Patent Application
20120219582
The present invention relates to a intranasal spray-type tuberculosis vaccine, using a paramyxovirus, particularly a type 2 human parainfluenza virus vector (hPIV2).
Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis belonging to the genus Mycobacterium. Tuberculosis is currently one of the most common infectious diseases in the world. More than eight million people develop tuberculosis and about two million people die every year in the world. The reason for this is that the bacilli de Calmette et Guerin (BCG) vaccine which is a widely used tuberculosis vaccine is ineffective for adults and accordingly, an effect of tuberculosis prevention can be obtained only in younger generations by vaccination in infancy and childhood, and even implementation of additional vaccinations cannot prevent tuberculosis effectively in adults. More than 20,000 people develop tuberculosis every year in Japan (about 20 patients/one hundred thousands people), thus Japan is a middle-level tuberculosis prevalence country.
The BCG vaccine used for prevention of tuberculosis originates from Mycobacterium bovis. The Mycobacterium bovis loses toxicity to human by successive culture for a long period of time, whereupon bacteria with only antigenicity are used as the BCG vaccine. When inoculated with the live attenuated bacteria, humans can acquire immunity preventing infection of Mycobacterium tuberculosis. The BCG vaccine is the only vaccine for tuberculosis prevention that is widely accepted as having an efficacy for prevention of infantile tuberculosis.
The efficacy of BCG vaccine lasts for dozen years or so, and prevention of adult tuberculosis relies upon an increase in natural immunity. However, natural immunity cannot suppress the onset of adult tuberculosis. It is known that adulthood BCG vaccination has a lower or almost no efficacy for adult tuberculosis.
In consideration of the aforementioned state of affairs, the development of several adult tuberculosis vaccines has been advanced. For example, under development are MVA85A vaccine (vaccine expressing 85A antigen of acid-fast bacterium on vaccinia virus Ankara), Aeras-402 vaccine (vaccine expressing 85A antigen and TB10.4 on type 35 adenovirus) and M72F (otherwise known as GSK TB101: a vaccine developed by GSK (Glaxo Smith Kline plc) and using a protein of Mycobacterium tuberculosis with GSKAS02 Adjuvant) and the like. However, no adult tuberculosis vaccine has been hitherto known that has a sufficient efficacy.
The present invention was made in view of the foregoing circumstances and an object thereof is to provide a intranasal spray-type tuberculosis vaccine, which has efficacy in prevention of tuberculosis.
HPIV2 is a virus belonging to Family Paramyxoviridae and has genome that is a single negative-stranded RNA having about 15000 bases. The nucleocapsid protein (NP) is combined with this nucleic acid to be formed into a helically symmetric ribonucleoside protein complex (ribonucleocapsid, RNP). M protein, one of proteins encoded by the virus, interacts with an intracytoplasmic domain of a viral glycoprotein, lipid bilayer envelope and RNP, and is important for budding of viral particle. Recombinant virus of which M protein gene has been deleted by gene manipulation can infect cells to express host viral proteins but cannot bud. The present inventors found that M protein-deficient PIV2 could be used as a vector to express exogenous protein in a target cell (non-patent document 1).
Meanwhile, the inventors found that an α antigen derived from acid-fast bacteria had efficacy in prevention and treatment of allergic diseases (Patent Document 1). The α antigen expression vector was constructed by using pcDNA-α-K having a CMV promoter and a TPA signal peptide, and the effect in asthmatic mouse models was investigated. The inventors further found that PIV2 vector expressing α antigen has efficacy against allergic diseases, since PIV2 vector has high ability of α antigen induction (Patent Document 2).
The inventors examined whether or not the above-described inventors' technique was applicable to a tuberculosis vaccine. The inventors found that the expression vector made by integrating the α antigen into hPIV2 (rhPIV2) deficient in M protein or F protein exhibited efficacy for prevention of tuberculosis, regarding infection prevention of tubercle bacillus against which conventional methods had almost no efficacy, whereupon the inventors basically completed the present invention.
