blood of healthy individuals, and were added to the culture solution on the 1st, 4th and 7th days of cell culture. Cells were cultured by adding GM-CSF to a final concentration of 50 ng / mL and IL-4 to a final concentration of 25 ng / mL. Cells with 8 days of culture were infected with hPIV2 / ⁇ F into which GFP gene had been introduced so that the multiple infectivity (MOI) was 25, 50, 100.
- MOI multiple infectivity
- Bone marrow was collected from the B57BL / 6 femur according to the ethical regulations, and the residue was removed and cultured (RPM-1640 medium, 10% FBS). The culture medium was changed every 2 days of culture and added so that the final concentration of GM-CSF was 20 ng / mL) and the final concentration of IL-4 was 20 ng / mL. Cells with 8 days of culture were infected with hPIV2 / ⁇ F into which GFP gene had been introduced so that the multiple infectivity (MOI) was 25, 50, 100.
- MOI multiple infectivity
- dendritic cells 2 days after infection, the infection efficiency of dendritic cells was examined using flow cytometry with GFP fluorescence as an index. CD11c positive cells were used as dendritic cells.
- hPIV2 / ⁇ F can infect human dendritic cells and mouse dendritic cells to express genes, and human dendritic cells have higher infection efficiency than mouse dendritic cells. was found to be expensive.
- Example 4 Maturation of human dendritic cells by hPIV2 / ⁇ F
- hPIV2 / ⁇ F can efficiently introduce genes into human dendritic cells.
- MOI 25
- the maturation ability of dendritic cells was examined for the expression of CD40, CD80, and CD86, which are co-stimulatory factors for antigen-presenting cells.
- the homing ability to the secondary lymphoid organs was evaluated using the increased expression of CCR7 as an index.
- LPS lipopolysaccharide
- dendritic cells were able to induce co-stimulatory factor expression to an extent comparable to LPS due to infection with hPIV2 / ⁇ F, and CCR7 expression was increased (FIG. 4).
- Example 5 Induction of Major Histocompatibility Complex (MHC, HLA in Human) and Human Cytokine Complexes in Human Dendritic Cells by hPIV2 / ⁇ F Presentation of antigen on MHC is important for antigen-specific immune induction .
- the antigen needs to be presented to the MHC class I or II molecule.
- CD8 positive T cells recognize antigens on MHC class I molecules and induce cytotoxic T cells and the like.
- CD4-positive T cells recognize and activate antigens on MHC class II molecules.
- Dendritic cells can present antigens with MHC class I and MHC class II molecules.
- hPIV2 / ⁇ F is the same as in Examples 3 and 4 with respect to the increased expression of MHC class I molecules (HLA-A molecules in humans) and MHC class II molecules (HLA-DR molecules in humans) of human dendritic cells.
- the increase in expression was examined using human HLA-A molecules for class I molecules and human HLA-DR molecules for MHC class II molecules as indicators.
- HLA-A molecules and HLA-DR molecules also increased the expression to the same extent as the positive control LPS (FIG. 5).
- IL-6 and IL-12 cytokines were examined.
- the expression of hPIV2 / ⁇ F was comparable to that of LPS in human dendritic cells, and that of IL-12 was lower than that of LPS but increased in expression in IL-12 (FIG. 5).
- Example 6 Inactivation of hPIV2 / ⁇ F and Hemagglutination Activity and Immunity Induced by Inactivated hPIV2 / ⁇ F
- BPL propiolactone
- HPIV2 / ⁇ F was recovered by ultracentrifugation.
- the same volume of hPIV2 / ⁇ F was also concentrated by ultracentrifugation, and the final volume was suspended in the same volume of PBS solution.
- Inactivated vectors and control vectors were infected with hPIV2 F-expressing cells and cultured for 10 days. Further, the supernatant was infected with new hPIV2 F-expressing cells and cultured for 10 days. Furthermore, the same operation was performed. In total, the vector was cultured in F-expressing cells. Similar results were obtained by virus measurement with TCID50.
- control vector showed strong fluorescence and cytopathy, but 0.004%, 0.005%, 0.0075%, 0.009%, 0.01%, 0.01%, 0.012% None of the vectors inactivated at 0.025% and 0.05% showed GFP fluorescence and no cell degeneration was observed. That is, it was confirmed that the genome was completely inactivated by BPL treatment (FIG. 6A).
- the inactivated vector showed the same 512 HA value as that of the live hPIV2 / ⁇ F control vector that had not been inactivated (FIG. 6B). This indicates that the HN protein of the inactivated vector does not lose any hemagglutination ability, that is, the three-dimensional structure is maintained and the function is not lost. Therefore, it was speculated that only the genome of the vector was inactivated by the inactivation treatment.
- the vector was inactivated by 0.012% BPL and formalin treatment by a conventional method, and raw hPIV2 / ⁇ F / GFP, BPL inactivated hPIV2 / ⁇ F / GFP and formalin-treated hPIV2 / 2 ⁇ 10 7 vector particles of ⁇ F / GFP were administered twice (every other week) from the nasal area of B6 mice, and alveolar lavage fluid was collected one week after administration, and antibody titers against the vectors (IgG1, IgG2 and IgA) ).
