EP0349594A1 - Okklusionskörper aus rekombinanten bazillenviren in impfstoffen und biologischen insektiziden - Google Patents
Okklusionskörper aus rekombinanten bazillenviren in impfstoffen und biologischen insektizidenInfo
- Publication number
- EP0349594A1 EP0349594A1 EP88904294A EP88904294A EP0349594A1 EP 0349594 A1 EP0349594 A1 EP 0349594A1 EP 88904294 A EP88904294 A EP 88904294A EP 88904294 A EP88904294 A EP 88904294A EP 0349594 A1 EP0349594 A1 EP 0349594A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- polyhedrin
- recombinant
- amino acid
- foreign
- protein
- 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.)
- Withdrawn
Links
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
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- C07K2319/00—Fusion polypeptide
- C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
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- C07—ORGANIC CHEMISTRY
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- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
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Definitions
- Baculoviruses contain double-stranded, circular DNA molecules, which range from 60-110 x 10 daltons.
- the prototype of the Baculoviridae family is AcNPV, which has a genome of approximately 82-88 x 10 daltons (Miller, L. K. ,
- Two forms of virus are 30 produced as a result of wild-type AcNPV infection, occluded and non-occluded virions.
- the apparent role of the occlusion body in the virus life cycle is to provide stability outside the host insect by protecting the virus from inactivating environmental actors.
- Ingested occlusion bodies dissolve in the alkaline environmen of the midgut, releasing virus particles for another round of infection, late after viral replication.
- the occlusion body of NPV consists predominantly of a single, approximately
- polyhedrin 29,000 dalton molecular weight polypeptide, known as polyhedrin (Vlak, J. M. and Rohrmann, G. F. , supra) . This protein forms the paracrystalline lattice around the virions, and is present as a multimer. Polyhedrin is produced in enormous amounts during the course of viral infection, late after viral replication. As there is no evidence of gene amplification (Tjia, S. T. , et al., 1979, Virology 99: 399-
- polyhedrin promoter is an extremely efficient one.
- Cloning and sequencing the mutant polyhedrin gene can b accomplished by any technique known in the art (Maniatis, T. , 0 et al. , 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, New York) .
- the polyhedrin DN may be cleaved by restriction enzyme digestion, DNase 5 digestion, physical shearing, etc.
- Identification of the polyhedrin DNA can be accomplished in a number of ways, including, but not limited to, nucleic acid hybridization, comparison of restriction digestion patterns with known restriction maps, and mRNA selection through nucleic acid hybridization followed by jLn vitro translation.
- the polyhedri 0 DNA, or total baculovirus DNA is inserted into the cloning vector which is used to transform appropriate host cells so that many copies of the polyhedrin sequences are generated. This can be accomplished by ligating the polyhedrin DNA into a cloning vector with complementary cohesive termini, with or 5 without first ligating linkers onto DNA termini in order to generate desired restriction sites, or blunt-end ligation, homopolymeric tailing, etc. Any of a large number of vector- host systems may be used. Vector systems may be either plasmids or modified viruses, but the vector system must be 0 compatible with the host cell used. Recombinant molecules ca be introduced into cells via transformation, transfection, or infection. Identification of a cloned polyhedrin gene can be achieved by any technique known in the art. Such techniques
- __ include, but are not limited to, screening for expression of o the gene by colony blot analysis (Huynh, T.V. , et al. , 1985, DNA Cloning: A Practical Approach, Vol. 2, Glover, D.M. -24- present invention to express a foreign peptide on or within occlusion body.
- colony blot analysis Huynh, T.V. , et al. , 1985, DNA Cloning: A Practical Approach, Vol. 2, Glover, D.M. -24- present invention to express a foreign peptide on or within occlusion body.
- a baculovirus that produces a truncated polyhedrin protein which still forms occlusion bodies, can analyzed by cloning and sequencing its polyhedrin gene. Regi that are non essential for crystallization may be identified comparison of the truncated sequence to that of other known polyhedrins.
- the corresponding region of the full length polyhedrin gene could be replaced, or heterologous sequences could be inserted into the appropriate region of the truncat gene, in order to express a new antigenic determinant.
- One example of a mutant which has been isolated and may be analy in such a fashion is a mutant AcMNPV which may differ from other polyhedrins by a small deletion of 20-30 amino acids. This AcMNPV produces tetrahedral rather than polygonal occlusion bodies, which contain a 31 kD polyhedrin protein rather than the 33 kD wild-type polyhedrin.