Thus, a intranasal spray-type tuberculosis vaccine according to a first invention comprises a gene encoding an α antigen derived from Mycobacteria (Ag85B), an analog thereof or a mutant thereof having a function similar thereto (hereinafter called “α antigen or the like”), said gene being integrated into a paramyxovirus gene deficient in an M gene, F gene or HN gene.
Furthermore, a method of prevention or treatment of tuberculosis, according to a second invention, comprises integrating a gene encoding an α antigen derived from Mycobacteria, an analog thereof or a mutant having a function similar thereto into a paramyxovirus gene deficient in an M gene, F gene or HN gene, and nebulizing a human or mammals for intranasal administration.
The paramyxovirus represents virus belonging to Family Paramyxoviridae. The paramyxovirus represents a class of virus that has minus stranded RNA as a genome and virions ranging from 150 nm to 350 nm and presents a multiform appearance. The paramyxovirus virions are enveloped and have two types of glycoproteins (F and HN) arranged on the surface of the envelope. The paramyxovirus includes parainfluenza virus (PIV), bovine parainfluenza virus-3, Sendai virus, mumps virus, canine distemper virus, rinderpest virus, morbillivirus, peste-des-petits-ruminants virus, Newcastle disease virus, Avian paramyxovirus type 2-9, Hendra virus, Nipah virus, bovine respiratory syncytial virus, human respiratory syncytial virus, human metapneumovirus, and the like.
It is preferred that the α antigen is derived from Mycobacterium kansasii or Mycobacterium bovis BCG. Furthermore, it is preferred that the analog of the α antigen is an 85 complex-forming protein 85A or 85 complex-forming protein 85C.
According to the present invention, a vaccine having a high efficacy in prevention and treatment of adult tuberculosis can be provided.
The vaccine according to the invention is a intranasal spray-type tuberculosis vaccine and has a high administration easiness while utilizing a high protein expression signature of paramyxovirus central to the virus (particularly, hPIV2) in respiratory infections. Although recombinant vaccines of MVA85A and Aeras402 currently under development are injection products, the intranasal spray-type tuberculosis vaccine according to the invention is an aerosolized agent and has a higher expression level of target protein than injection solution administration. Induction of local immunity in the lung as a disease expression site is more important than systemic immunization in tuberculosis prevention. A high cell-mediated immunity induction in the lung is obtained by the use of a paramyxovirus vector into which a protein gene having a cell-mediated immunity (Th1) skewing function is inserted, whereby it can be considered that a higher vaccine efficacy against tuberculosis bacteria as compared with other vectors. Furthermore, the vector based on the vaccine has a high safety particularly for adults in virus pathogenicity.
Furthermore, a high yield and reduction in costs can be realized by the use of cultivated cell and nebulization in the production aspect.
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Embodiments according to the present invention will be described with reference to the drawings. The technical scope of the invention should not be limited by the embodiments, which can be practiced in various forms without changes in the scope of the invention. The technical scope of the invention covers the range of equivalence.
Human paramyxovirus (particularly, hPIV2) used in the present invention has an exceedingly low pathogenicity and a high protein expression. Accordingly, it is possible to prepare and use a paramyxovirus gene into which a gene encoding α antigen or the like as foreign protein is integrated.
However, the hPIV2 vector becomes a safer hPIV2 vector that does not produce infectious hPIV2 by use of rhPIV2 deficient in an M gene, F gene or HM gene although infection and expression of protein can equivalently be carried out, following an adenovirus vector from which composite protein genes of viruses have been deleted and to which the genes have been supplied from cells, plasmid or the like thereby to supply a safer nongrowing virus vector. Thus, genes encoding α antigen are integrated into hPIV2 having deletion of the M gene, F gene or HM gene in the present invention. The α antigen or the like may be inserted into any location of rhPIV2 genome. Since an expression level of foreign protein is increased, it is preferable to insert the α antigen or the like into 5′ distal end side of an antisense virus genome. The antisense virus genome is a single-stranded RNA and has a structure of leader (promoter sequence), NP, V/P, M, F, HN, L and trailer from the 5′ end to 3′ end. Accordingly, it is preferable that the gene encoding α antigen or the like is located shortly in the rear of the leader (promoter sequence) or in the vicinity of 5′ end side between NP V/P or the like.
