CA2075896A1 - Production of antigens by self-assembly of polypeptide components - Google Patents
Production of antigens by self-assembly of polypeptide componentsInfo
- Publication number
- CA2075896A1 CA2075896A1 CA002075896A CA2075896A CA2075896A1 CA 2075896 A1 CA2075896 A1 CA 2075896A1 CA 002075896 A CA002075896 A CA 002075896A CA 2075896 A CA2075896 A CA 2075896A CA 2075896 A1 CA2075896 A1 CA 2075896A1
- Authority
- CA
- Canada
- Prior art keywords
- polypeptides
- process according
- particles
- btv
- proteins
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
- C12N2710/14141—Use of virus, viral particle or viral elements as a vector
- C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
- C12N2720/00011—Details
- C12N2720/12011—Reoviridae
- C12N2720/12111—Orbivirus, e.g. bluetongue virus
- C12N2720/12123—Virus like particles [VLP]
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- C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
- C12N2720/00011—Details
- C12N2720/12011—Reoviridae
- C12N2720/12311—Rotavirus, e.g. rotavirus A
- C12N2720/12322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C12N2720/00011—Details
- C12N2720/12011—Reoviridae
- C12N2720/12311—Rotavirus, e.g. rotavirus A
- C12N2720/12334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- Life Sciences & Earth Sciences (AREA)
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- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Antigens are produced by self-assembly of polypeptide components.
The production of bluetongue virus antigens (BTV) in the form of assembled particles comprising separate polypeptide components, particularly proteins VP2, VP3, VP5 and VP7 is described.
The production of bluetongue virus antigens (BTV) in the form of assembled particles comprising separate polypeptide components, particularly proteins VP2, VP3, VP5 and VP7 is described.
Description
W O 91100914 P ~ /GB90/01049 -- 1 -- 2~75 ~
Production Of Anti~ens By Self-Assembly of Poly~e~tide Components This inventiGn relates to the production of antigens by self-assembly of polypeptide components and particularly, but not exclusively to the production of bluetongue virus antigens (3TV) in the form of assembled particles comprising separate polypeptide components.
Many virus particles, including bluetongue virus (BTV) consist of a nucleic acid (DNA or RNA) packaged together with a plurality of polypeptide components. The latter contribute to the antigenic properties of the virus and constitute the key components of vaccines and diagnostic test kits. Virus assembly involves highly specific interactions between the component protein species within the matrix of the host cell. As such viral morphogenesis provides a model for the study of cellular processes of macromolecular interaction.
To date, only single-shelled virus~ e particles or cores have been assembled following the expression of one or two viral genes. Thus empty, single-shelled virus particles have been assembled for a number of different viruses from their appropriate structural proteins both in vivo (e.g. Hepatitis B) and in vitro (e.g. Rotavirus). In each of these examples the p~rticles were organised from a single species of polypeptide. Recently empty polio virus particles containing three different protein species have been synthesized in vivo, however, the coding information for these proteins was provided by translation of a single mRNA.
SUBSTITUTE SHEET
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`o~
W O 91/00914 PCT~GB90/01049 --2-- 2 ~
Viral structures which contain multiple polypeptide species encoded by separate genes (e.g. BTV) present a more difficult and challenging objective. Formation of such structures, and evaluation of the interactions of their protein components can be attempted in vitro, but this may introduce artifacts. Synthesis of the individual proteins in eukaryotic cells by an expression system which mimics the natural situation of macromolecular interactions would be desirable, but hitherto, this has proved unatainable in practice.
Gene cloning and expression have proved to be powerful tools for assessing the structural and/or functional roles of individual proteins. The natural progression for future studies will be the examination of multi-protein 'structures' such as viruses or enzyme complexes. The assembly of virus-like particles following the synthesis of their component proteins is an area which presents a number of opportunities. For example, the stages of viral assembly, the contributions of individual components to that process, and the sites and nature of viral protein interactions can be studied. An increased understanding of viral morphogenesis may aid the development of antiviral agents which specifically interfere with the assembly process. Intact virus-like particles could also prove useful as vaccines if epitoPes are presented in authentic conformations.
It has now been found that recombinant baculoviruses may be used to overcome these problems. Thus it has been found that by expressing.
in susceptible insects or insect cells, proteins having a capacity to self-assemble, the polypeptides are able to for~ assembled antigen particles, which in many instances resemble the native viruses themselves, both in morphology and antigenic properties.
SUBSTITUTE SHEET
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- 3 ~
Thus according to the invention there is provided a process for the production of antigen particles by self-assembly of separate polypeptides, which comprises transforming a host with at least one recombinant expression vector wherein at least one of the vectors has a DNA insert coding for one or more of said polypeptides whereby each of the polypeptides is expressed, and self-assembly of said polypeptides occurs with formation of said antigen particles.
The process of the invention is not restricted to any particular expression system and the host cells may be prokaryotic or eukaryotic (including, e.g. yeast or animal cells). Preferably however, the host comprises of susceptible insect or insect cells and said expression vector in a baculovirus expression vector. -~
Preferably, susceptibie insects or insect cells are infected with a baculovirus expression vector having a DNA insert coding for each of said polypeptides.
More specifically, the present invention relates to the stimultaneous expression of major Bluetongue virus (BTV~ structural proteins in insect cells using multiple recombinant baculoviruses. The expressed polypeptides assembled to ~orm non-infectious, double-shelled, virus-like particles of the same size and appearance as authentic BTV
virions. Antibodies raised to the expressed particles contained very high titres of neutralizing activity against the homologous BTV
serotypes. These results indicate the potential of this technology for the study of complex multiprotein structures, as well as the development of a new generation of viral vaccines.
There are numerous reports of high level expression of foreign proteins by recombinant baculoviruses (reviewed Luckow & Summers), and insect cells have been demonstrated to provide an appropriate environment SUBSTITUTE SHEET
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W O 9]/00914 PCr/GB90~0l~49 2~for the accurate expression of foreign polypeptides. However, until recently a maior limitation of baculoviruses has been that simultaneous expression of sever~l proteins within a single cell requires co-infection with two recombinant viruses, each containing a single foreign gene.
Success by this approach is dependent upon achieving an efficient infection at a multiplicity of 5 or more pfu/cell (plaque forming units) for each virus. This requires large amounts of high titre virus stocks and means that scaling up to large oultures, should it be required, is impractical. To overcome these problems multiple expression vectors which allow the construction of recombinant viruses expressing a number of foreign gene products si~ultaneously have been designed and constructed.
Typical vectors incorporate two copies ,of the polyhedrin promoter and transcription termination se~uences, although other duplicated promoters may also be used.
One system utilising such a vector is the subject of International Patent Application W089/01518 and is particularly useful in carrying out the method of the present invention.