- a 96-well plate was coated with 4 ⁇ g / mL inactivated hPIV2, and the antibody titer was measured.
- IgG1, IgG2 and IgA antibodies were induced in the alveolar lavage fluid, although BPL inactivation-treated hPIV2 / ⁇ F / GFP was inferior to live hPIV2 / ⁇ F / GFP. Little increase in antibody to formalin-treated hPIV2 / ⁇ F / GFP was seen in the alveolar lavage fluid (FIGS. 6C, D and E). This indicates that the formalin treatment shows low induction of antibodies against the hPIV2 protein having a normal steric structure in the alveolar lavage fluid.
- Example 7 Maturation of mouse dendritic cells by hPIV2 / ⁇ F and expression of major histocompatibility complex (MHC, HLA in human) and cytokine induction
- Bone marrow was collected from the femur of B57BL / 6 mice and cultured for 1 day.
- GM-CSF was added to the culture solution at 25 ng / mL, and the cells were cultured.
- Infected vector hPIV2 / ⁇ F which retains the GFP gene and was treated with 0.05% BPL, was cultured on cells on the 8th day of culture so that the multiple infectivity (MOI) was 25.
- MOI multiple infectivity
- Example 8 Construction of hPIV2 / ⁇ F in which HPI gene of hPIV2 and antigen gene were fused
- the M2e antigen peptide of the universal influenza virus (N-terminal-SLLTEVETPIRNEWGCRCNDSSDD-C-terminal (SEQ ID NO: 1)) was added to the C-terminal tail of HPI protein of hPIV2.
- a gp-100 antigen peptide N-terminal-KVPRNQDWL-C-terminal (SEQ ID NO: 2)
- the construction was based on the rule of 6 rule so that the total number of genomes, which is important for constructing hPIV2, is a multiple of 6. Virus recovery was attempted by the Reever Genetics method.
- hPIV2 / ⁇ F / HN-M2e and hPIV2 / ⁇ F / HN-gp-100 obtained by fusing the M2e antigen peptide of universal influenza virus or the gp-100 antigen peptide of skin cancer malignant tumor to the HN gene are recovered. (Fig. 8).
- N-terminal-SLLTEVETPTRNEWERCRCSDSDSD-C-terminal (SEQ ID NO: 7)), (N-terminal-SLLTEVETLTRNGWGCRCSDSSDDD-C-terminal (SEQ ID NO: 8)), WT-1 N-terminal-ALLPAVPSL-C terminal (SEQ ID NO: 3),
- a gene encoding N-terminal-CYTWNQMNL-C-terminal (SEQ ID NO: 4), N-terminal-CMTWNQMNL-C-terminal (SEQ ID NO: 5), N-terminal-RMPNAPYL-C-terminal (SEQ ID NO: 6) is also introduced.
- Example 9 Construction of packaging cell expressing fusion product of F gene and antigen gene and construction of hPIV2 / ⁇ F Universal type at 3 ′ end side of hPIV2F gene on plasmid for F expression constructed to produce packaging cell Influenza virus M2e antigen peptide (N-terminal-SLLTEVETPIRNEWCGCRCNDSSDD-C-terminal: SEQ ID NO: 1), WT-1 antigen peptide (N-terminal-ALLPAVPSL-C terminal (SEQ ID NO: 3)), WT-1 antigen peptide (N-terminal-CYTWNQMNL A gene encoding a -C terminus (SEQ ID NO: 4)) or WT-1 antigen peptide (N terminus-RMPNAPYL-C terminus (SEQ ID NO: 6)) was introduced to construct packaging cells. One or more antigens were introduced. Using the cells, F-deficient hPIV2 was recovered by a conventional reverse genetics method.
- the vector retaining the antigen inside the vector could be recovered.
- Vectors expressing multiple antigens could also be recovered. It was also found that the vector is easier to recover in the system in which the antigen is expressed inside than the outside of the vector envelope.
- M2e antigen peptide N-terminal-SLLTEVETPTRNEWERCSDSDSD-C-terminal (SEQ ID NO: 7)
- WT-1 peptide N-terminal-CMTWNQMNL-C-terminal (SEQ ID NO: 5)
- Example 10 Construction of a vector capable of expressing an antigen both inside and outside the F-deficient hPIV2 envelope
- collection of a vector capable of expressing an antigen inside and outside the vector envelope was performed. Indicated.
- the recovery of F-deficient hPIV2 retaining the antigen at both the inside and outside of the vector was examined. Whether the vector recovered in Example 8 retaining the M2e antigen (SEQ ID NO: 1) is infected with F packaging cells obtained by fusing two M2e genes to the F gene shown in Example 9, and the vector can be recovered. Examined.
- FIG. 10 shows that two types of proteins that react with the anti-M2 antibody were detected by Western blotting of the vector collected from 3 mL of the virus-infected culture supernatant. These molecular weights correspond to the molecular weights obtained by adding the M2e antibody to the F protein (cleaved F protein) and the HN protein, and it was confirmed that the vector holding the antigen outside and inside the envelope could be recovered.
- FIG. 10 also shows that the M2e bunt fused to the F protein is darker than the HN protein, and that the vector contains more F protein and antigen supplied from the package cell to the trans.