In order that M gene that is a matrix protein and F gene composing a membrane protein or HN gene may be knocked out, exemplified are a method of removing all or part of gene encoding M protein, F protein or HN protein, a method of inserting a stop codon into any location of gene encoding M protein, F protein or the HN protein, and the like.
The replication-deficient PIV2 which does not carry out multiple-stage growth in cells and tissues which do not express the genes can be prepared by a method of transiently introducing plasmid expressing M protein, F protein or HN protein thereby to express the gene or by developing cells expressing M gene, F gene or HN gene thereby to proliferate PIV2 which is deficient in M gene, F gene or HN gene on the cells. Viruses that are completely absent of M gene, F gene or HN gene such that the viruses cannot autonomically replicate are called ΔM/rhPIV2 or rhPIV2(ΔM), ΔF/rhPIV2 or rhPIV2(ΔF) or ΔHN/rhPIV2 or rhPIV2(ΔHN) respectively.
In the present invention, an α antigen (Ag85B) is one of proteins that are common in acid-fast bacilli and is identified as antigen 85 complex constituting protein 85B that is one of antigen 85 complexes. The antigen 85 complex includes antigen 85 complex constituting protein 85A having molecular weight of about 30 to 32 kD (Ag85A: Infect. Immun. 57: 3123-3130, 1989), antigen 85 complex constituting protein 85B having molecular weight of about 30 to 32 kD (Infect. Immun. 58(2). 550-556 (1990). Accession No. X53897), and antigen 85 complex constituting protein 85C (Ag85C: Infect. Immun. 59: 5205-3212, 1991). These proteins are major secreted proteins of acid-fast bacilli. These secreted proteins are known to have a gene-amino acid sequence with a high homology transcending species and cross reactivity with monoclonal antibody (Microbiol. Rev. 56: 648-661, 1992). An analog of the α antigen (antigen 85 complex constituting protein 85B: Ag85B) includes antigen 85 complex constituting protein 85A (Ag85A), antigen 85 complex constituting protein 85C (Ag85C).
In the present invention, the gene encoding α antigen derived from acid-fast bacilli or the analog thereof indicates a gene capable of expressing α antigen protein or analog thereof such as the antigen 85 complex constituting protein 85A or antigen 85 complex constituting protein 85C. More specifically, a gene is exemplified that is in the form of an expression vector and includes a gene encoding an α antigen or analog thereof. As a gene encoding the α antigen are exemplified Mycobacterium kansasii (Infect. Immun. 58: 550-556, 1990), Mycobacterium bovis BCG (J. Bacteriol. 170:3847-3854, 1988), Mycobacterium avium (Infect. Immun. 61: 1173-1179, 1993), Mycobacterium intracellulare (Biochem. Biophys. Res. Commun. 196: 1466-1473, 1993), Mycobacterium leprae (Mol. Microbiol. 6: 153-163. 1992) and the like. All of these genes can be used in the present invention. Of these, as genes encoding an α antigen of Mycobacterium kansasii is exemplified a DNA having the base sequence from positions 390 to 1244 of SEQ ID NO: 1 and as genes encoding an α antigen of Mycobacterium bovis BCG is exemplified a DNA having the base sequence from positions 121 to 975 of SEQ ID NO: 3. Other DNAs include a mutant DNA that hybridizes to a DNA with a sequence complementary to this DNA under a stringent condition and a mutant DNA comprising a DNA encoding a protein having an amino acid sequence in which one or more than one (preferably, several) amino acids have been substituted, deleted and/or added to the amino acid sequence of the protein encoded by this DNA, wherein the mutant encodes a protein having the same function as the Mycobacterium kansasii-derived or Mycobacterium bovis BCG-derived α antigen. The aforementioned same function as the Mycobacterium kansasii-derived or Mycobacterium bovis BCG-derived α antigen indicates achieving the same preventive efficacy against the infection of Mycobacterium kansasii.
