JPH06509228A - improved vaccines - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] improved vaccines Technical field The present invention relates to improved vaccines, and specifically to rabies virus, bovine hyperthermia virus, and virus (i.e. BEFV) and varicella stomatitis virus (i.e. VSV). rhabdoviruses, including human measles virus and mumps virus, Human and bovine respiratory syncytial virus, bovine rinderpest virus, canine distemper virus in dogs virus, Newcastle disease virus of chickens and various rose influenza viruses Works to treat infections caused by rosemyxoviruses, including Regarding Kuching.

Background technology Rabies is a central nervous system disease of great importance for human and livestock medicine. . The disease causes fatal brain meningitis with no cure once its symptoms appear. wake up Vaccination before or after exposure to the virus can help fight this infection. This is the only way to Various vaccines approved for use in humans, animals and livestock All of them are manufactured from killed viruses. Until now Colo subunit vaccines are not used commercially.

The causative agent of rabies is Lyssavirus, which belongs to the rhabdovirus genus. Ru. Rabies virus particles are approximately 12,000 molecules surrounded by a protective sheath of N protein. Ribonucleoproteins (RNPs) consisting of leotide-length negative-strand (-) RNA molecules Contains. This RNP is bound to two other proteins (L and Ml). and forms a transcription complex with this structure. This transcription complex is a transmembrane glycoprotein (G) and an internal matrix protein (M2) Surrounded by a lipid bilayer membrane. G protein is found on the surface of virus particles It can form spikes. Although the number and function of proteins can vary, other Rhabdoviruses and baramyxoviruses also have similar structures.

The above overview regarding rabies can be found in Brehoud et al. (1989), VirologY 1. 73:390-399 and Brehoud et al. (1990), Virol, ogy 1 78, 486-497. Some other rhabdoviruses and roses A summary of the structure of kumisoviruses can be found in rThe Rhabdoviruses J ( (1987) (E. R. Wagner, Blenheim Press, New York), Emerging (1985) rVirologyJ (edited by BN Field, (pp. 1157-1178), Bunner et al. (1985) rImmunochem istry of VirusesJ (Volume 1, MH Buisha Fan Fist Edited by Genmotel and A.R. Neurath, Elsepier Press, Ams. Terdam, pp. 367-388) and Ober and Norby (1985) rImmunochesistry of VirusesJ Edited by Bui van Regenmotel and A.R. Neuras, 241-2 (page 64).

Prehoud et al. (1989) solved the problems associated with vaccination against rabies virus. It is clear that this is far from a decision. Vaccines are related to vaccine quality and effectiveness. rabies diagnosis, epidemiology and surveillance have improved. Although advances have also been made in this disease, the disease remains a threat to human and animal populations. continues to be powerful.

Types of vaccines already developed or considered candidates include: Includes:

(i) Inactivated rabies virus preparations These vaccines are currently used in humans and livestock. It is used. These are safe unless the virus is incompletely inactivated. used for. The main problem with these vaccines is their production cost.

(II) Attenuated live vaccine These vaccines have been used for oral vaccination of wild animals. such purpose For example, a candidate vaccine may be useful for target species as well as for non-target animals that can occasionally be infected. A prerequisite for this is that it is disease-free. Candidate vaccines also provide immunity. It also needs to be genic and genetically stable. In recent years, several New attenuated virus strains with mutations are being produced.

(tti) recombinant poxvirus These include recombinant poultry poxvirus and recombinant vaccinia virus. . Both have been shown to be effective vaccines. However, the living group Whether the release of modified poxviruses into the environment presents other risks to wildlife. I don't know.

1) Subunit protein or subunit structure Virus subunit protein Vaccines derived from protein or subunit structures have been proposed. death However, the cost of these vaccines is high. Several subunit vaccines have been proposed. These include Penmansor (1985), Ann, In5t, P. a5teur Virol, G- described in 136E2 167-173 M2 complex and Moraine et al. (1984), Nature (Lond, ) 30 8:457-460. . Liposomal vaccines are described by Belin et al. (1985) Dev, 5tand, Bio 160:483-491, and is a ribonucleoprotein-based protein. Cutting is based on Dietskjold et al. (1983) Proc, Natl, Acac. l, Sci, IJs^80 Near 0-74 and Tsu et al. (1991) Proc. Natl, @Acad, Sci, US A 88:2001-2005. G-ta expressed in insect cells protein has been tested in mice as a vaccine (Brehoud et al. (1989) ,the above〕.

Regarding VSV, this virus contains five structural proteins, Each of these proteins plays a role in VSV replication, assembly, and germination. It is known that it does its job. These include those mentioned above for rabies virus. A large polypeptide that forms an RNP transcription complex similar to that of the viral RNA genome. merase protein (L), nucleocabund protein (N) and linta Contains protein (NS). Also, the spikes on the virus envelope It also includes glycoproteins (G) that form and interact with receptors on sensitive cells. . A matrix protein that is thought to be similar to the M2 protein of the rabies virus. Proteins (M) are also present, which are assembled on the inner surface of the cell plasma membrane and form particulates. It is thought to enable binding with G proteins and RNP complexes during morphogenesis. ing. A summary of the structure and morphogenesis of VSV is given by Butniak and Belz (1 991) Proc, Natl, Acad, Sci, IjS^88:13 79-1383 and rThe Rhabdoviruses J (1987X Edited by R.R. Wagner, Blenheim Press, New York). Ru.

v SV infects not only humans (horses, cows, and pigs are also infected, and is different from conventional rabies vaccines). As mentioned above, past vaccines used inactivated or killed viruses. virus or a weakened virus. Conversion from asymptomatic type to toxic type The same problem applies to the VSv vaccine. to G protein Immunity could be achieved by subunit vaccines based on Unit vaccines have not been well studied (Cox et al. (1977) Inf Ection and Immunity 1' 6:754-759; Francoi (1984) J, Virol, 51:208). N protein and Vaccines based on combinations of G proteins were also developed by Dietznjord et al. (1983). X above).