- Example 11 Vaccine effect of hPIV2 / ⁇ F / HN-M2e against influenza virus
- the preventive effect of hPIV2 / ⁇ F / HN-M2e against influenza RP8 strain in which influenza M2e antigen (SEQ ID NO: 1) was expressed in a fusion form with HN protein was examined. It was.
- As hPIV2 / ⁇ F / HN-M2e live hPIV2 / ⁇ F / HN-M2e and 0.05% BPL-treated inactivated hPIV2 / ⁇ F / HN-M2e were used.
- HN exists in the membrane of the viral envelope of the N-terminal region, and the C-terminal region exists outside the membrane.
- the test was conducted using 5-week-old female BALB / c in accordance with the ethical rules. Under anesthesia, 2x10 8 vectors (converted to TCID50) by nasal administration of live hPIV2 / ⁇ F / HN-M2e, inactivated hPIV2 / ⁇ F / HN-M2e, live hPIV2 / ⁇ F / M2, inactivated hPIV2 / ⁇ F / GFP 20 ⁇ l was administered. The administration was repeated 2 weeks later. Two weeks after the administration, 16,000 Influenza virus PR8 strains (LD50 was 1,000 or less) were administered to the anesthetized mice of the vector administration group and the control group by 20 ⁇ l from the nasal route. From the day of administration, daily body weight measurement and appearance observation were performed. For mice, mice that died or lost 30% of body weight compared to the time of influenza virus inoculation were considered dead.
- Fig. 11 shows the change in body weight (percentage) for 10 days after influenza virus inoculation and the results of death cases. It was very surprising that the inactivated hPIV2 / ⁇ F / HN-M2e group (4 cases) had no weight loss, no puffing, no movement and no feeding in all cases after influenza virus administration. It was the same as before the virus administration.
- M2 is a membrane protein consisting of 97 amino acid residues, which forms a tetramer and functions as a proton channel.
- inactivated hPIV2 / ⁇ F / HN-M2e takes a form in which M2e is fused with an HN membrane protein and induced with a highly immunogenic HN membrane protein, thereby increasing the antigenicity against M2e, It is considered that a higher vaccine effect was obtained. In fact, an example in which antigenicity is enhanced in M2e fused to Neisseria Meningitidis outer membrane complex (OMPC) has been reported.
- OMPC Neisseria Meningitidis outer membrane complex
- live hPIV2 / ⁇ F / HN-M2e did not show a vaccine effect against influenza virus.
- inactivated hPIV2 / ⁇ F / HN-M2e showed a very high vaccine effect proves that the inactivated vector system functions extremely effectively as a vaccine system against influenza virus.
- Inactivated hPIV2 which retains hemagglutination activity, can induce vaccine effects without the addition of an adjuvant, that M2e fused with a viral structural protein functions sufficiently as an antigen for vaccines, and that M2e is an hPIV2 membrane. It has been confirmed that M2e can be imparted with a high immunity effect by fusing with a protein, and that it does not involve transcription / replication of a virus-constituting protein and thus has low toxicity to a recipient.
- Example 12 Vaccine effect of hPIV2 / ⁇ F / HN-gp-100 on B-16 melanoma cells
- the preventive tumor suppressive effect of ex vivo on h-16V / HN-gp-100 on B-16 melanoma cells was examined.
- DC cells were collected from the mouse femur, cultured in vitro, added with a vector, and examined for tumor suppressive effect by ex vivo returning to the mouse.
- bone marrow was collected from the femur of a B57BL / 6 mouse, the residue was removed and cultured (RPMI-1640 medium, 10% FBS). The medium was changed to the culture medium every 2 days of culture, and GM-CSF (20 ng / mL) and IL-4 (20 ng / mL) were added. Differentiation into DC was confirmed after 8 days of culture.
- the control group had the largest tumor volume. The most effective was the live hPIV2 / ⁇ F / HN-gp-100 treatment group, and no tumor growth was observed during the measurement period. No tumor growth was observed in either the live hPIV2 / ⁇ F / GFP group or in some mice. This was expected to be due to the effect of enhancing the overall immune function of DC by infection with live hPIV2 / ⁇ F / GFP.
- live hPIV2 / ⁇ F / HN-gp-100 showed a higher tumor growth inhibitory effect than live hPIV2 / ⁇ F / GFP.
- inactivated hPIV2 / ⁇ F / HN-gp-100 and inactivated hPIV2 / ⁇ F / GFP and control were compared, inactivated hPIV2 / ⁇ F / HN-gp was compared with inactivated hPIV2 / ⁇ F / GFP and control.
- -100 showed a clear tumor growth inhibitory effect.
- the tumor suppressive effect of inactivated hPIV2 was smaller than that of live, but this was lower in gene transfer efficiency of hPIV2 into DC than in epithelial cells, and mouse cells were infected / transferred compared to human cells.
- the inefficiency is considered to be a factor, and the effect is expected to increase if the amount of vector added is increased.
- Example 13 Vaccine effect of hPIV2 / ⁇ F / HN-gp-100 and WT-1 on B-16 melanoma cells
- inactivated hPIV2 / ⁇ F / HN-gp-100 and inactivated hPIV2 / ⁇ F / WT-1 The therapeutic effect of the introduced sequence of WT-1: N-terminal-RMPNAPYL-C-terminal (SEQ ID NO: 6) was examined. The back of B57BL / 6 mice was shaved and 2 ⁇ 10 6 B-16 melanoma cells were transplanted into the mouse skin.