The α antigen of Mycobacterium bovis BCG comprises a mature protein of 285 amino acids, and this amino-acid sequence has a higher amino-acid homology (100%) than the α antigen of Mycobacterium kansasii due to Mycobacterium tuberculosis. It is considered that a higher protective efficacy of tuberculosis incidence will be achieved by introduction of the α antigen.
A specific method of obtaining a mutant DNA includes the following one. Thus, a colony hybridization in the presence of 50% formamide, 4×Denhardt, 5×SSPE (SSPE solution: EDTA sodium phosphate (SSPE), 1×Denhardt: 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% bovine serum albumin), 0.2% SDS, 100 μg/mL ssDNA, and 12.5 ng of a probe (12.5 ng of a purified cDNA fragment having a base sequence from positions 390 to 1244 of SEQ ID NO: 1 or a base sequence from positions 121 to 975 of SEQ ID NO: 3 labeled with [α-32P] dCTP (Amersham) using the BcaBest DNA labeling kit (Takara) at 45° C. for 14-16 hours, the filter is washed in 1×SSPE and 0.5% SDS solution at 45° C./30 min, then in 0.1×SSPE and 0.5% SDS solution at 65° C./1 hour to eliminate the background completely, which is then exposed to an X-ray film (Fuji) at −80° C. for 72 hours to detect the positions of the corresponding colonies and to isolate the single colony, thus the mutant DNA can be obtained. These mutants are those for which amino acid sequences encoded thereby normally have a homology of 60% or greater with the amino acid sequence of the α antigen protein, and preferably a homology of 75% or greater. In the case of genes encoding the α antigen other than Mycobacterium kansasii or Mycobacterium bovis BCG, they may be mutants thereof as well.
As genes encoding the antigen 85 complex-forming protein 85A which is an analog of the α antigen, there can be mentioned genes derived from the Mycobacteria similar to various Mycobacteria described above for the α antigen gene. More specifically, there can be mentioned a DNA encoding the antigen 85 complex-forming protein 85A derived from Mycobacterium tuberculosis (Infect. Immun. 57: 3212-3130, 1989). For genes encoding the antigen 85 complex-forming protein 85C as well, there can be mentioned a gene derived from various Mycobacteria, and more specifically, there can be mentioned a DNA encoding the antigen 85 complex-forming protein 85C derived from Mycobacterium tuberculosis (Infect. Immun. 59: 3205-3212, 1991). For these DNAs as well, as described antibody, they may be a mutant DNA that hybridizes to this DNA under a stringent condition, or a mutant DNA comprising a DNA encoding a protein having an amino acid sequence in which one or more than one (preferably, several) amino acid residues have been substituted, deleted and/or added to the amino acid sequence of the protein encoded by this DNA, wherein said mutant encodes a protein having the same function as the antigen 85 complex-forming protein 85A or the antigen 85 complex-forming protein 85C.
The above DNAs can be cloned by a method such as RT-PCR reaction on mRNA derived from Mycobacteria using as PCR primers appropriate DNA segments based on the sequence information of Genebank, etc. Chemical synthesis is also possible based on the amino acid sequence information. Furthermore, the above DNA mutants may easily be obtained using, for example, site-directed mutagenesis, PCR method, a common hybridization method, or the like.
In the present invention, the α antigen protein derived from Mycobacteria or derivatives thereof such as antigen 85 complex-forming protein 85A or antigen 85 complex-forming protein 85C or mutant proteins thereof can be used for the prevention of tuberculosis. As such α antigens, there can be mentioned proteins encoded by genes encoding the above-mentioned α antigen. More specifically, there can be mentioned an α antigen which is encoded by the DNA having the base sequence derived from Mycobacterium kansasii described in SEQ ID NO: 1 and which has the amino acid sequence of SEQ ID NO: 2 or an α antigen which is encoded by the DNA having the base sequence derived from Mycobacterium bovis BCG described in SEQ ID NO: 3 and which has the amino acid sequence of SEQ ID NO: 4. The latter base sequence and amino acid sequence can be obtained by visiting this URL (http://www.ncbi.nlm.nih.gov/nuccore/X62397?ordinalpos=1& itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum).