Regarding BEFV, this virus causes acute infections in cattle and buffalo. the current Vaccines produced to date include live, attenuated viruses or inactivated whole viruses. is included. Such vaccines have not proven to be commercially successful to date. The risk of incomplete inactivation and reversal to the toxic form described above is unknown. are also exposed. Australian Patent Specification 61356/90 and Walker et al. (1991) J, Gen Virol, 72:67-74. BEFV contains envelope glycoprotein (G), nucleoprotein (N), and matrix. protein (M2), polymerase-related protein (M+) and large polymerase -se protein (L). Australian patent specification 61356/90 is describes the use of subunit vaccines based on G proteins. but Such vaccines are not commercially produced.

Both baramyxovirus and rhabdovirus are ordered mononegavirales (O Virus families (respectively (Paramyxoviridae and Rhabdov) Iridae (Rhabdoviridae). these viruses Families share broadly similar structures, genome organization, and methods of gene expression and replication. have. Both families of viruses contain 3′ proteins involved in nucleation of particle assembly. and a single-stranded (−) sense RNA genome incorporating the 5′-terminal domain and the nuclear protein proteins, matrix proteins, polymerase proteins, glycoproteins and and at least five encoded RNA-dependent RNA polymerases. Contains viral genes. These corresponding proteins are found in viruses of both families. They play the same universal role in replication. Structure of paramyxovirus particles binds to matrix proteins and germinates through the cell plasma membrane. Therefore, glycoproteins are released in a process similar to that described above for rabies virus. Includes an RNP complex that incorporates. Paramyxovirus, measles virus, epidemic parotitis virus, parainfluenza virus, respiratory syncytial virus, chicken Newcastle disease virus, rinderpest virus and canine cyst virus Contains important human and veterinary pathogens, including: Various anti-paramyxoviruses inactivated vaccines, live attenuated vaccines, recombinant vaccines and subunit vaccines. Protein vaccines have been described and these are similar to rhabdovirus vaccines. has properties similar to those described above. Rhabdovirus and Nokuramictuvirus For a discussion of the similarities, see Pringel (1991) Arch, Virol, 1. 17:137-140, and describes the structure and paramyxovirus of paramyxoviruses. A summary of virus vaccines is given by Ray and Compans (1990) rIma+ Unochemistry of VirusesJ Volume ■ Edited by Van Regenmotel and A.R. Neuras, El Sepia Pres. 217-236).

Recently, synthetic viruses have been developed by expressing viral structural genes in cultured eukaryotic cells. It has been shown that it is possible to construct virus-like particles (VLPs). So Synthetic VLPs of poliovirus have been constructed using the technique described above. Urakawa et al. (1 989) J. Gen. Virol. 70:1453-1463. The complete polycistronic nature of poliovirus within the polyhetrin gene of the virus. It is reported that RNA was inserted.

Insect cells infected with this recombinant baculovirus contain poliovirus polyproteins. were synthesized and processed to produce large quantities of empty VLPs. These synthetic keys The capsids contained no RNA and were not infectious, but were otherwise similar to natural pores. It was similar to Riovirus. Using a similar method, bluetongue virus Wrench and Roy (1990) J, Virol, 64:1530-1536 ; French et al. (1990) J, Virol, 64:5695-5700) , hepatitis B virus (Takehara et al. (1988) J. Gen. Virol. 69:2763-2777) Oyohi bovine immunodeficiency virus (Rasmussen et al. 990) VirologY 178:435-451). Viral VLPs and (nucleoid particle) CLPs have been constructed. So far, this The particles formed by this method are generated by protein-protein interactions. 0 and did not contain viral nucleic acid (RNA or DNA) Using related techniques, cDNA clones representing whole genome samples of several viruses have been developed. Infectious virus has been recovered from the loan. This method allows eukaryotic cells to Infectious cDNA in a plasmid vector containing a suitable promoter for expression. A has been cloned. Infection of eukaryotic cells by such vectors is infectious leading to the production of viruses. Poliovirus (Lacaniero and Baltimo) A (1981) Science 214:916-919), Sindbas (Si ndbus) virus (Rice et al. (1987) J, Virol, 61:3 809), porcine vesicular disease virus (Inouni et al. (1990) J, Gen, Vir ol, 71:1835-1838) and Sparrow-spotted Mosaic Virus (Alquist et al. (1984) Proc.

Natl, ^cad, Sci, USA 81 Near 066-7070) This general method has been used for several human, plant and animal viruses. It is used. This method can act directly as metsenger RNA. Applies only to RNA viruses with positive sense (+) genomic RNA. child The method uses paramyxoviruses with negative (-) sense RNA genomes and labs. This does not apply to viruses.

Disclosure of invention The object of the present invention is to contain negative sense (-) RNA genomes and therefore infectious particles or cDNA clones representing only genome fragments or whole genome samples. Regarding paramyxoviruses and rhabdoviruses that are unable to create defective particles. The goal is to provide an effective and completely non-infectious vaccine. Also, these u Viruses require assembly elements located on RNA for particle formation; Furthermore, VLPs can be formed only by protein-protein interactions. I can't. The method of the invention therefore includes the following steps.

(i) Rhabdovirus or paramyxovirus genome fragments or modified Construct a DNA molecule that corresponds to the genome. The DNA molecule has a small 3° domain. It can consist of at least one oligozyme domain and optionally a 5° domain. , cohesive ends can be incorporated.

(ii) Place the DNA obtained in step (i) into the cloning site of a eukaryotic expression vector. and transfect eukaryotic cells with the vector containing the above genomic construct. At the same time, the M2 protein, M, protein, and N protein of the rabies virus are released. rhabdoviruses, including those with functions similar to protein and G protein; Vector containing cloned genes of paramyxovirus structural proteins transfect the same eukaryotic cells.

(i!1) M1 protein from cells transfected in step (if) and N protein sheath to form a ribonucleoprotein complex (i ) and the RNA genome transcribed from the DNA molecule constructed by M2 protein. The virus consists of an internal matrix and a lipid envelope containing G proteins. Soot-like particles (VLPs) are obtained.

(fv) The VLP obtained in step (tii) is included in a vaccine.

A general structure for a range of DNA constructs suitable for use in the present invention is illustrated in Figure 1. The general method for producing the desired VLPs is schematically summarized in Figure 2.