- Example 14 Insertion of a foreign gene into each site of hPIV2 / ⁇ F
- Two M2e genes (SEQ ID NO: 1) are linked, and the F membrane anchor region of hPIV2 and the proteins in the membrane and intracellular regions are encoded on the C-terminal side.
- a gene was ligated, that is, a ligation product with a packaging signal to a vector was constructed, and inserted into hPIV2 / ⁇ F to examine whether a foreign gene product was incorporated into the vector.
- HPIV2 R1, intervening sequence and R2 were added to the 3 ′ side of the gene construct by a conventional method, and Not I sequences were arranged at both ends thereof.
- a vector plasmid was constructed in which two M2e were linked by insertion into the NothI site of hPIV2 / ⁇ F and a fusion protein of an F membrane anchor region and an intramembrane region protein was incorporated (FIG. 14A).
- hPIV2 / ⁇ F a NotI restriction site is introduced in the upstream region of the NP gene. A foreign gene can be inserted into this region and the foreign gene can be expressed alone.
- the vector was recovered from F-expressing cells by a conventional method. Samples collected from the culture supernatant were infected with F-expressing Vero cells (1 ⁇ 10 6 cells). Cells were collected 8 days after infection, and Western blotting was performed using an anti-M2 antibody.
- a vector plasmid is constructed in which the insertion site of the foreign gene is NP-P, PM, M-HN, and HN-L other than the N gene front. Furthermore, a vector plasmid using RSV F protein and RSV modified F protein gene as a transgene is constructed.
- the present invention is useful as a vaccine for preventing or treating diseases.
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Abstract
Description
[1] 抗原ポリペプチドをコードする遺伝子がパラミクソウイルス科のウイルス遺伝子に組み込まれているウイルスベクターであって、該抗原ポリペプチドはウイルスの構造蛋白質あるいはその一部との融合蛋白質として発現するウイルスベクター:
[2] 融合蛋白質がウイルスのHN蛋白質、F蛋白質、およびM蛋白質から選ばれる1または2以上あるいはその一部との融合蛋白質である、[1]に記載のウイルスベクター:
[3] 抗原ポリペプチドをコードする遺伝子が、
1)HN遺伝子の3’末端側に配置され、抗原ポリペプチドがベクターエンベロープの外部に発現される、
2)F遺伝子の3’末端側に配置され、抗原ポリペプチドがベクターエンベロープの内部に発現される、または
3)HN遺伝子の3’末端側およびF遺伝子の3’末端側にそれぞれ配置され、抗原ポリペプチドがベクターエンベロープの外部と内部の両方に発現される、
ことを特徴とする[1]または[2]に記載のウイルスベクター。
[4]抗原ポリペプチドとウイルスパッケージングシグナルであるFまたはHN膜蛋白質の膜・細胞内ドメインとを融合させ、ベクターエンベロープ上に抗原ポリペプチドが保持されることを特徴とする[1]または[2]に記載のウイルスベクター:
[5] ウイルスがF蛋白質欠損型パラミクソウイルス科のウイルスである、[1]~[4]のいずれかに記載のウイルスベクター:
[6] ウイルスが核酸不活化処理されたものである、[1]~[5]のいずれかに記載のウイルスベクター:
[7] 核酸不活化処理がウイルスエンベロープの構造を極力変化させない(実質的に変化させない)ものである、[6]に記載のウイルスベクター:
[8] 抗原ポリペプチドがインフルエンザウイルスのM2e蛋白質、またはその断片である、[1]~[7]のいずれかに記載のウイルスベクター:
[9] 抗原ポリペプチドが、強毒型インフルエンザウイルスを含むインフルエンザウイルス、パラインフルエンザ3型ウイルス、RSウイルス、ヘンドラウイルス、SARSウイルス、ニパウイルス、ラッサウイルス、デングウイルス、ウエストナイルウイルス、ヒトメタニューモウイルス、エボラウイルス、ハンタウイルス、エイズウイルス、C型肝炎ウイルス、ラッサウイルス、ヒトパピローマウイルス、風疹ウイルス、ロタウイルス、ノロウイルス、クリミア-コンゴ出血熱ウイルス、ヘルペスウイルス、サイトメガロウイルス、およびパピローマウイルスから選ばれるウイルスの抗原ペプチド、A群β(ベータ)型溶連菌、Mycobacterium tuberculosis、コレラ菌、およびマイコプラズマから選ばれる細菌の抗原ペプチド、またはRSVのF蛋白質、癌抗原gp100、MUC1、NY-ESO-1、MelanA/MART1、TRP2、MAGE、CEA、CA125、HER2/neu、WT1、およびPSAからなる群から選ばれるいずれか1または2以上、あるいはこれらの断片である、[1]~[7]のいずれかに記載のウイルスベクター:
[10] 抗原ポリペプチドをウイルスの構造蛋白質あるいはその一部との融合蛋白質として発現するパラミクソウイルス科のウイルスベクターの製造方法であって、融合蛋白質をコードする遺伝子がウイルス遺伝子に組み込まれているパラミクソウイルス科のウイルスをVero細胞と共培養し、そして培養上清からウイルス粒子を単離する、
の各工程を含む方法:
[11] 融合蛋白質がウイルスのHN蛋白質、F蛋白質、およびM蛋白質から選ばれる1または2以上あるいはその一部との融合蛋白質である、[10]に記載の方法:
[12] 融合蛋白質がウイルスのHN蛋白質またはF蛋白質の細胞内領域から選ばれる融合蛋白質である[10]または[11]に記載の方法:
[13] 抗原ポリペプチドが、
1)HN蛋白質のC末端側に融合され、ベクターエンベロープの外部に発現される、
2)F蛋白質のNあるいはC末端側またはHN蛋白質のN末端側に融合され、ベクターエンベロープの内部に発現される、または
3)HN蛋白質のC末端側およびF蛋白質のNあるいはC末端側またはHN蛋白質のN末端側にそれぞれ融合され、ベクターエンベロープの外部と内部の両方に発現される、
ことを特徴とする、[10]~[12]のいずれかに記載の方法:
[14] 抗原ポリペプチドが、ウイルスのHN蛋白質またはF蛋白質の膜・細胞内領域と融合し、ベクターエンベロープ上に抗原ポリペプチドが包含されることを特徴とする、[10]~[12]のいずれかに記載の方法:
[15] F遺伝子を欠損したパラミクソウイルス科のウイルスを、パラミクソウイルス科のウイルスのF遺伝子または抗原と融合したF遺伝子を発現するVero細胞と共培養し、そして培養上清からウイルス粒子を単離することを含む、[10]~[14]のいずれかに記載の方法:
[16] ウイルスを核酸不活化処理する工程をさらに含む、[10]~[15]のいずれかに記載の方法:
[17] 核酸不活化処理がウイルスエンベロープの構造を極力変化させない(実質的に変化させない)ものである、[16]に記載の方法:
[18] 抗原ポリペプチドがインフルエンザウイルスのM2e蛋白質、またはその断片である、[10]~[17]のいずれかに記載の方法:
[19] 抗原ポリペプチドが、強毒型インフルエンザウイルスを含むインフルエンザウイルス、パラインフルエンザ3型ウイルス、RSウイルス、ヘンドラウイルス、SARSウイルス、ニパウイルス、ラッサウイルス、デングウイルス、ウエストナイルウイルス、ヒトメタニューモウイルス、エボラウイルス、ハンタウイルス、エイズウイルス、C型肝炎ウイルス、ラッサウイルス、ヒトパピローマウイルス、風疹ウイルス、ロタウイルス、ノロウイルス、クリミア-コンゴ出血熱ウイルス、ヘルペスウイルス、サイトメガロウイルス、およびパピローマウイルスから選ばれるウイルスの抗原ペプチド、A群β(ベータ)型溶連菌、Mycobacterium tuberculosis、コレラ菌、およびマイコプラズマから選ばれる細菌の抗原ペプチド、またはRSVのF蛋白質、癌抗原gp100、MUC1、NY-ESO-1、MelanA/MART1、TRP2、MAGE、CEA、CA125、HER2/neu、WT1、およびPSAからなる群から選ばれるいずれか1または2以上、あるいはこれらの断片である、[10]~[18]のいずれかに記載の方法。
[20] 抗原遺伝子と融合されたパラミクソウイルス科のウイルスのF遺伝子を含み、前記抗原とF蛋白質との融合蛋白質を発現するVero細胞。
2)本発明では、融合させたウイルスベクターの構造蛋白質、特に膜蛋白質自体が免疫原性をもつため、当該蛋白質に融合された免疫原性の低い抗原ポリペプチドに対する高い免疫を誘導できる。すなわち、ベクター自身がアジュバント活性を有し、投与の際にアジュバントの添加が不要または通常より少量である。