Thus, in carrying out the process of the invention, the so-called "multiple expression system" which is the subject of International Patent Application W089/01518 is advantageously used to express the aforementioned separate polypeptides. The procedures described in W089/01518 utilize a plasmid designated pAcVC3 which has been deposited at the National Collection of Industrial Bacteria under Accession No.
NCIB12516.
pAcVC3, contains duplicated copies of the polyhedrin transcriptional machinery from Autographa caZi~ornica nuclear SUBSTITUTE SHEET
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--: . : . ' . ~ . . , WO 91/00914 PCI`/GB90/01049 Spodoptera Jru~iperda insect cells. In pAcVC3, B unique enzy~e restriction site located downstream of e~ch ~ro~ote~ allows for th~
insertlon of two foreign genes, each of whieh will be placed under the control of its o~n copy of the polyhedrin transcrlprlonal machinary. The promoters are present in opposite orientations to mini~iæe the possibility of homologous sequence recombination and exci~ion Or one or other of the forei~n genes.
Thus in carrying out the method of the prese~t invention, each of the DNA inserts which codes for one of said separate polypeptides preferably comprises part of a polypeptide expression ~equence (P~S) w~erein each P~S includes (i) a transcriptional promoter~ ~1) a D.~A
~equence coding ~or one of s~id polypeptides and (iii) a transcrip~ional termination ~te. Pr~f~ably, re~pective PESs c~ntain DNA sequences (ii) coding for different polypeptides are arranged in the oppo~ite sense on separate strands Or DNA.
The promoters used in the method o~ the invention are not critical, but preferably e~pression of eac~ of said DNA inserts is under the control of a so-called "late" or "very late" promoter such as the AcNPV polyhedrin or plO promoters.
Applyin~ these technique~ to viruses with structures derived from the translation products of more than one mRNA, co-expre~sion of different ~enes has eD~bled e~senti&lly `complete' empty particles to be ~s3e~bled.
In c~ry~n~ ou~ the mothod of the 1nvention, ~cco~d~g to one embod~ent, ~he L3 ~nd M7 genes of bluetongue viru~ (BTV), which code for the two major structural core proteins VP3 and VP7 respectively, may be inserted into ~ baculovirus du~l expression transfer vector and a recombinant bac~lovirus expressing bot~ foreign 8~nes isolated followlng fn v~vo recombination with wild-type AcNP~ DNA.
SUBS~ITUTE SHEET
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BTV is an arthopod-borne virus causing disease in sheep and cattle. It is prevalent in many areas of the world, and is of economic importance to the livestock industry. The virus is a member of the Or~btv~r~s genus in the fa~ily Reovtr~dae, with ten segments of double-stranded RNA`located in a core particle consisting of two major proteins VP3 and VP7 together with three ma~or proteins VPl, VP4 and VP6.
An outer capsid of two major proteins VP2 and VP5 surrounds the core. The function(s) of the core proteins have not been determined, and little is known about their interactions and stoichio~etric arran~ements.
Appreciation of such factors is obviously important in order to understand the morphogenesis of a complex virus such as BTV. Recent cryoelectron microscopy studies suggests that the core of BTV consists of a nucleoprotein centre surrounded by two distinct protein layers, each of which is composed of a single polypeptide species. Immunogold analysis indicates that VP7 forms the outer-most layer, which is attached to a framework of VP3.
Utilising such a procedure, Spodoptera fr~giperda cells infected with such a recombinant have been shown to synthesize spherical core-like particles which are the same in size, appearance and stoichiometric arrangement of VP3 to VP7 as authentic BTV cores. This result is significant not just in the context of increasing understanding of BTV morphogenesis, but also for gene expression per se.
The correct assembly of a complex structure in vivo by a recombinant baculovirus expressin~ multiple foreign genes indicates the potential of this technique to approach fundamental questions in cell biology involvlng multimolecular structures.
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W O 91/00914 ,~ PCT/GB90/01049 ~) ' ' - , As indicated, the method of the invention is of particular use in producing antigenically active particles derived from bluetongue virus (BTV) proteins, but may be used in production of other antigen particles capable of self-assembly. These include bluetongue vlrus proteins other than VP3 and VP7.
In a particularly preferred emboidment which is further described and claimed in our copending International Patent Application No. (claiming priority from GB 8915572.5), the antigenically active particles includes at least one of proteins VP2 and VP5.
VP2 and VP5 are termed "outer" proteins in the bluetongue virus and have the role of binding receptors on lost cells and mediating in the passage of virions into the host cell's interior.
VP2 is an important antigen as it is effective in raising neutralizing antibodies and can be used as a vaccine component. By constructing a VP2-containing antigen particle according to the invention superior vaccines can be produced that can be used without adjuvants.
Production of assembled antigen particles according to the invention can be used to produce antigen for use in the manufacture of vaccines and components of diagnostic test kits.
So-called "chimeric" antigens may be produced according to the invention wherein the individual polypeptides of the assembled particles may be of different serotypes of a common virus species or they may be antigens derived from different virus species. Examples include the different serotypes of bluetongue virus. Thus one of the polypeptides may ba derived from one serotype and one from another.
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The production of antigen in the form of self-assembled particles, further permits the purification of the antigen particles by techniques that make use of characteristic features of the particles such as density and/or size. Thus the particleq produced by the method of the invention may be more readily purified by sucrose-density centrifugation than the individual polypeptide components thereof.
In the following examples Example 1 describes the synthesis of 9TV core particles fro= these components, utilizing a du ~ recombinant baculovirus containing the L3 and M7 genes which encode the VP3 and VP7 polypeptides. Example 2 describes the production of BTV core-like particles by co-expression of VP2, VP3, VP5 and VP7.
EXAMPLES
The construction of recombin~nt expression vector pAcVC3.BTV-10.7.BTV-17.3 is illustrated in Figure 1.
The initial step for the expression of the BTV genes was to synthesize cDNA copies of the double stranded RNA L3 and M7 segments.
Althou~h these were isolated from different serotypes (17 and 10 respectively), the L3 gene is very highly conserved with an amino acid homology of greater than 99%.
Homopolymeric tails introduced to aid the cloning procedure were removed by limited 9al31 exonuclease digestion before insertion of the genes into the pAcVc3 transfer vector. Recombinant baculoviruses were prepared by the established procedure of co-transfecting S. ~r~giperda cells with the dual expression plasmid DNA and wild-type AcNPV DNA.
Progeny viruses were titrated using confluent monolayers of 5. ~rug~perda cells and putative recombinants selected on the basis o~
their polyhedrin negative phenotype (ca 0.1% fequency). After successive rounds of plaque purification of high titre viral stock was ~:
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prepared. S, Jrugiperda cells infected with the recombinant baculovirus synthesized two unique proteins species in place of the 29kDa polyhedrin protein seen in wild-type AcNPV infected cells (Figure 2A~.