Regarding step (i) of the method of the invention, the 5° and 3′ domains are isolated from rhabdoviruses or or derived from sequences in the 5′ and 3′ non-coding regions of the paramyxovirus genome. can do. However, you can delete the 5' domain if necessary.

The insectozyme domain(s) can be derived from any of the known insectozyme structures. can be constructed, some of which have been described by Haserotsuf and Gerlach (1988). Nature 334:585-591. ribozyme domain (s) are transcribed in a eukaryotic cell in step (1i) of the method. The foreign parts of the RNA genome construct (such as vector sequences and poly(A) tails) The assembly of the particles is described in steps (quickly) after being cut at appropriate positions within the particle. ) would guarantee that it would be possible. Ribozyme domains assemble into VLPs It may be cleaved from the RNA transcript before being assembled, or it may be cut during the assembly process. may be included in the transcript provided that it does not prevent ribozyme dome Each of the ins is located outside the 3' and 5° domains! Can be interposed Creotide sequences may be inserted between the domains of the construct. Alternatively, ribo Zyme domain(s) as shown in Figure 1. (can also be placed inside the

Filler domains do not have characteristics that do not prevent VLP formation. Any nucleotide sequence may be constructed. Preferably filler domains , (-) rhabdovirus adjacent to the 5” non-coding region of the RNA genome or Consists of a fragment derived from a part of the L protein coding region of paramyxovirus will. The filler domain is the genome to be expressed at stage (ffi) It must be of sufficient size (approximately 1000 nucleotides or more) to enable the formation of LP. will guarantee that.

Specific examples of DNA constructs suitable for the formation of rabies VLPs are illustrated in Figures 3-8.

The exemplified DNA sequences are described as single-stranded molecules in the sense in which the constructs are transcribed. do. A double-stranded DNA molecule that may be required in some constructs is a second strand of anti-complementary sequence. will be incorporated.

Figures 3.4 and 5 show preferred embodiments containing two ribozyme domains (R1 and R2). Figure 2 illustrates the structural organization and sequence of the NA construct (TB-2). In this example are the 5′ and 3′ terminal regions of the genome of rabies virus (P■ and CVS strains). The 5° and 3′ domains are derived from known nucleotide sequences. R1 domain The protein was designed to target a site within the (-) RNA transcript of the TB-2 DNA construct. It is measured. The R1 ribozyme in the transcript cleaves the RNA and cuts the 5° The foreign portion of the transcript is removed so that the terminus corresponds to that of the rabies virus genome. will guarantee that it will be done. Similarly, the R2 ribozyme domain is TB-2 DNA The construct is designed to target a site within the (-) RNA transcript. R2 The ribozyme cuts the RNA so that the 3' end of the transcript is part of the rabies virus genome. Exogenization of the 3° region of the transcript (including the R2 domain) to approach that of the 3° end This will ensure that the part is removed. Filler domains within the TB-2 construct 1135 located at the 5′ end of the rabies virus (CVS strain) L protein gene. Derived from the known nucleotide sequence of a nucleotide region.

Figures 6.7 and 8 show a preferred embodiment incorporating one lipozyme domain (,R) The organization and sequence of the A construct (TB-1) is illustrated. In this example, the rabies virus (PV and 078 strains) in the corresponding 5' and 3' terminal regions of the genome. The 5' and 3' domains are derived from the cleotide sequence. R Lipozyme Domei The protein is designed to target a site within the (-) RNA transcript of the TB-I DNA construct. It is measured.

The R ribozyme cuts the RNA and the 3' end of the transcript becomes the rabies virus genome. The foreign part of the 3' region (including the R domain) of the transcript approaches the 3' end of the will ensure that it is removed. The filler domain in the TB-1 construct is 1167 nucleotide at the 5' end of canine disease virus (CVS strain) L protein gene Derived from the nucleotide sequence of the otide region.

Regarding step (il) of the method of the invention, the modified genome or genome fragment and the virus Any suitable vector may be used to express the rus structural proteins. This will be understood by those skilled in the art. This includes, for example, eukaryotic systems (e.g. boxwidth). mammalian cells using rus, papillomavirus or retroviral vectors; or in yeast cells). However, the preferred expression system is Spodobutella fu. What can be obtained from Spodoptera frugiperda Using baculovirus vectors to infect insect host cells such as It is.

Regarding the method of the present invention, by using insect cell culture and a baculovirus expression system, It is known that large amounts of protein can be easily produced at a relatively low cost. this This is described in Cameron et al. (1989) TIBTECH 7:66-70. . Baculovirus is a large NA virus that infects insects. infection cycle In the later stages of the virus, baculovirus expresses several proteins in extremely large amounts. child Genes that express these proteins (e.g. polyhetrin and plo) can be cultured. is not essential for baculovirus replication in It has been used as a dressing site. Such recombinant baculoviruses are foreign viruses. nuclear or viral proteins, often in a form that closely resembles the natural protein, Express to high levels. Many animal viral proteins are plo or polyhedo It is expressed in a baculovirus system under the control of the Lin promoter. virus An example of applying the baculovirus system to protein expression is Emery (1991) Re. Views in Medical Virology: Described in 11-17 It is. An example of using the baculovirus system to express rhabdovirus proteins is Bairei et al. (1989) VirologY 169:323-331, Brehau Do et al. (1989) virology 173:390-399 and Brehoud et al. 1990) Virology 178:486-497, and rose For myxovirus proteins, Van Wijk-Kohling et al. (1987 ) Virology 160:465-472 and Bialard et al. (1989) describe the expression of G proteins. rabies G protein The gene was cloned and the baculovirus Autographa californii Nuclear polyhedrosis of the mosquito (Autographa californica) in the baculovirus transfer vector pAcYMl derived from inserted into. Using this recombinant transfer vector and AcNPV DNA, subodopte Co-transfection of La frugiberda cells with high levels of rabies G-tan Recombinant baculovirus expressing protein was recovered from the cells.