In addition to the α antigen having this amino acid sequence, a mutant protein comprising an amino acid sequence in which one or more than one (preferably, several) amino acid residues have been substituted, deleted and/or added to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, said protein having the same function as the α antigen may be used. In the case of genes encoding the α antigen other than Mycobacterium kansasii or Mycobacterium bovis BCG, a mutant protein comprising an amino acid sequence in which one or more than one (preferably, several) amino acid residues have been substituted, deleted and/or added to the amino acid sequence and which has the same function as the α antigen as well can be used. Furthermore, for the antigen 85 complex-forming protein 85A or the antigen 85 complex-forming protein 85C as well, the antigen 85 complex-forming protein 85A derived from Mycobacterium tuberculosis (Infect. Immun. 57: 3123-3130, 1989), the antigen 85 complex-forming protein 85C derived from Mycobacterium tuberculosis (Infect. Immun. 59: 3205-3212, 1991) and the like can be used. Other the analogs of these α antigen proteins, mutants similar to those of the α antigen protein mentioned above can be used.
These proteins may be produced by a recombinant DNA technology using genes encoding them or by chemical synthesis. Alternatively, Mycobacteria such as Mycobacterium kansasii, Mycobacterium bovis BCG may be cultured in a suitable medium, and the proteins may be purified from the culture medium by a known purification method (Scand. J. Immunol. 43: 202-209, 1996; J. Bacterial. 170: 3847-3854, 1988; Hiroshima J. Med. Sci. 32: 1-8, 1983; J. Bacterial. 170: 3847-3854, 1988).
In the present invention, when the gene encoding Mycobacterium-derived α antigen, analogs thereof or mutants thereof are used as tuberculosis vaccine, they are used in the form of hPIV2 containing the gene encoding Mycobacterium-derived α antigen, analogs thereof or mutants thereof. An hPIV2 genome deficient in M, F or HN gene can be used as the hPIV2. An rhPIV2 vector expressing α antigen etc. is constructed by integrating a gene encoding the α antigen etc. into the M, F or HN gene-deleted hPIV2(rhPIV2) genome.
This expression vector can generally be administered as an aerosolized agent to mammals including human. The aerosolized agent can be prepared by conventional methods. For example, after having been dissolved into a suitable solvent (a buffer solution such as PBS, normal saline, stabilizing agent or the like), the expression vector is filter-sterilized, if necessary and is then caused to fill an aseptic container, thereby being prepared as the aerosolized agent. A commonly used carrier or the like may be added to the aerosolized agent, if necessary. In order to use the expression vectors thus obtained as the tuberculosis vaccine, the aerosolized agent can be administered under normal use of the aerosolized agent.
A dose of tuberculosis vaccine can vary depending upon patient's weight or the like. As a vaccine, about 1×103 to 1×1010 viruses, preferably about 1×104 to 1×109 viruses are normally used as the expression vector per dose. The expression vector is preferably administered twice to five times at intervals of several days to several weeks.
The present invention will be described concretely by way of examples as follows. However, the present invention should not be limited to the examples.
Next, the following will describe a method of recovering virus particles from the plasmid including the antisense rhPIV2 genome cDNA inserted downstream of T7 promoter.
When the transfected cells were co-cultured with Vero cells 5 to 7 times at intervals of 48 hours, a cytopathic effect (CPE) was confirmed with 90% efficiency. In this case, a large amount of recombinant virus particles were obtained in use of hPIV2. However, virtually no recombinant virus particles were detected in use of rhPIV2 (Δ289) and rhPIV2 (Δ119). This shows that hPIV2 did not bud when M protein was deficient.
Instead of Vero cells, co-cultivation was then carried out by the use of Cos cells expressing hPIV2-M (Cos cells to which hPIV2-M that was a vector expressing M protein was transfected). As a result, a large amount of recombinant virus particles were detected in use of rhPIV2 (Δ289) and rhPIV2 (Δ119).
As the method of preparing rhPIV2 using the T7 promoter has been described above, rhPIV2 prepared using any vector (phage vector, plasmid vector, etc.) may be used.