The sizes of the expression proteins agree with those expected for VP3 and VP7 calculated from their amino acid compositions (103,226 KDa and 385,4O KDa respectively). Conflrmation that these expressed proteins represented authentic BTV proteins was provided by Western blot analysis with antisera raised to BTV-10 virus particles (Figure 2B).
VP3 is a group specific antigen and is recognised by antisera to all BTV serotypes. Elec~ron micrographs of S. Jrugiperda cells infected with the recombinant showed large aggregates of foreign material in the cytoplasm (Figure 3) which, under high magnification, appeared to consist of spherical particles. .
This expressed material was isolated by lysing the cells with NP40, and purification on a discontinuous sucrose gradient. When examined under the electron microscope the material was found to consist of empty core-like particles whose size and appearance were essentially identic~
to authentic BTV core particles prepared from BTV-infected BHK cells (Figure 3).
How precisely the expressed particles resembled their viral counterparts was further appraised by calculation of the VP3 and VP7 stoichimetry. S. rrugtperda cells infected with the recombinant baculovirus were pulse-labelled at 24 h p.i. with [35S]methionine and, after purificstion on discontinuous sucrose gradients, the labelled particles were dissociated, and the components separated by SDS-PAGE. The bands of VP7 and VP3 were located by autoradiography, excised and counted . . .
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in a liquid scintilation cocktail containing a tissue solubilizer. Since the number of methionine residues in each protein is known their stoichiometry could be calculated. After averaging the results from two different experiments, a value of 15 VP7 molecules to 2 VP3 molecules was obtained. This exactly matches the stoichiometry of these components in BTV core particles as determined by labelling with [14C] amino acids.
Recent evidence from our laboratory indicates that 780 copies of VP7 are present per core particle, which based upon a 15:2 molecular arrangement, denotes the cores contain lO~ copies of VP3.
The synthesis of empty BTV core particles from the major core proteins VP3 and VP7 reveals some important aspects of BTV morphology. It can be concluded from the fact that core-like particles were synthesized that their formation is not dependent on the presence of the three minor core proteins, nor the BTV double-stranded RNA. In addition, the three BTV non-structural proteins (NSl, NS2, NS3) are not required to assist, or direct the assembly of the VP3 and VP7 proteins to form stoichiometrically correct empty particles.
The results obtained provide the opportunity for more detailed research of viral morphogenesis and the interactions of structural proteins, as well as testing antivirals that may inhibit morphogenic pathways.
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Example 2 When used to immunize animals, the single-shelled core-like particles produced according to Example l do not protect against subsequent ~TV infection as the epitopes that elicit neutralizing antibodies are present on the outer capsid protein VP2. Since a recombinant baculovirus expressing VP2 has been constructed co-infection experiments were initially undertaken with the VP3 ane VP7 du~l vector and the VP2 vector in an attempt to determine if VP2 became associated with the core-like particles. No VP2 was detected on particles purified from insect cells expressing VP2, VP3 nd VP7 ~results not shown).
Similar results were obtained in a parallel study using the VP3 and VP7 vector together with a recombinant baculovirus expressing VP5. Since neither of the capsid proteins attached individually to the core-like particles, a dual recombinant baculovirus was constructed which would express both VP2 and VP5 simultaneously.
The manipulations for the construction of VP2-VP5 recombinant plasmid are shown in Fig 4. They involved excision of the L2 and M5 genes from their pAcYMI single expression transfer vectors and insertion into the ~amHI and BgIII sites (respectively) o~ the multiple expression vector pAcVC3. Recombinant baculoviruses were prepared by the established procedure of co-transfecting S Jrugiperda insect cells with the recc~binant plasmid DNA and infectious wild-type AcNPV DNA. Progeny viruses were titrated using confluent monolayers of S.frug~perda ceZ~s and putative recombinants were selected on the basis of their polyhedrin-negative phenotype (ca 0.1% frequency). After successive rounds of plaque purification, a high titre viral stock was prepared.
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S. Jrug~perda cells infected with the recombinant baculovirus synthesized two unique protein species in place of the 29KDa polyhedrin protein seen in wild-type AcNPV infected cells (Fig.5A). The sizes of the expressed proteins agree with those expected or VP2 and VP5 calculated from their amino acid compositions (i.e., 111,112 Da and 59,136 Da respectively). Since the levels of expression were below that which could be determined by staining, confirmation that the expressed proteins represented authentic BTV proteins was provided by Western blot analyses using antisera raised to BTV-10 virus particles (Fig.5B).
Although the outer capsid of BTV is composed of VP2 and VP5, no capsid like structures were detected in insect cells infected with the VP2-VP5 recombinant baculovirus. To assess the interaction of these proteins with the BTV core-like particles, insect cells were co-infected with both du~l recombinant baculoviruses (in order to co-express VP2, VP3, VP5 and VP7).
The cells were harvested at 48 hours post-infection, lysed with the non-ionic detergent Nonidet P40, and particles purified to homogeneity by centrifugation on discontinuous sucrose gradients. When examined under the electron microscope, empty double-shelled particles were observed consisting of a core surround by a thick outer capsid (Fig.
6A, large arrow). The diameters of the largest particles were estimated to be of the order of 85 nm, i.e., comparable to those of BTV (Fig. 6B).
Some simple core-like particles were also observed in the preparation (Fig, 6A, thin arrows). Their diameters were estimated to be of the order of 65 nm. A range of intermediate structures were PlSo observed, apparently with varying amounts of the outer capsid proteins attached.
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J ~ 8 These may reflect different stages in particle asse~bly. Interestingly, the centre areas of both types of particles (cores, and virus-like particles) exhibited an icosahedral configuration. The smaller size of the central area of the virus-like particles is presumably due to the presence and density of the outer capsid proteins. The icosahedral configuration of the centre was also apparent in several authentic BTV
particles where stain had penetrated the particles. The purified expressed particles were analysed by SDS-PAGE and Western Immunoblot and shown to contain large amounts of VP2 and VP5 (Fig. 5), in addition to VP3 and VP7~
The autheniticity of the expressed empty double-shelled virus particles was assessed by their immunogenicity and haemagglutinating acitvity. Guinea pig sera raised against purified core-like particles and double-shelled virus-like particles were tested for their neutralizing activity against BTV-10. As expected, sera raised to the cores exhibited no neutralizing activity while in a 50% plaque reduction test substantial neutralization was demonstrated by the sera raised to the double-shelled particles at a dilution of 1:10,000. Monospecific sera raised to VP2 gave titres of <500. Purified double-shelled particles also exhibited haemagglutinating titers (Table 1), comparable to those observed with authentic virus. Purified cores did not haemagglutinate. VP2 has been demonstrated to be the haemagglutinating protein in authentic bluetongue virus. These data are supported by the inhibitory effect of monospecific sera raised to VP2 on the haemagglutination activity of the double-shelled particles. Monspecific sera raised to the other component proteins (VP3, VP5 and VP7) had essentially no effect (Table 1). Unlike authentic BTV, the virus-like particles were non-infectious when assayed in ~ammalian cells.