The previously mentioned Prehoud et al. (199) is the nucleus of Autographa californica. It is derived from polyhedrosis virus and contains the complete N protein of rabies virus. Production of baculovirus expression vector (AcNPV3) containing the coding region It is described as follows. The rabies gene is controlled by the ACNPV polyhedrin promoter. Under your command! This was then transformed into Spodoptera by the induced recombinant baculovirus. was expressed at high levels in P. frugiberda cells. Baculovirus expression systems are, for example, Smith et al. (1985) Proc, Natl, Acad, Sci, USA 82:8404-8408, Miller et al. (1986) Genet Amame@eng ineering:Pr1nciples and Methods 8:27 7-298, Po'/ (1986) Virus Res, 5F43-59, Matsuura et al. (1987) J, Gen, Virol, 67 +1515-1 529, Luknow et al. (1988) Biotechnolog ■ 6:47-55, Kang (1988)^dv, Virus Res, 35: 177-192, Bishop and Posey (1990) Adv, Gene T echnol, 1 +55-72 and Miller et al. (1988) Ann, Re. v”1crobio1.42F177- 199 was also reported.

The nature of rhabdovirus and rosemyxovirus particles regarding the formation of synthetic VLPs. formation of virus requires viral structural proteins, but not complete genomic RNA. It is known that it does not. For example, animal or cell culture rhabdoviruses or Defective interference occurs in the process of Tahahara myxovirus infection. rferingXD 1) Particles are generally formed. DI particles are virus structural particles. Genomes that can contain all of the proteins but are deleted and therefore defective Contains. Defective genomes replicate in the absence of intact non-defective viruses with DI particles make something impossible. Aspects regarding the properties of DI particles are discussed in Haung and Baltimore. (1977) Comprehensive Virology 10 Near 3-1 16, Holland et al. (1980) Comprehensive Virolog Y 16:137-192, Bellault (1981) Curr op, njc robiol, Immunol, 93:151-207 and Holland ( 1985) rVirologyJ (edited by BN Field, Rapan Pu. (Res., New York, pp. 77-99). Also, Batniak and and Beltz (1991) Proc, Natl, Acad, Sci.

USA 88:1379-1383, all of the five ~・”SV structural proteins vaccinia virus vector to express all from the cloned cDNA, A. When cells are simultaneously infected with VSVDI particles and VSVDI particles, replication of VSVDT particles occurs. It is also known to obtain

Regarding steps (ili+) and (tv) of the method of the invention, rhabdovirus TB-2 and and TB-I DNA constructs and by using the methods described and exemplified. , synthetic rhabdovirus or baramyxovirus defective or infectious virus-like Particles (VLPs) could be produced within insect cells. produced in this way VLPs do not require helper viruses or DI particles and helper cells. Probably. After assembly and release from the cells, the VLPs can be purified using methods known in the art. It can be used as a suitable vaccine in combination with suitable adjuvants. So Adjuvants such as Quil A and other saponins, TSCOM, Freund's incomplete or complete adjuvant, e.g. Vanserow (1987) v et, Bull 57:881-896. be caught.

The use of VLPs as described above allows them to exist in a form that closely resembles the natural structure. Contains all of the important immunogenic proteins of the naturally occurring virus will be understood. Caused by rhabdovirus or baramyxovirus When used in vaccines to be administered to subjects susceptible to a disease or disease, VLPs In much the same way as the currently used vaccines that incorporate inactivated viruses. will cause immunity. However, rhabdovirus or baramyxovirus particles Unlike vaccines that incorporate children, there is a possibility that infectious viruses may be present or There is no possibility of reversal to toxicity. This is because the VLP of the present invention use only fragments of the virus and not the genes encoding complete viral proteins. This is because you can do a lot of things.

The principles and methods described for the construction of VLPs based on rabies virus can be applied to other Loose (-) sense non-dividing RNA viruses (specifically rhabdoviruses and rhabdoviruses) It will also be appreciated that it can also be applied to myxoviruses). Basically, the requested The VLPs contain the 3' terminal sequence of the rhabdovirus or baramyxovirus genome. The expressed RNA is cleaved to create the required 3° domain. at least one ribozyme domain to ensure that the expressed The size of subgenomic RN A that is sufficient to aggregate particle formation (approximately 1000 nuclei) Contains essential assembly elements including filler domains that ensure that It will contain a suitable modified genome or genome fragment. 3′ domain and complete The fully or incompletely base-paired 5' domain and the expressed RNA A second gluepozyme domain that ensures that it is cleaved to generate the required 5′ end. may be incorporated into this construct. Then for all VLP formation, eukaryotic Inside the cell, transcripts of the required DNA construct are obtained from homologous rhabdoviruses or rhabdoviruses. The required structural proteins of myxovirus will be expressed simultaneously.

Preferred embodiments of the invention The method of the invention is described in the following examples, which are intended to illustrate the invention. and is not intended to limit the scope of the invention.

Abbreviations and definitions , AcNPV: Autographa caliphus lunica nuclear polyhedrosis virus BE FV: Bovine hypersexual fever virus cDNA: complementary deoxyribonucleic acid CLP: Nucleoid particle C~rS: Challenge virus standard DI, defect interference DNA: deoxyribonucleic acid EDTA: ethylenediaminetetraacetic acid PAC; E-polyacrylamide gel electrolyte Aerophoresis PBS diphosphate buffered saline PCR: Polymerase chain reaction PFU: Plaque forming unit PMSF: Fluorinated phenylmethylsulfonyl poly(A) polyadenylic acid PV Pasteur virus SDS sodium nidodecyl sulfate Tris:) Lis(hydroxymethyl)aminomethane R: TB-I Ribozyme domain R1 of the DNA construct: Ribozyme 1 of the TB-2 DNA construct Domain R2: Ligzyme 2 domain RNA of TB-2 DNA construct: Liponucleic acid RNP: liponucleoprotein rpm/number of revolutions per minute RT: room temperature VSV: Varicella stomatitis virus VLP: virus-like particle w/w: weight percentage Materials and general experimental techniques Synthetic oligonucleotides (see Figure 9) are deprotected and purified products. Center for Molecular Biology and Biotechnology Supplied by Gee (Australia, Brisbane, St. Lucia).