3)本発明のウイルスベクターでは、抗原とアジュバントが同一の細胞に導入されるため、ペプチドとアジュバントを混合投与したときにそれぞれが異なる細胞に取り込まれることによる非効率的な免疫誘導を低減させることが可能となる。
4)本発明で用いられるヒトパラインフルエンザウイルスは、マイナス一本鎖ウイルスで、末端が3リン酸で修飾されている(5’-PPP)。この末端の3リン酸とこれに続く数ベースのシングル鎖RNA部分は、IFITという細胞内シグナルの基質となり、抗ウイルス活性を有する1型インターフェロン等を誘導し、DC細胞成熟等に寄与する(Abbas YT., Nature, Vol. 494, Issue 7435, pp60-64 (2013);Saito T. et al., J. Exp. Med. Vol.205, No.7, pp1523-1527 (2008))。
5)本発明では、低濃度のβプロピオラクトン等を用いて核酸不活化が行われるため、ウイルスエンベロープ蛋白質の立体構造が維持され、これら蛋白質の機能を維持した状態でウイルスゲノムのみが不活化される。それゆえ、ベクターは生ウイルスに近い細胞吸着性を有し、上述した構造による免疫誘導能・DC細胞成熟化能を発揮できる。
6)上記した利点から、本発明のウイルスベクターは、従来ヒトにおいて十分な効果が期待できなかったウイルスベクターとは異なり、ヒトにおいても高い効果を期待できる。
7)抗原導入を遺伝子レベルで行うことにより、不活化後に抗原導入を行う必要がなく、不活化後の抗原導入に伴う問題(低い抗原導入効率、エンベロープ構造の破壊等)を回避できる。
8)本発明のウイルスベクターは、ベクターエンベロープの外部、内部、又はその両方に抗原を配置できるように構成することが可能であり、これにより複数抗原のベクターへの様々な導入が可能となる。
以上のとおり、本発明によれば、効率的なウイルス不活化処理により、樹状細胞等に対しライブウイルスベクターと遜色のない免疫誘導が可能な不活化ベクターを取得でき、パラミクソウイルス科ウイルスを利用した不活化ベクターのワクチン化に途を開くことが可能となる。
96ウエルプレートにVero細胞およびNIH3T3細胞がベクター感染時に単層になるように培養した。GFP遺伝子を導入したhPIV2/ΔFを原液から108まで希釈した。当該ベクターは、感染性ベクターを生じないので1次感染した細胞でしかGFPの蛍光を呈しない。当該希釈ウイルスを細胞培養液の10分の1量を添加し、3日間培養した。
(ヒト樹状細胞の回収)倫理規定に基づき健常人の抹消血よりCD14陽性細胞を回収し、細胞培養1日目、4日目、7日目に培養液にGM-CSFが50ng/mL、IL-4が25ng/mLとなるように添加し、細胞の培養を行った。培養8日の細胞にGFP遺伝子を導入したhPIV2/ΔFを多重感染度(MOI)が25、50、100になるように感染し、感染3日後の細胞中のhPIV2のN遺伝子領域部分について定量的PCRを行い、ゲノムのコピー数を測定した。
倫理規定に基づき健常人の抹消血よりCD14陽性細胞を回収し、細胞培養1日目、4日目、7日目に培養液にGM-CSFの終濃度が50ng/mL、IL-4の終濃度が25ng/mLとなるように添加し、細胞の培養を行った。培養8日の細胞にGFP遺伝子を導入したhPIV2/ΔFを多重感染度(MOI)が25、50、100になるように感染させた。
実施例3においてhPIV2/ΔFは効率よくヒト樹状細胞に遺伝子導入できることが確認された。樹状細胞が免疫提示細胞として有効に機能し、効率的な免疫を誘導するためには、当該細胞が成熟化しリンパ器官へのホーミングすることが極めて重要である。そこで、MOI=25でhPIV2/ΔFをヒト樹状細胞に2日間感染させ、樹状細胞の成熟化能を、抗原提示細胞の補助刺激因子であるCD40、CD80、CD86の発現について調べた。二次リンパ器官へのホーミング能についてはCCR7の発現上昇を指標にして評価した。陽性コントロールとして、TRL4を活性化し成熟化およびリンパ器官へのホーミングを非常に効率よく引き起こすことが知られているLPS(リポ多糖)を培地中に1μg/mLとなるように添加した。評価は指標抗体によりフローサイトメトリーにより行った。
抗原特異的な免疫誘導にはMHC上への抗原の提示が重要である。そのためには、MHCクラスI分子またはII分子に抗原が提示される必要がある。CD8陽性T細胞は、MHCクラスI分子上の抗原を認識し細胞障害性T細胞等を誘導する。CD4陽性T細胞は、MHCクラスII分子上の抗原を認識し、活性化する。
hPIV2/ΔFの蛋白質の構造と機能を保持したまま、ゲノムのみを不活化すべく、アルキル化剤のβ―プロピオラクトン(BPL)による不活化を検討した。無血清培地で培養したGFP遺伝子保持hPIV2/ΔFの培養液に、0.004%、0.005%、0.0075%、0.009%、0.01%、0.012%、0.025%、0.05%となるようにBPLを添加し、4℃で24時間処理した。その後37℃で30分間処理しBPLを不活性化した。超遠心にてhPIV2/ΔFを回収した。コントロールとして、同容量のhPIV2/ΔFも超遠心にて濃縮し、最終容量を同量のPBS溶液に懸濁した。不活化ベクターとコントロールベクターをhPIV2のF発現細胞に感染させ、10日間培養した。さらに、上清液を新たなhPIV2のF発現細胞に感染させ、10日間培養した。さらに、同操作を行った。合計、3回のベクターをF発現細胞で培養させた。TCID50によるウイルスの測定でも同様の結果を得た。