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Several interesting conclusions regarding BTV morphogenesis can be drawn from the results described herein. The outer capsid proteins VP2 and VP5 do not attach individually to the core-like particles. This suggests that these proteins may interact before attaching to the core, or alternatively they may bind sequentially until a complete particle is produced. As with the formation of core-like particles in insect cells, the addition of the outer-capsid is not dependent on the presence of the BTV non-structural proteins (NSl. NS2, NS3), or viral double-stranded RNA, or the minor proteins VPl, VP4, VP6.
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W O 91/00914 ~ PCT/GB90/0~049 TABLE 1 Haemagglutination an~lysis of BTV double-shelled virus-like particles SubstrateHaemagglutination titer Single-shelled core-like particles <2 Double-shelled virus-like particles 2048 Sera testedHaemagglutination-Inhibition titer Preimmune rabbit 16 Rabit anti VP2 >1024 Rabbit anti VP7 2 Preimmune mouse 4 Mouse anti VP5 8 Mouse anti VP3 32 S. frugiperda cells infected with the appropriate recombinant baculoviruses were lysed with Nonidet P-40 and double-shelled virus-like particles, or core-like particles, were isolated on discontinuous sucrose gradients. the haemagglutination titer of this material was assayed at 4 C using 0.25~ rabbit erythrocytes as the indicator. Titers are expressed as the reciprocal of the highest serial dilution that gave complete haemagglutination. Antisera raised to baculovirus expressed BTV
proteins were used in haemagglutination-inhibition tests. The inhibition titers are expressed as the reciprocal of the highest serial dilution of sera that gave complete inhibition of haemagglutination.
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Figure l. Construction diagram of the dual expression transfer vector showing the appropriate manipulations for the insertion of the BTV L3 and M7 genes.
Figure 2. Expression of the BTV core proteins VP3 and VP7 in insect cells by the recombinant baculovirus, and confirmation of their authenticity by Western blot analysis. S. rrugiperda cells were infected at a multiplicity of lO pfu/cell with recombinant virus, wild-type AcNPV, or were mock infected. Cells were harvested at 48 h post-infection, washed with PBS and lysed at 4C in 50 mM Tris-Hcl pH 8.0, 150 mM NaCI, 0.5% NP40. The expressed particles were purified by banding at the interface of a 30% w/v, 50X w/v discontinuous sucrose gradient (in 0.2 M Tris-HCI pH 8.0) after centrifugation at 8s,000 rpm for 3 h. Authentic BTV virions and core particles prepared from BTV-infected BHK cells are included for comparison. Proteins were separated by SDS-PAGE and stained with Coomassie blue (A), or were electroblotted onto immobilon membrane, and reacted with rabbit anti BTV-lO ~erum (B). Bound antibody was detected with an aIk~1ine phosphatase conjugate by the standard method.
Figure 3. Electron micrographs of empty BTV core particles synthesized in insect cells by a recombinant baculovirus expressing both msjor BTV core proteins VP3 and VP7 (A) sections of S.
~r~perda cells infected with the recombinant (l), or wild-type AcNPV virus (2). (B) purified expressed particles ~3) compared with authentic BTV core particles (4).
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Figure 4C Constr~ction of the baculov~rus expression transfer vector containing the L2 and M5 gene~ o~ BTV serotype 10. The cloning, genetic manipulations, and individual expression of these gene~ have previously been described. The L2 and M5 genes were excised from their single baculovirus expression transfer vector (pAcYMl) and ligated into the BglII and BamHI sites respectively o~
the multiple expression vector pAcVC3.
Figure 5. E2pression of the four major BTV structural proteins VP2, VP3, VP5 and YP7 in insect cells by recombinant baculoviruses, and confirmation of their authenticity by Westèrn Immunoblot analysis.
S. frugiperda cells were infected at a multiplicity of 5 pfu/cell with either the recombinant baculovirus expressing VP2 and VP5, or the recombinant expressing Vp3 and VP7, or were co-infected with both recombinant viruses. Mock and wild-type AcNPV infected cells acted as controls. Cells were harvested at 48 h post-infection, washed with PBS and lysed at 4 C in 50 mM Tris-HCI pH8.o, 150 mM
NaCI, 0.5% NP40. The expressed particles (both single-shelled core-like particles and double-shelled virus-like particles) were purified by banding at the interface of a 30% w/v, and 66% w/v discontinuous sucrose gradient (in 0.2 M Tris-HCI,pH8.0) after centrifugation at 85,000 g for ~ h. Authentic BTV virions prepared from BTV-infected BHK cells are included for comparison. Proteins were separated by SDS-PAGE and stained with Coomassie Blue (A), or were electroblosted onto Immobilon membrane and reacted with rabbit SUBSTITUTE SHEET
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BTV-10 antiserum (B). Since VP2 co-migrstes with VP3, its presence in the purified double-shelled virus-like particles and authentic BTV virions was confirmed by reacting with antisera raised to expressed YP2 (C). Bound antibody was detected with an aIkaline phosphatase conjugate using standard methods.
Figure 6. Electron micrographs of baculovirus expressed particles.
Empty BTV double-shelled virus-like particles are shown in (A) compared with authentic BTV particles (B). The high magnification micrographs (x30,000) show the appearance of expressed core-like particles composed of VP3 and VP7 (C), and the double-shelled particles with VP2 and VP5 attached to VP3~and VP7 (D).
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Production Of Anti~ens By Self-Assembly of Poly~e~tide Components This inventiGn relates to the production of antigens by self-assembly of polypeptide components and particularly, but not exclusively to the production of bluetongue virus antigens (3TV) in the form of assembled particles comprising separate polypeptide components.
Many virus particles, including bluetongue virus (BTV) consist of a nucleic acid (DNA or RNA) packaged together with a plurality of polypeptide components. The latter contribute to the antigenic properties of the virus and constitute the key components of vaccines and diagnostic test kits. Virus assembly involves highly specific interactions between the component protein species within the matrix of the host cell. As such viral morphogenesis provides a model for the study of cellular processes of macromolecular interaction.
To date, only single-shelled virus~ e particles or cores have been assembled following the expression of one or two viral genes. Thus empty, single-shelled virus particles have been assembled for a number of different viruses from their appropriate structural proteins both in vivo (e.g. Hepatitis B) and in vitro (e.g. Rotavirus). In each of these examples the p~rticles were organised from a single species of polypeptide. Recently empty polio virus particles containing three different protein species have been synthesized in vivo, however, the coding information for these proteins was provided by translation of a single mRNA.