- M5 Another AcMNPV mutant, termed M5, has been described (Carstens, E.B., 1982, J. Virol. 43:809-818; Carstens, E.B., al., 1986, J. Virol. 58:684-688). M5 has a single point -23- expected given the similar functional and structural roles of these proteins in the different viruses. One might expect th the conservation of certain regions is essential for crystal formation. However, there are small regions where the amino acids
- regions of the polyhedrin g which are variable and which encode alpha-helical domains may be inserted into or replaced by a sequence encoding a foreig epitope that is also alpha-helical and which may be
- amphipathic so that a recombinant protein is produced which allows both the OB and the epitope within it to retain their structural integrity.
- polyhedrin gene sequence which is both variable and encodes an alpha-helical region, is that encoding amino acids 37-49; see the discussio on . o ⁇ infra and section 5.1.1.4.).
- amino acid sequence comparisons of a number of baculovirus polyhedrin proteins reveal a high degree of sequence homology among these differe proteins (FIG. 2) .
- HSSNPV Heliothis zea
- 35 mori (BmMNPV) and Autoqrapha californica (AcMNPV) polyhedrin proteins are 80-85% homologous.
- the sequence homology is -21- may be especially useful in an embodiment of the invention employing recombinant OBs in a vaccine formulation, since they would readily provide for presentation of the foreign epitope to the host immune system.
- Portions of the polyhedrin gene which encode hydrophilic regions are prime candidates for insertion into or replacement by a heterologous gene sequence, since the foreign epitope inserted therein is thus likely to b immunogenic in a vaccine formulation.
- the heterologous gene sequence can be inserted into the polyhedrin gene sequenc so that the heterologous gene either interrupts or replaces al or a portion of nucleotide residue numbers 1 to 12, or 142 to -20- integrity of the crystalline lattice.
- the identification of modifiable domains can be accomplished by comparing sequence of cloned polyhedrin genes, analyzing polyhedrin genes encod truncated polyhedrin proteins, and by structural analysis of
- 5 polyhedrin amino acid sequences Exposing the heterologous gene product on the surface of the OB may be preferable (but not required) for formulation in vaccines. Surface domains the OB may be more amenable to alteration without concomitan destruction of crystal integrity than internal domains.
- the Q identification of polyhedrin surface domains can be accomplished by hydrophilicity analysis of polyhedrin, and b generating and characterizing monoclonal antibodies to OBs. Segments of the sequence which are hydrophilic and hypervariable regions are prime candidates for insertion int 5 or replacement with the foreign sequence of interest. Howev sequences of the polyhedrin gene which encode hydrophobic regions of the polyhedrin protein may also be inserted into replaced by heterologous sequences.
- hydrophobic and hydrophilic regions of the polyhedrin amino acid sequence and the corresponding regions of the gene sequence which encode the hydrophilic and hydrophobic region should be identified. Since the hydrophilic regions of the amino acid sequence are likely to be external domains of the crystal, and, furthermore, are likely to be external domains the polyhedrin monomer upon crystal dissolution, such region -19- According to one embodiment of the invention, the recombinant OB which contains an immunogenic determinant of a pathogenic microorganism can be used in vaccine formulations.
- the recombinant OBs which expose an active site of an enzyme can b used as immobilized enzyme in appropriate procedures.
- the recombinant OBs of the presen invention have uses in immunoassays and as expression vectors.
- the production of recombinant polyhedrin crystals can also facilitate the isolation.of the component heterologous gene product in substantially pure form.
- the method of the invention may be divided into the following general stages solely for the purpose of description (a) identification of modifiable domains encoded by the polyhedrin gene, (b) identification and characterization of immunodominant peptides for expression on or within recombinan occlusion bodies, (c) construction of recombinant polyhedrin genes, (d) selection _>f recombinant occlusion bodies, (e) verification of expression of foreign epitopes on or within th recombinant occlusion body, and (f) determination of immunopotency of foreign epitopes expressed on or within recombinant occlusion bodies.
- Exposing new antigenic determinants on the surface of within the OB requires identifying regions of the polyhedrin protein that can be modified without affecting the formation O stability of the lattice. Such segments can be altered by th insertion of new epitopes without interfering with the -18- Figure 16A.
- This vector contains an altered polyhedrin gene in which the polyhedrin sequence between amino acid residue numbers 43 through 50 were replaced with an epitope of influenza hemagglutinin.