All recombinant DNA cloning and analysis techniques require restriction enzyme digestion, sample preparation, including oxidation, ligation, transformation, DNA preparation and DNA electrophoresis. Tsuku et al. (1989) rMolecular Cloning: A Lab oratory Manual J (2nd edition, Cold Spring Harbor ・Laboratory Press, New York) and Purpal (1988) rA pr actual Guide to Mo1ecular CloningJ (Willey, New York).

Methods for nucleotide sequence analysis are described by Sanger et al. (1977) Proc, Natl. Acad, Sci, USA 74:5463-5467. Ru.

Protein analysis techniques including 5DS-PAGE and immunoplotting are widely used. Howd et al., 1989. Virology 173:390-399.199 0. Performed as described in Virology 178:486-497 .

Culture of Subodobutella frugiperda cells, construction of shuttle and transfer vectors. Baculovirus expression, including generation, selection and analysis of recombinant baculoviruses and recombinant baculoviruses Instructions for using the system are given in Prehoud et al., 1989. Virology 173:390 -399.1990. Described in Virology 178:486-497 I went as I said.

Examples of preferred embodiments Example 1: Construction of a plasmid vector containing the TB-2 DNA molecule Synthetic oligonucleotide primers and rabies genomic RNA templates were used in the following steps. TB-2 DNA is constructed by several consecutive PCRs.

(i) Rabies virus (CVS strain) genome, primer LL (Figure 9) and inversion A single-stranded cDNA copy of the necessary part of the rabies L protein gene using copy enzyme was prepared, then primers L1 and L2 (Figure 9) and polymerase chain reaction (P by amplifying a double-stranded DNA molecule of the desired nucleotide sequence using Thus, a plasmid vector containing a filler domain was obtained. Then the DNA Clone the molecule into the SmaI site of a suitable plasmid vector (e.g. pUC118). It has become an online version.

This recombinant plasmid containing filler domain was named pFILL. p The complete nucleotide sequence of the recombinant DNA insert within FILL was determined. It was found that this corresponds to that of the filler domain shown in FIG.

(if) Using pFILL obtained in step (i) as a template, primers TB5A and TB3 Filler domains were extended by PCR using A (Figure 9).

(ffi) by PCR using primers TB5B and TB3B (Figure 9). The PCR product obtained in step (it) was extended.

(Juice) by PCR using primers TB5C and TB3C (Figure 9). ffi) was extended.

(v) After BAh H1 digestion, the PCR product obtained in step (residue) is transferred to an appropriate plasmid. Vector (e.g. pBluescript KS+, Stocitagene. Jora). Plasmid vector containing the TB-2 construct It was named pTB2.

The complete nucleotide sequence of the recombinant insert in pTB2 was determined and is shown in Figure 5. It was found that it corresponds to that of the described TB-2 construct.

Example 2 Construction of a plasmid vector containing the TB-I DNA molecule The following steps TB-2' DNA construct to TB- I DNA was induced.

(i) Transducing the pTB-2 plasmid using primers TBI and TB3C (Figure 9). It was used as a template for CR.

(it) step by PCH using primers TB5C and TB3C (Figure 9). The PCR product obtained in (i) was extended.

(tit) After BamH1 digestion, the PCR product obtained in step (if) is added to an appropriate midvector (e.g. pBluescript KS+, Stratagene, USA It was cloned into U.Jora). Plasmid vector containing the TB-1 construct - was named pTBl. Complete nucleotide sequence of the recombinant insert in pTBl was determined and found that it corresponds to that of the TB-I DNA construct shown in Figure 8. has become clear.

Cut the plasmid vectors pTB2 and pTBl with Xbal or pGEM Expression Transcription Kit (Promega, Australia, NSW, Rose) ) and [”53UTP (Ama/Yani International Limited)] was used as a template for in vitro transcription with T3 RNA polymerase.

In vitro transcription was performed at 37°C or 28°C. 6% polyacrylamide-urea sequence The products were analyzed by electrophoresis in a determination gel. Plasmi with known sequence prepared in a standard dideoxynucleotide sequencing reaction and run on a molecular weight ladder. and then flowed into the adjacent lane. After electrophoresis, the gel is fixed, dried, and autotransferred. Visualized by geography. In vitro transcription at 37°C or 27°C to the product of cis-acting cleavage at the site targeted by the ribozyme domain. resulting in RNA transcripts of corresponding size. from plasmid pTB2 at 37°C. The results of the obtained transcripts are shown in FIG. at the 5° end of the transcript. The short product of ribozyme R1 cleavage extends from the T3 transcription start to the R1 cleavage site. Appears as a discrete band of 93 nucleotides (A) corresponding to the expected sequence. Ta. The short product of ribozyme R2 cleavage at the 3′ end of the transcript is When using a fragmented plasmid as a template, 66 nucleotides and 67 nucleotides appeared as a double band (B). This is a single base run-on (in vitro transcript (known to often occur at the 3' end) and from the R2 cleavage site to Xba1 Corresponded to the predicted sequence up to the site. As expected, the uncut plug When Sumid is used as a template, the expected product is large and transcription termination is Band B did not occur since it can be of various sizes depending on the nature. rabies The long cleavage products of R1 and R2 corresponding to the subgenomic RNA are located in band C. was identified. Similar results were obtained for TB-2 at 28°C, although the transfer efficiency was lower. The fruit was obtained. As expected, in vitro translocation using Xbal-cleaved pTB1 The photo shows only large products slightly smaller in size than Band B and Band C above. Brought.

These results indicate that both the 5' and 3' ribozyme domains are active and that the DN Efficiently cleave the transcript derived from the A construct to obtain the desired subgenomic RNA fragment. was shown to be generated. The truncated apozyme domain that occurred at 28°C was found in insects. The cells were shown to be active at ambient temperature.

It was extracted from recombinant plasmids pTB2 and pTBl by digestion with BamHl. TB-2 and TB-I DNA inserts were obtained, BamH1 digested, and dephosphorylated. Oxidized transfer vector pAcYM1 (NERCIVEM, Offford, UK) baculovirus (AcNPV) by subcloning into derivatives of baculovirus (AcNPV ) A transfer vector was constructed. Contains TB-2 and TB-I DNA inserts The transfer vectors were named pAcTB2 and p,A,cTB1, respectively. did.