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Viral structures which contain multiple polypeptide species encoded by separate genes (e.g. BTV) present a more difficult and challenging objective. Formation of such structures, and evaluation of the interactions of their protein components can be attempted in vitro, but this may introduce artifacts. Synthesis of the individual proteins in eukaryotic cells by an expression system which mimics the natural situation of macromolecular interactions would be desirable, but hitherto, this has proved unatainable in practice.
Gene cloning and expression have proved to be powerful tools for assessing the structural and/or functional roles of individual proteins. The natural progression for future studies will be the examination of multi-protein 'structures' such as viruses or enzyme complexes. The assembly of virus-like particles following the synthesis of their component proteins is an area which presents a number of opportunities. For example, the stages of viral assembly, the contributions of individual components to that process, and the sites and nature of viral protein interactions can be studied. An increased understanding of viral morphogenesis may aid the development of antiviral agents which specifically interfere with the assembly process. Intact virus-like particles could also prove useful as vaccines if epitoPes are presented in authentic conformations.
It has now been found that recombinant baculoviruses may be used to overcome these problems. Thus it has been found that by expressing.
in susceptible insects or insect cells, proteins having a capacity to self-assemble, the polypeptides are able to for~ assembled antigen particles, which in many instances resemble the native viruses themselves, both in morphology and antigenic properties.
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Thus according to the invention there is provided a process for the production of antigen particles by self-assembly of separate polypeptides, which comprises transforming a host with at least one recombinant expression vector wherein at least one of the vectors has a DNA insert coding for one or more of said polypeptides whereby each of the polypeptides is expressed, and self-assembly of said polypeptides occurs with formation of said antigen particles.
The process of the invention is not restricted to any particular expression system and the host cells may be prokaryotic or eukaryotic (including, e.g. yeast or animal cells). Preferably however, the host comprises of susceptible insect or insect cells and said expression vector in a baculovirus expression vector. -~
Preferably, susceptibie insects or insect cells are infected with a baculovirus expression vector having a DNA insert coding for each of said polypeptides.
More specifically, the present invention relates to the stimultaneous expression of major Bluetongue virus (BTV~ structural proteins in insect cells using multiple recombinant baculoviruses. The expressed polypeptides assembled to ~orm non-infectious, double-shelled, virus-like particles of the same size and appearance as authentic BTV
virions. Antibodies raised to the expressed particles contained very high titres of neutralizing activity against the homologous BTV
serotypes. These results indicate the potential of this technology for the study of complex multiprotein structures, as well as the development of a new generation of viral vaccines.
There are numerous reports of high level expression of foreign proteins by recombinant baculoviruses (reviewed Luckow & Summers), and insect cells have been demonstrated to provide an appropriate environment SUBSTITUTE SHEET
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W O 9]/00914 PCr/GB90~0l~49 2~for the accurate expression of foreign polypeptides. However, until recently a maior limitation of baculoviruses has been that simultaneous expression of sever~l proteins within a single cell requires co-infection with two recombinant viruses, each containing a single foreign gene.
Success by this approach is dependent upon achieving an efficient infection at a multiplicity of 5 or more pfu/cell (plaque forming units) for each virus. This requires large amounts of high titre virus stocks and means that scaling up to large oultures, should it be required, is impractical. To overcome these problems multiple expression vectors which allow the construction of recombinant viruses expressing a number of foreign gene products si~ultaneously have been designed and constructed.
Typical vectors incorporate two copies ,of the polyhedrin promoter and transcription termination se~uences, although other duplicated promoters may also be used.
One system utilising such a vector is the subject of International Patent Application W089/01518 and is particularly useful in carrying out the method of the present invention.
Thus, in carrying out the process of the invention, the so-called "multiple expression system" which is the subject of International Patent Application W089/01518 is advantageously used to express the aforementioned separate polypeptides. The procedures described in W089/01518 utilize a plasmid designated pAcVC3 which has been deposited at the National Collection of Industrial Bacteria under Accession No.
NCIB12516.
pAcVC3, contains duplicated copies of the polyhedrin transcriptional machinery from Autographa caZi~ornica nuclear SUBSTITUTE SHEET
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--: . : . ' . ~ . . , WO 91/00914 PCI`/GB90/01049 Spodoptera Jru~iperda insect cells. In pAcVC3, B unique enzy~e restriction site located downstream of e~ch ~ro~ote~ allows for th~
insertlon of two foreign genes, each of whieh will be placed under the control of its o~n copy of the polyhedrin transcrlprlonal machinary. The promoters are present in opposite orientations to mini~iæe the possibility of homologous sequence recombination and exci~ion Or one or other of the forei~n genes.
Thus in carrying out the method of the prese~t invention, each of the DNA inserts which codes for one of said separate polypeptides preferably comprises part of a polypeptide expression ~equence (P~S) w~erein each P~S includes (i) a transcriptional promoter~ ~1) a D.~A
~equence coding ~or one of s~id polypeptides and (iii) a transcrip~ional termination ~te. Pr~f~ably, re~pective PESs c~ntain DNA sequences (ii) coding for different polypeptides are arranged in the oppo~ite sense on separate strands Or DNA.
The promoters used in the method o~ the invention are not critical, but preferably e~pression of eac~ of said DNA inserts is under the control of a so-called "late" or "very late" promoter such as the AcNPV polyhedrin or plO promoters.
Applyin~ these technique~ to viruses with structures derived from the translation products of more than one mRNA, co-expre~sion of different ~enes has eD~bled e~senti&lly `complete' empty particles to be ~s3e~bled.
In c~ry~n~ ou~ the mothod of the 1nvention, ~cco~d~g to one embod~ent, ~he L3 ~nd M7 genes of bluetongue viru~ (BTV), which code for the two major structural core proteins VP3 and VP7 respectively, may be inserted into ~ baculovirus du~l expression transfer vector and a recombinant bac~lovirus expressing bot~ foreign 8~nes isolated followlng fn v~vo recombination with wild-type AcNP~ DNA.
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BTV is an arthopod-borne virus causing disease in sheep and cattle. It is prevalent in many areas of the world, and is of economic importance to the livestock industry. The virus is a member of the Or~btv~r~s genus in the fa~ily Reovtr~dae, with ten segments of double-stranded RNA`located in a core particle consisting of two major proteins VP3 and VP7 together with three ma~or proteins VPl, VP4 and VP6.
An outer capsid of two major proteins VP2 and VP5 surrounds the core. The function(s) of the core proteins have not been determined, and little is known about their interactions and stoichio~etric arran~ements.