- Figure 16B The construction of two transfer vectors pAV15-InHem43 and pAV15InHem-50. These contain the epitope influenza hemagglutinin at positions 43 and 50, respectively of the polyhedrin sequence.
- Figure 17A and 17B The construction of vector pAV17 InHem-1 in which the epitope of influenza hemagglutinin is located after amino acid residue number 1 of polyhedrin. Th plasmid encodes a unique SphI restriction site at the initiation codon of polyhedrin.
- Figure 18 The construction of vector pAV17b InHem-2 which the epitope of influenza hemagglutinin is located afte amino acid residue number 2 of polyhedrin.
- FIG. 19 The construction of pBRX13.
- This plasmid can be used to insert a coding sequence for any epitope into the polyhedrin gene spanning the coding region for amino aci residues 36-50.
- the resulting recombinant polyhedrin gene c be excised from pBRX13 and cloned into a transfer vector.
- the present invention relates to recombinant baculoviruses which encode fusion polyhedrin proteins capabl of forming occlusion bodies containing foreign peptides.
- Th recombinant OBs of the present invention comprise crystalliz polyhedrin fusion proteins which bear the heterologous gene product on the surface of or within the occlusion body.
- the recombinant OBs are formed by replacing regions of the baculovirus polyhedrin gene that are nonessential for occlus body formation with sequences encoding foreign peptides.
- Th present invention is also directed to vector/host systems wh can express the recombinant polyhedrin gene in different hos including but not limited to, cultured cells, larvae, or microorganisms. -17- identical. Staining procedures for the isozymes are described infra.
- FUM Fumarate Hydratase
- LDH Lactate Dehydrogenase
- EST Esterase.
- Figure 15 The construction of a transfer vector containing a foreign DNA sequence encoding amino acids 98-106 of the influenza hemagglutinin inserted at a specific Hpall site within the Autographa polyhedrin gene.
- This transfer vector can be used to produce recombinant Autographa viruses containing the influenza sequence via in vivo recombination, which express the foreign sequence under the control of the transfer vector
- FIG. 8 A linear genomic map of the plague-purifie strain, HzS-15.
- the genomic map was made with virion purifi DNA digested either singly or with combinations of BamHI, Ps and SstI. Any ambiguities in the map were resolved by doubl digestions of cloned BamHI and PstI fragments.
- the genomic of Knell and Summers (1984, J. Gen. Virol. 65:445-450) was u as a reference since the restriction endonuclease banding pattern for BamHI was identical for both isolates.
- FIG. 9 Structural proteins of HzSNPV Elcar"' isola and plaque purified strains. Sucrose gradient purified viri were electrophoresed for 4.5 hours on a 12% SDS-polyacrylami gel. The position and size of major wild-type proteins are labeled on the left, while unique proteins found in several the plaque-purified strains are labeled on the right.
- FIG. 10 Cell growth curves for clonally isolated cell strains derived from IPLB-HZ1075. Three defined regions of a tissue culture flask (25 cm 2) were counted at 48 hour intervals for a total of 8 days. Points on the graphs represent the average of the three counted areas with the err bars indicating 1 standard deviation. Letters in the lower left corner of each graph correspond to the nomenclature of t specific cell strain.
- FIG. 11 Comparison of isozyme banding patterns between all IPLB-HZ1075 derived cell strains and Heliothis ze larvae with isozymes FUM, LDH, and EST. Cell and larval extracts were electrophoresed in a 5% polyacrylamide gel (95% acrylamide, 5% bis-acrylamide) in either TBE or TC buffer and stained for the appropriate enzyme. Staining for FUM and for
- LDH confirm that the cell strains were derived from the parental IPLB-HZ1075 (W + ) cell line and that they are ultimately derived from Heliothis zea larvae.
- the difference in the EST gels suggest that all of the strains are not -15- sequence) , Rl (EcoRI) , Sst (SstI) , Sma (Smal) , Bm (BamHI) Xb
- Phe2.61ac which contains a functional E ⁇ coli beta- galactosidase gene.
- Phe2.61ac can be used to insert other foreign genes or fragments thereof within the Heliothis polyhedrin gene, which can then be transferred to the Helioth virus genome by in vivo recombination.
- the following Q abbreviations are used in the figure: Xba (Xbal) , Bam (BamHI) , Kpn (Kpnl) , Sma (Smal) , Oligo (Oligonucleotide) , and MCS (Multiple Cloning Site) .