Recombinant baculoviruses expressing TB-2 and TB-1 subgenomic RNAs To obtain recombinant transfer vector (pAcTB2 or pAcTBl) DNA ( 1t, tg) and baculovirus AcRP23-1 acZ virus DNA (10 Lipofunication of Spodobutella frugiperda cells with a mixture of 0 Tl g’) did. Kitts et al. screened recombinant baculoviruses as previously described. 　1990. Nucleic Ac1d Re5earch 18:5667 -5672 and Brehoud et al., 1992.　Virology, in press), A high titer (ie >107 PFU/ml) stock was prepared. TB-2 and Recombinant baculoviruses containing the TB-I DNA construct were transformed into Ac and TB, respectively. 2 and Ac, TBI.

Example 5. Mixed infection of Spodobutera frugiberda cells and purification of rabies VLPs ( f) Wild-type baculovirus (AcNPV), JJJ rabies subgenome Recombinant baculovirus expressing RNA (Ac, TB2 or Ac, TBl) , or (if　　Rabies N　9　:/ha’) Quality (AcNPV3), M1 Ta protein (AcNPVMl), M2 protein (ACNPvM2) and G protein A recombinant baculovirus (preliminary protein) expressing protein (AcNPV2) Howd et al., 1989. Virology 173:390-399.199 0. Vir.

1ogy 178, 486-497) or (if) rabies N/M1 protein (A, cNPVN/Ml) and M2/G protein (A, cNPVM2/G) or a recombinant dual-expressing baculovirus expressing (t v) Recombinant dual expression baculoviruses AcNPVN/Ml and AcNPVM2/ A recombinant baculovirus (Ac.G) expressing rabies subgenomic RNA as well as rabies subgenomic RNA.

Spodobutella frugiberda cells (5X Four identical cultures of 10' cells) were co-infected at a multiplicity of IPFU/cell.

The cultures were incubated at 38D for 3 days. Collect the culture supernatant from the culture. , centrifugation for 10 minutes at 400 rpm at 4°C in a JA20 rotor (Beckman). Purified by separation. 27000 rpm at 4°C in 5W280-tar (Beckman) Particles were pelleted from the supernatant for 1 h at m. The pellet was soaked in TD buffer (0 , 8mMh Li-HCl, 150m, M NaCL 5mM KCL 0.7m Resuspend in M Na2HPO4, 10mM EDTA, pH 7,4) and TD buffer. Centrifugation is performed in a two-step sucrose gradient of 10 to 40% (W/W) created in the solution. It was further purified by Collect the bands located at the interface of the gradient, 5W40 by centrifugation for 1 hour at 30,000 rpm at 4°C in a rotor (Beckman). The particles were pelleted. The pellets were resuspended in TD buffer and stored at -20°C. . As illustrated in Examples 7 and 8 below, this method can reduce rabies virus. A medium in which both protein and TB-2 or TB-1 subgenomic RNA are expressed. VLPs were purified only from nutrients.

Using the recombinant baculovirus and wild-type baculovirus described in Example 5 above, Spodobutella frugiperda cells (1,5X10 Four identical cultures of 6 cells) were co-infected. After 3 days of infection, remove the supernatant medium from the plate. the monolayer was rinsed three times with phosphate buffered saline (PBS) and RIPA buffer ( 1% Triton X-100, 1% sodium dodenyl sulfate, pH 7,4) 150μ Cells were lysed in I. A portion of the protein sample was added to dissociation buffer (2,3% SDS, 10% glycerol, 5% β-mercaptoethanol, 62.5mM hlysuM CI, 001% bromophenol blue, pH 6,8) for 10 minutes, Cell lysis preparations obtained as described in Example 6 above and carried out above. Gradient purified particle preparations obtained from culture supernatants as described in Example 5 were prepared using 5DS-PA Presence of rabies virus proteins by GE and immunoplotting analyzed.

Proteins separated by electrophoresis in 10% 5DS-polyacrylamide gels. The texture was electrically plotted on a nitrocellulose membrane. Plot the blocking solution (in PBS) 3% low-fat skim milk powder and 0.01% sodium azide) Mouse anti-CVS polyclonal antibody at a dilution of 11500 after incubation for and incubated overnight. After cleaning, Beloxida -se-conjugated anti-mouse IgG (Sigma Chemicals, St. Louis, MO, USA) ) was used to detect bound antibodies. 4-chloro-1-naphthol and 3,3゛ - Diaminobenzine tetrahydrochloride (Sigma Chemicals, St., Missouri, USA) Lewis) as a substrate for peroxidase, and the above plot was colored.

Analysis of cell lysate preparations by this technique can be performed in a single or multiplex containing the corresponding gene. is a lysate of a culture infected with a double recombinant baculovirus expression vector and a double recombinant baculovirus expression vector. Replacement baculovirus Ac NP VN/M 1 addressee, 4. AcNPVM2/ Cultures infected with recombinant baculovirus AcTB2 or AcTBl Rabies virus GSN, Ml and M2 proteins in the lysate obtained from resulted in the detection of wild-type baculovirus alternating g recombinant baculovirus Ac, Rabies virus protein was detected in cultures infected with TB2, Ac, and TBl. It wasn't served.

Gradient purified particle preparations analyzed using this technique showed that rabies virus N, M1 , M2 and G proteins are double recombinant baculoviruses AcNPVN/M1 and and AcNPVM2/G and recombinant baculovirus AcTB2 and was detected only in particles prepared from cultures infected with AcTBl. above fruit In the particle preparation obtained from the pond culture described in Example 5, there was no reaction with rabies antibodies. Not detected.

The results of these analyzes are summarized in Figure 11. These results indicate rabies N, Ml, M2. and G protein in a baculovirus expression vector containing the corresponding gene. It was shown to be synthesized in all infected cultures. However, rabies tongue The release of protein-containing particles stimulates TB-2 and TB-1 subgenomic RN A. This occurred only in cultures infected with expressed recombinant baculovirus. The following implementation These particles were also identified by electron microscopy as described in Example 8.

Example 8 Electron microscopy of rabies VLP preparations wild-type and recombinant Bacro The purified fraction obtained in Example 5 above derived from the culture mixedly infected with the virus was carbon-coated. Drop onto a covered grid, wash three times with TD buffer, and negative with 2% uracil acetate. stained. The sample was then examined using a Hitachi transmission electron microscope.