Appreciation of such factors is obviously important in order to understand the morphogenesis of a complex virus such as BTV. Recent cryoelectron microscopy studies suggests that the core of BTV consists of a nucleoprotein centre surrounded by two distinct protein layers, each of which is composed of a single polypeptide species. Immunogold analysis indicates that VP7 forms the outer-most layer, which is attached to a framework of VP3.
Utilising such a procedure, Spodoptera fr~giperda cells infected with such a recombinant have been shown to synthesize spherical core-like particles which are the same in size, appearance and stoichiometric arrangement of VP3 to VP7 as authentic BTV cores. This result is significant not just in the context of increasing understanding of BTV morphogenesis, but also for gene expression per se.
The correct assembly of a complex structure in vivo by a recombinant baculovirus expressin~ multiple foreign genes indicates the potential of this technique to approach fundamental questions in cell biology involvlng multimolecular structures.
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W O 91/00914 ,~ PCT/GB90/01049 ~) ' ' - , As indicated, the method of the invention is of particular use in producing antigenically active particles derived from bluetongue virus (BTV) proteins, but may be used in production of other antigen particles capable of self-assembly. These include bluetongue vlrus proteins other than VP3 and VP7.
In a particularly preferred emboidment which is further described and claimed in our copending International Patent Application No. (claiming priority from GB 8915572.5), the antigenically active particles includes at least one of proteins VP2 and VP5.
VP2 and VP5 are termed "outer" proteins in the bluetongue virus and have the role of binding receptors on lost cells and mediating in the passage of virions into the host cell's interior.
VP2 is an important antigen as it is effective in raising neutralizing antibodies and can be used as a vaccine component. By constructing a VP2-containing antigen particle according to the invention superior vaccines can be produced that can be used without adjuvants.
Production of assembled antigen particles according to the invention can be used to produce antigen for use in the manufacture of vaccines and components of diagnostic test kits.
So-called "chimeric" antigens may be produced according to the invention wherein the individual polypeptides of the assembled particles may be of different serotypes of a common virus species or they may be antigens derived from different virus species. Examples include the different serotypes of bluetongue virus. Thus one of the polypeptides may ba derived from one serotype and one from another.
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The production of antigen in the form of self-assembled particles, further permits the purification of the antigen particles by techniques that make use of characteristic features of the particles such as density and/or size. Thus the particleq produced by the method of the invention may be more readily purified by sucrose-density centrifugation than the individual polypeptide components thereof.
In the following examples Example 1 describes the synthesis of 9TV core particles fro= these components, utilizing a du ~ recombinant baculovirus containing the L3 and M7 genes which encode the VP3 and VP7 polypeptides. Example 2 describes the production of BTV core-like particles by co-expression of VP2, VP3, VP5 and VP7.
EXAMPLES
The construction of recombin~nt expression vector pAcVC3.BTV-10.7.BTV-17.3 is illustrated in Figure 1.
The initial step for the expression of the BTV genes was to synthesize cDNA copies of the double stranded RNA L3 and M7 segments.
Althou~h these were isolated from different serotypes (17 and 10 respectively), the L3 gene is very highly conserved with an amino acid homology of greater than 99%.
Homopolymeric tails introduced to aid the cloning procedure were removed by limited 9al31 exonuclease digestion before insertion of the genes into the pAcVc3 transfer vector. Recombinant baculoviruses were prepared by the established procedure of co-transfecting S. ~r~giperda cells with the dual expression plasmid DNA and wild-type AcNPV DNA.
Progeny viruses were titrated using confluent monolayers of 5. ~rug~perda cells and putative recombinants selected on the basis o~
their polyhedrin negative phenotype (ca 0.1% fequency). After successive rounds of plaque purification of high titre viral stock was ~:
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prepared. S, Jrugiperda cells infected with the recombinant baculovirus synthesized two unique proteins species in place of the 29kDa polyhedrin protein seen in wild-type AcNPV infected cells (Figure 2A~.
The sizes of the expression proteins agree with those expected for VP3 and VP7 calculated from their amino acid compositions (103,226 KDa and 385,4O KDa respectively). Conflrmation that these expressed proteins represented authentic BTV proteins was provided by Western blot analysis with antisera raised to BTV-10 virus particles (Figure 2B).
VP3 is a group specific antigen and is recognised by antisera to all BTV serotypes. Elec~ron micrographs of S. Jrugiperda cells infected with the recombinant showed large aggregates of foreign material in the cytoplasm (Figure 3) which, under high magnification, appeared to consist of spherical particles. .
This expressed material was isolated by lysing the cells with NP40, and purification on a discontinuous sucrose gradient. When examined under the electron microscope the material was found to consist of empty core-like particles whose size and appearance were essentially identic~
to authentic BTV core particles prepared from BTV-infected BHK cells (Figure 3).
How precisely the expressed particles resembled their viral counterparts was further appraised by calculation of the VP3 and VP7 stoichimetry. S. rrugtperda cells infected with the recombinant baculovirus were pulse-labelled at 24 h p.i. with [35S]methionine and, after purificstion on discontinuous sucrose gradients, the labelled particles were dissociated, and the components separated by SDS-PAGE. The bands of VP7 and VP3 were located by autoradiography, excised and counted . . .
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in a liquid scintilation cocktail containing a tissue solubilizer. Since the number of methionine residues in each protein is known their stoichiometry could be calculated. After averaging the results from two different experiments, a value of 15 VP7 molecules to 2 VP3 molecules was obtained. This exactly matches the stoichiometry of these components in BTV core particles as determined by labelling with [14C] amino acids.
Recent evidence from our laboratory indicates that 780 copies of VP7 are present per core particle, which based upon a 15:2 molecular arrangement, denotes the cores contain lO~ copies of VP3.
The synthesis of empty BTV core particles from the major core proteins VP3 and VP7 reveals some important aspects of BTV morphology. It can be concluded from the fact that core-like particles were synthesized that their formation is not dependent on the presence of the three minor core proteins, nor the BTV double-stranded RNA. In addition, the three BTV non-structural proteins (NSl, NS2, NS3) are not required to assist, or direct the assembly of the VP3 and VP7 proteins to form stoichiometrically correct empty particles.
The results obtained provide the opportunity for more detailed research of viral morphogenesis and the interactions of structural proteins, as well as testing antivirals that may inhibit morphogenic pathways.
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Example 2 When used to immunize animals, the single-shelled core-like particles produced according to Example l do not protect against subsequent ~TV infection as the epitopes that elicit neutralizing antibodies are present on the outer capsid protein VP2. Since a recombinant baculovirus expressing VP2 has been constructed co-infection experiments were initially undertaken with the VP3 ane VP7 du~l vector and the VP2 vector in an attempt to determine if VP2 became associated with the core-like particles. No VP2 was detected on particles purified from insect cells expressing VP2, VP3 nd VP7 ~results not shown).