B57BL/6マウスの大腿骨より骨髄を回収し、細胞培養1日目、4日目、7日目に培養液にGM-CSFが25ng/mLとなるように添加し、細胞の培養を行った。培養8日の細胞にGFP遺伝子を保持し、0.05%のBPLで処理した不活化ベクターのhPIV2/ΔFを多重感染度(MOI)が25になるように感染し、感染2日後の細胞中のCD40およびCD80の発現上昇による樹状細胞の成熟化、H-2KbおよびI-A/I-EによるMHCクラスIまたはIIの上昇、培養液中でのIL-6およびIL-12の発現を調べた。コントロールは、不活化をしていない(ライブ)hPIV2/ΔFを用いた。
hPIV2のHN蛋白質のC末端テール部分に万能型インフルエンザウイルスのM2e抗原ペプチド(N末端-SLLTEVETPIRNEWGCRCNDSSDD-C末端(配列番号1))または皮膚がん悪性腫瘍のgp-100抗原ペプチド(N末端-KVPRNQDWL-C末端(配列番号2))を挿入したベクターのプラスミドコンストラクトを構築した。当該構築はhPIV2にコンストラクト上重要とされる、ゲノムの総数が6の倍数になるように、ルールオブ6ルールに基づいて構築した。リーバースジェネティクス法により、ウイルスの回収を図った。
パッケージング細胞を作製するために構築したF発現用プラスミド上のhPIV2F遺伝子の3’末端側に万能型インフルエンザウイルスのM2e抗原ペプチド(N末端-SLLTEVETPIRNEWGCRCNDSSDD-C末端:配列番号1)、WT-1抗原ペプチド(N末端-ALLPAVPSL-C末端(配列番号3))、WT-1抗原ペプチド(N末端-CYTWNQMNL-C末端(配列番号4))またはWT-1抗原ペプチド(N末端-RMPNAPYL-C末端(配列番号6))をコードする遺伝子を導入し、パッケージング細胞を構築した。抗原は、一個あるいは複数個導入した。当該細胞を用いて、常法のリバースジェネテクス法によりF欠損型hPIV2の回収を行った。
実施例8および9において、ベクターエンベロープの内と外に抗原を発現させることができるベクターの回収を示した。本実施例では、ベクターの内と外の両部位に抗原を保持するF欠損型hPIV2の回収について検討を行った。M2e抗原(配列番号1)を保持する実施例8で回収したベクターを、実施例9に示したF遺伝子にM2e遺伝子を2個融合させたFパッケージング細胞に感染させ、ベクターが回収できるかを調べた。
インフルエンザM2e抗原(配列番号1)をHN蛋白質に融合型で発現させたhPIV2/ΔF/HN-M2eのインフルエンザRP8株に対する予防効果を調べた。hPIV2/ΔF/HN-M2eは、ライブhPIV2/ΔF/HN-M2eおよび0.05%BPL処理した不活化hPIV2/ΔF/HN-M2eを用いた。さらに、単独でM2の全体を発現するライブhPIV2/ΔF/M2も比較の対象として用いた。HNと融合したM2eは、HNがN末端領域のウイルスエンベロープの膜内に存在し、C末端領域は膜外に存在している。
hPIV2/ΔF/HN-gp-100のB-16メラノーマ細胞に対するex vivoによる予防的な腫瘍抑制効果について調べた。
次に、不活化hPIV2/ΔF/HN-gp-100と不活化hPIV2/ΔF/WT-1(WT-1の導入配列:N末端-RMPNAPYL-C末端(配列番号6))の治療効果について調べた。B57BL/6マウスの背中を剃毛し、2x106個細胞のB-16メラノーマ細胞をマウス皮内に移植した。4日後に移植B-16メラノーマの径が3mm程度になったところで、腫瘍内に70μLに調整した各7.0x106個の不活化hPIV2/ΔF/HN-gp-100および不活化hPIV2/ΔF/WT-1を、細胞移植後4日、7日、10日、13日目に合計4回投与した。腫瘍径をそれぞれ測定した。コントロールとして、不活化hPIV2/ΔF/GFPを投与した。投与初期には、70μLの容量は腫瘍容量を超えた容量であった。
M2e遺伝子(配列番号1)を2個連結し、そのC末端側にhPIV2のF膜アンカー領域と膜・細胞内領域の蛋白質をコードする遺伝子を連結、つまりベクターへのパッケージングシグナルとの連結産物を構築し、hPIV2/ΔFに挿入し、外来遺伝子産物がベクターに取り込まれるかどうかを検討した。
Claims (20)
- 抗原ポリペプチドをコードする遺伝子がパラミクソウイルス科のウイルス遺伝子に組み込まれているウイルスベクターであって、前記抗原ポリペプチドはウイルスの構造蛋白質あるいはその一部との融合蛋白質として発現するウイルスベクター。
- 融合蛋白質がウイルスのHN蛋白質、F蛋白質、およびM蛋白質から選ばれるいずれか1または2以上あるいはその一部との融合蛋白質である、請求項1に記載のウイルスベクター。
- 抗原ポリペプチドをコードする遺伝子が、
1)HN遺伝子の3’末端側に配置され、抗原ポリペプチドがベクターエンベロープの外部に発現される、
2)F遺伝子の3’末端側に配置され、抗原ポリペプチドがベクターエンベロープの内部に発現される、または
3)HN遺伝子の3’末端側およびF遺伝子の3’末端側にそれぞれ配置され、抗原ポリペプチドがベクターエンベロープの外部と内部の両方に発現される、