(D) A recombinant virus expressing rabies subgenomic RNA derived from wild-type baculovirus. Crovirus, or (ii) Recombinant Bakurou expressing rabies N, Ml, M2 and G proteins virus, or (iii) A group expressing rabies N/Ml protein and M2/G protein Recombinant dual expression baculovirus, In control preparations obtained from cultures co-infected with Only rod-shaped particles corresponding to the structure were observed. The particles have a relatively constant size ( 25Or+mx4Qnm) and shape (rod-like), and appears to be mostly intact. I was disappointed.

Recombinant baculoviruses expressing rabies N, Ml, M2 and G proteins and co-infection with a recombinant baculovirus expressing rabies subgenomic RNA. In the preparation obtained from the culture, two types of particles were observed (as illustrated in Figure 12). It was measured. One particle corresponds to the baculovirus particles observed in the control preparation. Ta. Another type of particle (never observed in the control preparation) is the diameter consists of an irregular structure with a diameter of approximately 70 to 85 nm. These particles often It shows a nucleus of child density and a brightly illuminated periphery, with numerous regular regularities throughout the periphery. New surface spikes or protrusions were protruding. These particles are rabies VLPs The electron-dense nucleus represents the ribonucleoprotein complex, and the brightly illuminated periphery The edges represent the lipid envelope of the virus, and the spikes represent the surface glycoproteins of the virus. Several types of rhabdovirus DI particles have been reported in the past to represent (rThe RhabdovirusesJ (19 87) (Edited by R.R. Wagner.

Plenum Press, New York).

Scope and deposit related to the invention The scope of the present invention is to use it for purposes other than vaccine components (i.e., as a diagnostic reagent). It also includes the above-mentioned DNA constructs themselves as well as VLPs that can be used.

Plasmid pAcTB2 was purchased from the Australian Government on 16 July 1992. Analytical Laboratories (AGALX Australia, N.N.I.S. ・Deposited with W, Suokin Estate Pimple), accession number 92/3 I was assigned 2589.

(Margin below) Drawing description Figure 1: Rhabdovirus or paramyxovirus sample for inclusion in VLPs Schematic diagram of several DNA constructs suitable for expression of genomic RNA. R1 and R2 are The 5' domain represents the appropriate pozyme domain, and the 5' domain is rhabdovirus or paramyelin. It is derived from the 5' end genome of the (-) sense RNA genome of the virus. 3° de The main is the (-) sense RNA genome of rhabdovirus or paramyxovirus. The filler domain is derived from the 3° terminal sequence of represents. Fl and F2 are part of the filler domain. Sl and 82 are intervening nuclei Represents the ocide sequence.

Figure 2: Using baculovirus expression system as an example of rabies virus VLP formation Schematic representation of the general process of rhabdovirus or paramyxovirus VLP formation figure.

Figure 3: Schematic diagram of the organization of the TB-2 subgenomic DNA construct. Baculovirus expression vector Structured as a double-stranded DNA molecule suitable for cloning into the BamH1 site of the vector represents. The R1 and R2 lipozyme cleavage sites hypothesize that transcription occurs in the direction described. and is active within the RNA transcript of this cloned DNA.

Figure 4l TB-2 showing functional domains and R1 and R2 ribozyme cleavage sites. Illustration of the sequences of transcripts of subgenomic DNA constructs.

Figure 5: Nucleotide sequence of TB-2 subgenomic DNA construct (ii copy [+ copy sense single-stranded DNA).

Figure 6: Schematic diagram of the organization of the TB-1 subgenomic DNA construct. Baculovirus expression vector Structured as a double-stranded DNA molecule suitable for cloning into the BamH1 site of the vector represents. The R ribozyme cleavage site is located at this location, assuming that transcription occurs in the direction described. It is active within the RNA transcript of the cloned DNA.

Figure 7. TB-1 subgenome D showing bifunctional domains and R lipozyme cleavage sites. Illustration of the sequences of the NA construct transcripts.

Figure 8: Nucleotide sequence of TB-1 subgenomic DNA construct (transcription [+] expressed as single-stranded DNA).

Figure 9 = Filler domain, TB-2 subgenomic DNA and induced TB-1 Synthetic oligonucleotide nuclei used for PCR in the construction of subgenomic DNA Creotide sequence.

Figure 10: Uncleaved and XbaI at 37°C using T3 RNA polymerase. 6% polyacrylamide, representing the product of in vitro transcription of cleaved pTB2 DNA. Autoradiograph of de-urea gel. Lipozyme as described in the above specification The cleavage products are identified as bands A, B and C.

Figure 1: Polyclonal anti-rabies mouse serum was used as described in the specification above. Cell lysates and particle preparations obtained from mixed infections with recombinant baculoviruses Immune plot of. Lane 1: Lysate of cells infected with wild-type AcNPV. lane 2: Recombinant two expressing rabies N/M1 protein and M2/G protein Lysate of cells infected with heavily expressed baculovirus. Lane 3: Rabies N, Ml, M Cells infected with a recombinant single-expressing baculovirus expressing 2 and G proteins. Cyst lysate. Lane 4: Rabies N/Ml protein and M2/G protein A combination containing the recombinant dual expression baculovirus and TB-2 DNA construct to be expressed. Particles prepared from culture supernatant of cells infected with recombinant baculovirus AcTB2.

Lane 5: Set expressing rabies N/Ml protein and M2/G protein Particles prepared from culture supernatant of cells infected with recombinant dual-expressing baculovirus.

Figure 12 = Electron microscopy image of rabies VLP preparation. As described in the above specification, rabies Recombinant baculovirus vector expressing N5M1, M2 and G proteins and Spodobutella fulgii using recombinant baculovirus expression vectors AC, TB2. Samples were prepared from culture supernatants obtained from mixed infections of Perda cells. Described in the above statement This microscopic image shows rabies VLPs (70-80 nm in diameter with surface projections). irregular particles) and recombinant baculovirus particles (40 x 250 nm rod-shaped particles) child).