Similar results were obtained in a parallel study using the VP3 and VP7 vector together with a recombinant baculovirus expressing VP5. Since neither of the capsid proteins attached individually to the core-like particles, a dual recombinant baculovirus was constructed which would express both VP2 and VP5 simultaneously.
The manipulations for the construction of VP2-VP5 recombinant plasmid are shown in Fig 4. They involved excision of the L2 and M5 genes from their pAcYMI single expression transfer vectors and insertion into the ~amHI and BgIII sites (respectively) o~ the multiple expression vector pAcVC3. Recombinant baculoviruses were prepared by the established procedure of co-transfecting S Jrugiperda insect cells with the recc~binant plasmid DNA and infectious wild-type AcNPV DNA. Progeny viruses were titrated using confluent monolayers of S.frug~perda ceZ~s and putative recombinants were selected on the basis of their polyhedrin-negative phenotype (ca 0.1% frequency). After successive rounds of plaque purification, a high titre viral stock was prepared.
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S. Jrug~perda cells infected with the recombinant baculovirus synthesized two unique protein species in place of the 29KDa polyhedrin protein seen in wild-type AcNPV infected cells (Fig.5A). The sizes of the expressed proteins agree with those expected or VP2 and VP5 calculated from their amino acid compositions (i.e., 111,112 Da and 59,136 Da respectively). Since the levels of expression were below that which could be determined by staining, confirmation that the expressed proteins represented authentic BTV proteins was provided by Western blot analyses using antisera raised to BTV-10 virus particles (Fig.5B).
Although the outer capsid of BTV is composed of VP2 and VP5, no capsid like structures were detected in insect cells infected with the VP2-VP5 recombinant baculovirus. To assess the interaction of these proteins with the BTV core-like particles, insect cells were co-infected with both du~l recombinant baculoviruses (in order to co-express VP2, VP3, VP5 and VP7).
The cells were harvested at 48 hours post-infection, lysed with the non-ionic detergent Nonidet P40, and particles purified to homogeneity by centrifugation on discontinuous sucrose gradients. When examined under the electron microscope, empty double-shelled particles were observed consisting of a core surround by a thick outer capsid (Fig.
6A, large arrow). The diameters of the largest particles were estimated to be of the order of 85 nm, i.e., comparable to those of BTV (Fig. 6B).
Some simple core-like particles were also observed in the preparation (Fig, 6A, thin arrows). Their diameters were estimated to be of the order of 65 nm. A range of intermediate structures were PlSo observed, apparently with varying amounts of the outer capsid proteins attached.
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WO 91/û0914 - PC~/GB90/01049 ~``~ .
J ~ 8 These may reflect different stages in particle asse~bly. Interestingly, the centre areas of both types of particles (cores, and virus-like particles) exhibited an icosahedral configuration. The smaller size of the central area of the virus-like particles is presumably due to the presence and density of the outer capsid proteins. The icosahedral configuration of the centre was also apparent in several authentic BTV
particles where stain had penetrated the particles. The purified expressed particles were analysed by SDS-PAGE and Western Immunoblot and shown to contain large amounts of VP2 and VP5 (Fig. 5), in addition to VP3 and VP7~
The autheniticity of the expressed empty double-shelled virus particles was assessed by their immunogenicity and haemagglutinating acitvity. Guinea pig sera raised against purified core-like particles and double-shelled virus-like particles were tested for their neutralizing activity against BTV-10. As expected, sera raised to the cores exhibited no neutralizing activity while in a 50% plaque reduction test substantial neutralization was demonstrated by the sera raised to the double-shelled particles at a dilution of 1:10,000. Monospecific sera raised to VP2 gave titres of <500. Purified double-shelled particles also exhibited haemagglutinating titers (Table 1), comparable to those observed with authentic virus. Purified cores did not haemagglutinate. VP2 has been demonstrated to be the haemagglutinating protein in authentic bluetongue virus. These data are supported by the inhibitory effect of monospecific sera raised to VP2 on the haemagglutination activity of the double-shelled particles. Monspecific sera raised to the other component proteins (VP3, VP5 and VP7) had essentially no effect (Table 1). Unlike authentic BTV, the virus-like particles were non-infectious when assayed in ~ammalian cells.
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Several interesting conclusions regarding BTV morphogenesis can be drawn from the results described herein. The outer capsid proteins VP2 and VP5 do not attach individually to the core-like particles. This suggests that these proteins may interact before attaching to the core, or alternatively they may bind sequentially until a complete particle is produced. As with the formation of core-like particles in insect cells, the addition of the outer-capsid is not dependent on the presence of the BTV non-structural proteins (NSl. NS2, NS3), or viral double-stranded RNA, or the minor proteins VPl, VP4, VP6.
SUBSTITUTE SHEET
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W O 91/00914 ~ PCT/GB90/0~049 TABLE 1 Haemagglutination an~lysis of BTV double-shelled virus-like particles SubstrateHaemagglutination titer Single-shelled core-like particles <2 Double-shelled virus-like particles 2048 Sera testedHaemagglutination-Inhibition titer Preimmune rabbit 16 Rabit anti VP2 >1024 Rabbit anti VP7 2 Preimmune mouse 4 Mouse anti VP5 8 Mouse anti VP3 32 S. frugiperda cells infected with the appropriate recombinant baculoviruses were lysed with Nonidet P-40 and double-shelled virus-like particles, or core-like particles, were isolated on discontinuous sucrose gradients. the haemagglutination titer of this material was assayed at 4 C using 0.25~ rabbit erythrocytes as the indicator. Titers are expressed as the reciprocal of the highest serial dilution that gave complete haemagglutination. Antisera raised to baculovirus expressed BTV
proteins were used in haemagglutination-inhibition tests. The inhibition titers are expressed as the reciprocal of the highest serial dilution of sera that gave complete inhibition of haemagglutination.
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Figure l. Construction diagram of the dual expression transfer vector showing the appropriate manipulations for the insertion of the BTV L3 and M7 genes.
Figure 2. Expression of the BTV core proteins VP3 and VP7 in insect cells by the recombinant baculovirus, and confirmation of their authenticity by Western blot analysis. S. rrugiperda cells were infected at a multiplicity of lO pfu/cell with recombinant virus, wild-type AcNPV, or were mock infected. Cells were harvested at 48 h post-infection, washed with PBS and lysed at 4C in 50 mM Tris-Hcl pH 8.0, 150 mM NaCI, 0.5% NP40. The expressed particles were purified by banding at the interface of a 30% w/v, 50X w/v discontinuous sucrose gradient (in 0.2 M Tris-HCI pH 8.0) after centrifugation at 8s,000 rpm for 3 h. Authentic BTV virions and core particles prepared from BTV-infected BHK cells are included for comparison. Proteins were separated by SDS-PAGE and stained with Coomassie blue (A), or were electroblotted onto immobilon membrane, and reacted with rabbit anti BTV-lO ~erum (B). Bound antibody was detected with an aIk~1ine phosphatase conjugate by the standard method.