ことを特徴とする請求項1または2に記載のウイルスベクター。 - 抗原ポリペプチドとウイルスパッケージングシグナルであるFまたはHN膜蛋白質の膜・細胞内ドメインとを融合させ、ベクターエンベロープ上に抗原ポリペプチドが保持されることを特徴とする請求項1または2に記載のウイルスベクター。
- ウイルスがF蛋白質欠損型パラミクソウイルス科のウイルスである、請求項1~4のいずれかに記載のウイルスベクター。
- ウイルスが核酸不活化処理されたものである、請求項1~5のいずれかに記載のウイルスベクター。
- 核酸不活化処理がウイルスエンベロープの構造を実質的に変化させないものである、請求項6に記載のウイルスベクター。
- 抗原ポリペプチドがインフルエンザウイルスのM2e蛋白質、またはその断片である、請求項1~7のいずれかに記載のウイルスベクター。
- 抗原ポリペプチドが、強毒型インフルエンザウイルスを含むインフルエンザウイルス、パラインフルエンザ3型ウイルス、RSウイルス、ヘンドラウイルス、SARSウイルス、ニパウイルス、ラッサウイルス、デングウイルス、ウエストナイルウイルス、ヒトメタニューモウイルス、エボラウイルス、ハンタウイルス、エイズウイルス、C型肝炎ウイルス、ラッサウイルス、ヒトパピローマウイルス、風疹ウイルス、ロタウイルス、ノロウイルス、クリミア-コンゴ出血熱ウイルス、ヘルペスウイルス、サイトメガロウイルス、およびパピローマウイルスから選ばれるウイルスの抗原ペプチド、A群β(ベータ)型溶連菌、Mycobacterium tuberculosis、コレラ菌、およびマイコプラズマから選ばれる細菌の抗原ペプチド、または癌抗原gp100、MUC1、NY-ESO-1、MelanA/MART1、TRP2、MAGE、CEA、CA125、HER2/neu、WT1、およびPSAからなる群から選ばれるいずれか1または2以上、あるいはこれらの断片である、請求項1~7のいずれかに記載のウイルスベクター。
- 抗原ポリペプチドをウイルスの構造蛋白質あるいはその一部との融合蛋白質として発現するパラミクソウイルス科のウイルスベクターの製造方法であって、融合蛋白質をコードする遺伝子がウイルス遺伝子に組み込まれているパラミクソウイルス科のウイルスベクターをVero細胞と共培養し、そして培養上清からウイルス粒子を単離する、
の各工程を含む方法。 - 融合蛋白質がウイルスのHN蛋白質、F蛋白質、およびM蛋白質から選ばれる1または2以上あるいはその一部との融合蛋白質である、請求項10に記載の方法。
- 融合蛋白質がウイルスのHN蛋白質またはF蛋白質の細胞内領域から選ばれる融合蛋白質である請求項10または11に記載の方法。
- 抗原ポリペプチドが、
1)HN蛋白質のC末端側に融合され、ベクターエンベロープの外部に発現される、
2)F蛋白質のCまたはN末端側に融合され、ベクターエンベロープの内部に発現される、または
3)HN蛋白質のC末端側およびF蛋白質のCあるいはN末端側またはHN蛋白質のN末端側にそれぞれ融合され、ベクターエンベロープの外部と内部の両方に発現される、
ことを特徴とする、請求項10~12のいずれかに記載の方法。 - 抗原ポリペプチドが、ウイルスのHN蛋白質またはF蛋白質の膜・細胞内領域と融合し、ベクターエンベロープ上に抗原ポリペプチドが包含されることを特徴とする、請求項10~12のいずれかに記載の方法。
- F遺伝子を欠損したパラミクソウイルス科のウイルスを、パラミクソウイルス科のウイルスのF遺伝子または抗原と融合したF遺伝子を発現するVero細胞と共培養し、そして
培養上清からウイルス粒子を単離することを含む、請求項10~14のいずれかに記載の方法。 - ウイルスを核酸不活化処理する工程をさらに含む、請求項10~15のいずれかに記載の方法。
- 核酸不活化処理がウイルスエンベロープの構造を実質的に変化させないものである、請求項16に記載の方法。
- 抗原ポリペプチドがインフルエンザウイルスのM2e蛋白質、またはその断片である、請求項10~17のいずれかに記載の方法。
- 抗原ポリペプチドが、強毒型インフルエンザウイルスを含むインフルエンザウイルス、パラインフルエンザ3型ウイルス、RSウイルス、ヘンドラウイルス、SARSウイルス、ニパウイルス、ラッサウイルス、デングウイルス、ウエストナイルウイルス、ヒトメタニューモウイルス、エボラウイルス、ハンタウイルス、エイズウイルス、C型肝炎ウイルス、ラッサウイルス、ヒトパピローマウイルス、風疹ウイルス、ロタウイルス、ノロウイルス、クリミア-コンゴ出血熱ウイルス、ヘルペスウイルス、サイトメガロウイルス、およびパピローマウイルスから選ばれるウイルスの抗原ペプチド、A群β(ベータ)型溶連菌、Mycobacterium tuberculosis、コレラ菌、およびマイコプラズマから選ばれる細菌の抗原ペプチド、または癌抗原gp100、MUC1、NY-ESO-1、MelanA/MART1、TRP2、MAGE、CEA、CA125、HER2/neu、WT1、およびPSAからなる群から選ばれる抗原ペプチドのいずれか1または2以上、あるいはこれらの断片である、請求項10~18のいずれかに記載の方法。
- 抗原遺伝子と融合されたパラミクソウイルス科のウイルスのF遺伝子を含み、前記抗原とF蛋白質との融合蛋白質を発現するVero細胞。
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