F　I　GURE　1 (A) R1-5l-5' domain-field-5-domain-3' domain-52 -R2 (B) 5' domain -R1-filler domain -R2-3' domain (C ) R1-8L-5° domain-filler domain-R2-3' domain (D) 5' domain-R1-filler domain-3' domain-82-R2(E)R1 -5' domain - Filler domain - 3' domain - R2 (F) 5' domain - F　-Rl-filler domain-R2-F2-3' domain (G) Sl-filler domain filler domain-3' domain-R2(F)Sl-filler domain-R2-3 'Domain (■) 5°,! In-F 1-Rl-Filler Tomei>-3”r "Main-32-R2 (J) R1-Sl-5' domain-filler domain-R 2-F2-3' domain F i g ur e 2 = rabies ■ LP construction VL Ps mouth FIGURE 5: Sequence description TB-2AτGTGTTTTCGCCGGTCT GA TGAGTCCGTG AGGACGAAACGC ding TAAGGAT C C1432F I GURE Total array description TB-1CG (jTAAGGA T CC” FIGURE 9 Nucleotide sequence of oligonucleotide used ICAGG C-cho TCCA GGAGTA 3' 711i-cho 15B: TB5C: 5’　GGCCGGATCCCrACGTCACCG　CTchohachinin CAAAT　X AACAAC entrance A AATG entrance G entrance A ACAAAτC■■`C60 Person ACTAGAGG Ding Ding CAGA Ding τ 31 77GXCTCAATC3+6 9 n33B: 5' AAACACATCG CCGGTGkCGCTTAACAACAA ACCAGAGAA　G入入入＾GACA　GCG入C`TTG　60 CAAAGCMA 3’ 69 B56 S’　GGCCGGATCC TO τAGCGTT　CGTCCTCACG　G ACTCATCAG ACCGGCGAAA ACACATbGCC60 GGTGACGCTT Enter A 3' 72TBI: Ll.

5' AGAGTGATAG A clove TTG CTCAATC3° 24L2° 5° TCGGTA Haccho Haccho to C OCTTAC 8 ACG 3° 24 countries International investigation report 1-'-1°21° PCTIAU92J003tl Poem PC7L15kn 101-Ryoen familiar of wtswwl +hm+) fjujy 19921 sep1m international investigation report Ka-1-N.

PCrllAUt21011311 This Annex 1iststhek+sown’A” publica cionlevelpatemfamily members@reliting 　1othe, patent document, +, ai, :h,,,,b ;,e,7en,ti,onrei n,,te;uorulrh,repo,rt, , The, A:s, t:: oam+ 0fflce u tn rio wa y　l1abl.

Form PCT/ISA/211X--1amly-Kjuly l9 921 s+v? +Continuation of front page (51) Int, C1,5 identification symbol Internal office reference number A61K 39/205 AFE 9284-4CC12N 7101 //(C12N 15109 C12R1:92) (72) Inventor Preford, Christoph Sheen Commonwealth of Australia 40 67 7 Sandford Street, Saint Lucia, Queensland /No.82 I

Claims (18)

[Claims] 1. Infections caused by rhabdoviruses and paramyxoviruses A method of producing a vaccine for treatment, the method comprising the steps of: (i) containing a 3' domain, a filler domain and a ribozyme domain; Complete genome, modified genome or genome of rhabdovirus or paramyxovirus construct a DNA molecule corresponding to the genome fragment, and (ii) convert the DNA obtained in step (i) into a true into the cloning site of a nuclear expression vector to create a vector containing the genomic construct. At the same time as transfecting eukaryotic cells with a Viral G protein, N protein, M1 protein and M2 protein Rhabdovirus or paramyxovirus structures, including those with functions similar to Transfection with a vector containing the cloned gene for the protein. (iii) wrapped in a sheath of N protein and M1 protein R transcribed from the DNA molecule constructed in step (i) of forming a protein complex NA genome, internal matrix consisting of M2 protein and G protein A virus-like particle (VLP) composed of a lipid envelope containing ) obtained from cells transfected with (iv) The VLP obtained in step (iii) is included in a vaccine. 2. In step (i), the DNA molecule further comprises a 5' domain and an expressed RN. Additional ribozyme domain to ensure that A is cleaved at its 5' end The method according to claim 1, characterized in that it contains. 3. In step (ii), rabies L protein or other rhabdovirus or or cloned genes corresponding to similar proteins in paramyxoviruses. The method of claim 1, further comprising a child. 4. Claim characterized in that the DNA molecule in step (i) has cohesive ends. The method described in 1. 5. The DNA molecule in step (iii) contains L protein derived from rabies virus. similar proteins of other rhabdoviruses or paramyxoviruses 2. The method of claim 1, comprising filler domains consisting of. 6. In step (ii), the eukaryotic expression vector is a baculovirus and the eukaryotic expression vector is a baculovirus. The spores are Spodoptera frugiperda. ) The method according to claim 1. 7. Claim 1 characterized in that in step (iii) the VLP is mixed with an adjuvant. The method described in. 8. A vaccine produced by the method according to claim 1. 9. Caused by rabies N protein or cystovirus or paramyxovirus similar proteins and rabies M1 protein or or a similar protein sheath from paramyxoviruses. Contains 3' domain and filler domain forming nucleoprotein complex RNA genome and rabies M2 protein or cystovirus or paramycosis Internal matrix of similar proteins from sow virus and rabies G-tan similar proteins derived from paramyxoviruses or paramyxoviruses. Virus-like particles (VLPs) containing a proteinaceous lipid envelope. 10. Rhabdovirus or paramyxovirus infection comprising the VLP of claim 9 Vaccine for. 11. 11. A vaccine according to claim 10, comprising an adjuvant. 12.3' domain, any RNA expressed from a DNA construct has its 3' end Contains a ribozyme domain and a filler domain to ensure that it is expressed at the end. DNA construct. 13. Additionally, any RNA expressed from the 5' domain and the DNA construct It also contains an additional ribozyme domain to ensure that it is expressed at its 5' end. 15. The DNA construct according to claim 14. 14. DNA construct described in Figure 1. 15. TB1. 16. TB2. 17. DNA construct Ac-TB2. 18. Plasmid pACTB2.