As shown in
Regarding construction of M gene deletion genome, ΔM/rhPIV2 which was completely deficient in M gene coding region including R1 and R2 regions of M gene was prepared. Primers were synthesized which connected M-gene-deficient upstream and downstream sequences. Multiple PCRs were carried out to construct ΔM/rhPIV2 from which the M gene coding region had been deleted completely. The primer sequence included 5′-tgaaggagat cattgactc ttaaagggac ttg-3′ (SEQ ID NO: 5) as a sequence including the restriction enzyme site, 5′-tggaacgttt agttttttta ttaaatttat catgtccagc ct-3′ (SEQ ID NO: 6) as an M gene upstream region sequence, 5′-ttaataaaaa aactaaacgt tccacaataa atcaacgttc ag-3′ (SEQ ID NO: 7) as an M gene downstream region sequence and 5′-ggattcttaa agcttggatg gaga-3′ (SEQ ID NO: 8). The total number of bases was adjusted so as to be multiples of 6.
Regarding construction of F gene deletion genome, ΔF/rhPIV2 was made from which an F gene coding region 1,884 bp including R1 and R2 regions completely deficient in F gene. Regarding the F gene-deleted genome, Bc11 restriction enzyme sequence is located in F structural gene region and Kpn1 restriction enzyme sequence is located in the HN gene. Complete deficiency of the F gene was carried out using the restriction enzyme sequence. Primers were constructed using sequences of 5′-actgatcaat ctaacaaaaa aactaaacgt tctaagcacg aacccttaag gtgtcgtaac gtctcgt-3′ (SEQ ID NO: 9) and 5′-acttgatagg acggtaccca ttgagcctca atg-3′ (SEQ ID NO: 10).
Regarding the system of transiently expressing and recovering ΔM/rhPIV2 or ΔF/rhPIV2 virus into which α antigen gene for tuberculosis vaccine was introduced and which was completely deficient in M or F gene, the virus was recovered by a method similar to the rhPIV2 (M-stop) virus recovery method or by another method.
When M protein or F protein of hPIV2 is constantly expressed in cells, it is considered that the protein has a cell toxicity. Accordingly, we constructed (1) the system which constantly expressed the protein, and (2) the system which inductively expressed the protein.
The construction was carried out using a vector which held a neomycin-resistant gene or a puromycin-resistant gene and into which an M or F gene sequence was introduced into the vector holding chicken β-actin promoter sequence and rabbit β-globin poly A sequence (CAG promoter).
The construction was carried out by removing stuffer sequences located in two loxP sequences respectively by an inducible expression system Cre recombinase, using an expression inducing system (pCALN vector). A foreign gene was insertable into an Swa1 restriction enzyme site of this vector. The blunt end by restriction enzyme Swa1 reaction was used to construct an expression vector. In the case of M gene, primers for PCR in which the PCR products by using the primers set contained M gene and Pme1 enzyme sites both 5′ and 3′ ends to make blunt ends by restriction enzyme Pme1 were used, whereby the M gene inducible expression vector was constructed.
Regarding an F gene inducible expression system, primers for PCR in which the PCR products by using the primers set contained F gene and Swa1 enzyme sites both 5′ and 3′ ends to make blunt ends by restriction enzyme Swa1 were used, whereby the F gene inducible expression vector was constructed. PCR primer sequences for the F gene recovery were 5′-aaatttaaat atattcagcc atgcatcacc tg-3′ (SEQ ID NO: 11) and 5′-aaatttaaat cttgtgatag atttcttaag atatcc-3′ (SEQ ID NO: 12).
The vectors were transfected into Vero cells using Lipofectamine 2000 (invitrogen) or the like and the cells were cultured with neomycin (1 mg/ml). Screening was carried out by the drug resistance against neomycin. Vero cells which were capable of inducible M gene expression were recovered. The cells were infected with an adenoviruses expressing Cre protein so that M proteins were expressed. The cells were infected with the M gene-deleted virus recovered in the previous embodiment so that non-proliferated viruses were recovered. Consequently, M-gene-deficient non-proliferated viruses were recovered.