Figure 3. Electron micrographs of empty BTV core particles synthesized in insect cells by a recombinant baculovirus expressing both msjor BTV core proteins VP3 and VP7 (A) sections of S.
~r~perda cells infected with the recombinant (l), or wild-type AcNPV virus (2). (B) purified expressed particles ~3) compared with authentic BTV core particles (4).
SUBSTITUTE SHEET
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W O 91J009l4 PCT/GB9OtO1049 ~ . I
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Figure 4C Constr~ction of the baculov~rus expression transfer vector containing the L2 and M5 gene~ o~ BTV serotype 10. The cloning, genetic manipulations, and individual expression of these gene~ have previously been described. The L2 and M5 genes were excised from their single baculovirus expression transfer vector (pAcYMl) and ligated into the BglII and BamHI sites respectively o~
the multiple expression vector pAcVC3.
Figure 5. E2pression of the four major BTV structural proteins VP2, VP3, VP5 and YP7 in insect cells by recombinant baculoviruses, and confirmation of their authenticity by Westèrn Immunoblot analysis.
S. frugiperda cells were infected at a multiplicity of 5 pfu/cell with either the recombinant baculovirus expressing VP2 and VP5, or the recombinant expressing Vp3 and VP7, or were co-infected with both recombinant viruses. Mock and wild-type AcNPV infected cells acted as controls. Cells were harvested at 48 h post-infection, washed with PBS and lysed at 4 C in 50 mM Tris-HCI pH8.o, 150 mM
NaCI, 0.5% NP40. The expressed particles (both single-shelled core-like particles and double-shelled virus-like particles) were purified by banding at the interface of a 30% w/v, and 66% w/v discontinuous sucrose gradient (in 0.2 M Tris-HCI,pH8.0) after centrifugation at 85,000 g for ~ h. Authentic BTV virions prepared from BTV-infected BHK cells are included for comparison. Proteins were separated by SDS-PAGE and stained with Coomassie Blue (A), or were electroblosted onto Immobilon membrane and reacted with rabbit SUBSTITUTE SHEET
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WO 91/00914 PCI'/GB90/01049 ,I
BTV-10 antiserum (B). Since VP2 co-migrstes with VP3, its presence in the purified double-shelled virus-like particles and authentic BTV virions was confirmed by reacting with antisera raised to expressed YP2 (C). Bound antibody was detected with an aIkaline phosphatase conjugate using standard methods.
Figure 6. Electron micrographs of baculovirus expressed particles.
Empty BTV double-shelled virus-like particles are shown in (A) compared with authentic BTV particles (B). The high magnification micrographs (x30,000) show the appearance of expressed core-like particles composed of VP3 and VP7 (C), and the double-shelled particles with VP2 and VP5 attached to VP3~and VP7 (D).
.
SUBSTITUTE SHEET
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Claims (15)
1. A process for the production of antigen particles by self-assembly of separate polypeptides, which comprises transforming a host with at least one recombinant expression vector wherein at least one of said vectors has a DNA insert coding for one or more of said polypeptides whereby each of the polypeptides is expressed, and self-assembly of said polypeptides occurs with formation of said antigen particles.
2. A process according to Claim 1 wherein said host comprises a susceptible insect or insect cell and said expression vector is a baculovirus expression vector.
3. A process according to Claim 2 wherein susceptible insects or insect cells are infected with a baculovirus expression vector having a DNA insert coding for each of said polypeptides.
4. A process according to Claim 3 wherein each DNA insert comprises part of a polypeptide expression sequence (PES) wherein each PES
includes (i) a transcriptional promoter, (ii) a DNA sequence coding for one of said polypeptides and (iii) a transcriptional termination site.
includes (i) a transcriptional promoter, (ii) a DNA sequence coding for one of said polypeptides and (iii) a transcriptional termination site.
5. A process according to Claim 4 wherein respective PESs containing DNA sequences (ii) coding for different polypeptides are arranged in the opposite sense on separate strands of DNA.
6. A process according to any of Claims 2 to 5 wherein expression of each of said DNA inserts is under the control of a polyhedrin promoter.
7. A process according to any preceding claim wherein said polypeptides are bluetongue virus (BTV) proteins.
8. A process according to Claim 7 wherein at least one of said bluetongue virus proteins is a structural core protein.
9. A process according to Claim ô wherein one of said polypeptides is BTV core protein VP3.
10. A process according to Claim 8 or Claim 9 wherein one of said polypeptides is BTV core protein VP7.
11. A process according to Claim 7 wherein at least one of said bluetongue virus proteins is a non-structural protein.
12. A process according to any preceding claim wherein said polypeptides include bluetongue virus VP2.
13. A process according to any preceding claim wherein said polypeptides include bluetongue virus VP5.
14. An antigen particle produced by a process according to any preceding claim.
15. The use of an antigen particle as claimed in Claim 12 in a diagnostic test or as a vaccine component.
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GB898915571A GB8915571D0 (en) | 1989-07-07 | 1989-07-07 | Production of antigens by self-assembly of polypeptide components |
GB8915571.7 | 1989-07-07 |
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AU (1) | AU667796B2 (en) |
CA (1) | CA2075896A1 (en) |
GB (1) | GB8915571D0 (en) |
WO (1) | WO1991000914A1 (en) |
ZA (1) | ZA905295B (en) |
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US5169784A (en) * | 1990-09-17 | 1992-12-08 | The Texas A & M University System | Baculovirus dual promoter expression vector |
EP0749694A1 (en) | 1995-06-20 | 1996-12-27 | Societe Des Produits Nestle S.A. | Treatment enzymatic of cocoa |
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AP114A (en) * | 1987-02-19 | 1991-02-01 | Oxford Virology Ltd | Production of bluetongue virus antigens using a baculovirus expression vector. |
IL87394A0 (en) * | 1987-08-12 | 1989-12-15 | Natural Environment Res | Expression vectors for the synthesis of proteins and plasmid replicons and sequence cassettes for use in constructing said vectors |
-
1989
- 1989-07-07 GB GB898915571A patent/GB8915571D0/en active Pending
-
1990
- 1990-07-06 AU AU59549/90A patent/AU667796B2/en not_active Ceased
- 1990-07-06 ZA ZA905295A patent/ZA905295B/en unknown
- 1990-07-06 CA CA002075896A patent/CA2075896A1/en not_active Abandoned
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GB8915571D0 (en) | 1989-08-23 |
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AU5954990A (en) | 1991-02-06 |
AU667796B2 (en) | 1996-04-18 |
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