F protein inducible expression cells or F protein regular expression cells were established in a manner similar to M protein expression cells or in another manner. Consequently, F protein inducible expression cells and F protein regular expression cells were obtained. F gene deleted replication-deficient viruses were recovered (
A signal sequence of TPA was added to 5′ of α antigen genes (αK) of Mycobacterium kansasii comprising the sequence from positions 390 to 1244 of SEQ ID NO: 1, and R1 of hPIV2, an intervening sequence and R2 were added to 3′ and inserted into a Not I site of rhPIV2 so that rhPIV2 into which α antigen genes were integrated (hereinafter, “rhPIV2-Ag85B” or “rhPIV2/Ag85B”) was constructed (see
A large amount of rhPIV2-Ag85B and rhPIV2-GFP were prepared by the method of embodiment 2.
Subsequently, regarding genes of α antigen gene (αK) BCG-Ag85B of Mycobacterium bovis BCG comprising the sequence from positions 121 to 975 of SEQ ID NO: 3, DNA having the sequence from positions 121 to 975 of SEQ ID NO: 3 was added to a start codon (ATG) and inserted into the Not I site of rhPIV2 so that rhPIV2 into which BCG-Ag85B was integrated (hereinafter, “ΔM/rhPIV2-Ag85B (BCG)” or “ΔF/rhPIV2-Ag85B (BCG)”) was constructed (see
More specifically, regarding Ag85B of BCG, a signal sequence region (1 to 40) in an amino acid sequence of SEQ ID NO: 4 was deleted and the sequence from positions 121 to 975 (ttctccc . . . ) which encodes the amino acids from positions 41 to 325 (FSR . . . ) was integrated.
When rhPIV2-GFP was intranasally administered to hamsters (IN: 5×105 viruses), fluorescence of GFP was detected in epithelial cells of trachea and lung as shown in
30 BALE/C mice were divided into three groups A, B and C with 10 mice per group, and the following treatments were conducted on the respective groups:
The rhPIV2-GFP was intranasally administered four times at the intervals of two weeks. An applied dose of rhPIV2-GFP by intranasal administration was 1×107 viruses/20 μL per administration.
An expression-type Ag85B-DNA was intranasally administered to an abdominal cavity twice at the intervals of two weeks, and rhPIV2-Ag85B (M-stop) was intranasally administered twice at the intervals of two weeks. An expression-type Ag85B-DNA by intranasal administration was 50 μg per mouse. Furthermore, an applied dose of rhPIV2-Ag85B (M-stop) by intranasal administration was 1×107 viruses/20 μL per administration.
The rhPIV2-Ag85B (M-stop) was intranasally administered four times at the intervals of two weeks, and an applied dose of rhPIV2-Ag85B (M-stop) by intranasal administration was 1×107 viruses/20 μL per administration.
In each group, highly-virulent tubercle bacillus (Mycobacterium tuberculosis Kurono strain) was intranasally infected one week after the last immunity. Lungs and spleens were removed 49 days after the start of experiment so that tubercle, a microscopic appearance of lesion cutout and the number of tubercle bacilli were confirmed.
The results were shown in
The tubercle bacillus is one of intracellular eubacteria that grow in phagocytes such as macrophage. Cell-mediated immunity (Th1) that induces activated macrophage and cytotoxic T cell (CTL) becomes central to the prevention of infection of tubercle bacillus but not humoral immunity which consists mainly of antibody. Ag85B is a single protein that presents bioactivity of Mycobacterium inducing Th1-type immune reaction without depending upon genetic background and promotes production of interferon γ (INF-γ) inducing activated macrophages important for tuberculosis prevention and differentiation of T cells into CTL.
The vector (rhPIV2-Ag85B) into which foreign genes were introduced as a secretion type was prepared for use as a tuberculosis vaccine, and expression of Ag85B was confirmed. Consequently, as shown in
When F expression cells were infected with GFP-PIV2(ΔF), expression and recovery of GFP-PIV2(ΔF) were found to be executable since increasing GFP fluorescence was recognized, as shown in
Furthermore, when the mice to which rhPIV2(M-stop), rhPIV2(ΔF) and rhPIV2(ΔM) had been administered were infected with Ag85B or BCG-Ag85B, lesion (tubercle) peculiar to lung tuberculosis due to highly-virulent tubercle bacillus (Mycobacterium tuberculosis Kurono strain) in the lungs was found to decrease significantly. Of these,
The hPIV2 prototype vector is specifically infected with an airway mucosa and has a negative nonsegmented single-stranded RNA as a genome. The RNA genome comprises hexanucleotide surrounded by a single nucleocapsid protein to be constructed into a helical ribonucleocapsidprotein (RNP). Since the hPIV2 prototype vector has the nonsegmented RNA as the genome, the hPIV2 prototype vector does not cause any intersegmental variation (discontinuous variation). Furthermore, the effects of transcription and replication are dramatically reduced when the nucleotide number of genomes is not any multiple of 6, whereupon gene mutation due to homologous recombination is highly unlikely. A risk such as accidental homologous recombination is unavoidable, since the above-mentioned MVA85A (vaccinia Ankara) and Aeras402 (adenovirus type 35) both of which are currently in the clinical test phase originate from DNA viruses respectively. In the embodiment, however, the hPIV2 vector originates from the RNA virus and the life cycle is completed within cell cytoplasm. Accordingly, differing from the above-mentioned two DNA vectors, the hPIV2 vector can prevent genome from being integrated into chromosome in the nucleus.
The vector from which expression of M, F and HN genes is eliminated by stop codon or genetic deletion is an RNP machine that has no risk of production of secondary infection particle and can intranasally introduce genes specifically into airway mucosa. The vector has a lower original pathogenicity than the above-mentioned DNA vectors, and it has not been reported that the vector is pathogenic to humans (adults). Thus, the vector has an exceeding safety. Furthermore, the vector is designed so that expression of foreign genes from the genome becomes maximum by maximizing the characteristic of transcription from the hPIV2 genome. Furthermore, the M, F or HN gene is considered to suppress transcription from the genome. Accordingly, the protein expression level of the vector from which products of M, F and HN genes have been deleted is exceedingly high. A high intensity of expression of GFP was confirmed when the rhPIV2 (M stop)-GFP into which GFP (green fluorescent protein) had been inserted was intranasally administered to hamsters (
Importance is given to induction of local immunity (cell mediated immunity) in the lung that is a disease expression site than to general immunity in tuberculosis prevention. In the embodiment, the nongrowing rhPIV2-Ag85B vector or rhPIV2-BCG-Ag85B vector into each of which cell-mediated immunity induction protein had been inserted was used as a vaccine. Since this vector is safe and can realize an intense expression of the cell-mediated immunity induction protein to the lung, the vector was considered to have an exceeding efficacy as an adult tuberculosis vaccine vector.
This vaccine vector was developed as a safe half-live attenuated intranasal spray-type tuberculosis vaccine. The α antigen (Ag85B) derived from atypical mycobacterium (Mycobacterium kansasii and Mycobacterium bovis BCG) encoded as a foreign antigen has a cell-mediated immunity inducing function. A high cell-mediated immunity (Th1) against tubercle bacilli was considered to be induced by highly expressing Ag85B in a lung that was a disease expression section. Actually, when hamsters were nasally infected with an rhPIV2 vector, intense expression of GFP which was a marker gene was found in airway epithelial cell and even in terminal bronchial (
The test for protecting against infection with use of mice (the tuberculosis prevention test) confirmed that rhPIV2-Ag85 had an intense tuberculosis preventing efficacy (
Furthermore, the similar preventive efficacy to rhPIV2-Ag85B was achieved from hgPIV2-Ag85B to which BCG-Ag85B was introduced instead of Ag85B.
Furthermore, there is a high possibility that each of Ag85A and Ag85c achieves the similar efficacy from the test similar to the foregoing test.
According to the above-described embodiment, it is clear that a noticeable efficacy in prevention of tuberculosis can be achieved by administering the expression vector in which the gene encoding the α antigen or the like has been integrated in the paramyxovirus (particularly, rhPIV2).
| Number | Date | Country | Kind |
|---|---|---|---|
| 2009 252218 | Nov 2009 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2010/069435 | 11/1/2010 | WO | 00 | 4/17/2012 |