- FIG. 5C The construction of transfer vectors which contain deletions in the amino terminal-coding region of the
- Heliothis polyhedrin gene These transfer vectors can be use to insert foreign genes within the polyhedrin gene and to produce H viruses via in vivo recombination.
- the following abbreviations are used in the figure: Xba (Xbal) , bam (BamHI) , kpn (Kpnl) , sma Smal) , sph (SphI) , bgl (Bgll) , and pst (PstI) Figure 6.
- HindiII restriction endonuclease analysis o
- Viral DNA was purified from band isolated virions as described in t methods. Viral DNAs were digested with Hindlll and fractionated on a 0.75% agarose gel. Most of the differences between the wild-type (W ) and plaque purified (1-25) strains
- FIG. 7 Comparison of the wild-type ElcarTM isolate and HzS-15 strain with enzymes BamHI, EcoRI, EcoRV, Hindlll,
- FIG. 4 Polyhedrin polylinker sequence.
- a syntheti polylinker gene sequence and its encoded amino acids are depicted. This gene segment encodes the Autographa californi polyhedrin gene extending from the amino terminus to the BamH site corresponding to amino acid 58. Restriction endonucleas digestion sites are indicated below the DNA sequence. The Pvul, Seal, Bell, and Xbal sites correspond to amino acids 9, 19, 27, and 46, respectively.
- Expression Vector An expression vector comprising a polyhedrin promoter and a heterologous gene sequence positioned under the control of the polyhedrin promoter so that the heterologous 0 gene is expressed in a suitable host.
- FIG. 1A Restriction map of the Heliothis polyhedri gene. A restriction endonuclease digestion map of the
- Numbers in parentheses represent the number of the nucleotide in Figure 1.
- Cassette Transfer Vector A transfer vector comprising a polyhedrin promoter and a restriction enzyme recognition site into which a heterologous gene sequence can be inserted under the control of the polyhedrin promoter, in which a polyhedrin promoter and the restriction site are flanked by sequences that are homologous to parent vector sequences. Heterologous gene sequences can be inserted into the cassette transfer vectors which can then be used to construct recombinant expression vectors via homologous recombination in vivo with a parent vector.
- TE 10 mM Tris-HCl, ImM EDTA, pH 7.6
- TBE 81.2 mM Tris, 20 mM boric acid, 1.5 mM
- OB Occlusion body, a paracrystalline protein matrix which occludes baculovirus virions.
- the paracrystalline protein matrix forms a refractile body which is polyhedral, cuboidal or spherical in shape.
- OB will also be used hereinafter to refer to lattices formed in vitro by the recrystallizatio of soluble polyhedrin.
- NPV Nuclear Polyhedrosis Viruses, a subgroup of the baculovirus genus in which the nucleocapsids are enveloped by a lipoprotein membrane singly (SNPV) o in multiples (MNPV) per common envelope. Up to 100 of these virion packages are embedded in an occlusion body, polyhedral to cuboidal in shape and 1-15 um in diameter.
- GV Granulosis Virus, a subgroup of the baculovirus genus in which one singly-enveloped nucleocapsid is embedded per occlusion body, round to ellipsoidal i shape, and 0.1-1 um in size.
- OBs recombinant occlusion bodies
- the recombinant viruses of the present invention can also- be used as expression vectors for the production of the foreign peptide(s) contained on the recombinant OB.
- the production of recombinant OBs can also facilitate the isolation of the component heterologous gene product in substantially pure form.
- the invention is demonstrated by way of examples in which recombinant baculoviruses were engineered to express recombinant occlusion bodies that present an influenza hemagglutinin epitope. These recombinant occlusion bodies
- Vaccines are presently available for diphtheria, pertussis, and tetanus (Warren, K. S., 1985, In Vaccines85, Lerner, R. A., R. M. Chanock, and F. Brown (eds.), Cold Sprin Harbor Laboratory, New York, pp. 373-376) .
- a vaccine consisting of the polysaccharide capsule of Hemophilu 0 influenzae was recently licensed, although it is ineffective in preventing disease in certain subgroups of the population (Granoff, D. M. and Munson, R. S., Jr., 1986, J. Infect. Dis.
- the present invention is directed to recombinant baculoviruses which encode fusion polyhedrin proteins capable of forming occlusion bodies containing foreign peptides.
- the recombinant baculoviruses of the invention are formed by insertion into or replacement of regions of the polyhedrin
- the present invention also relates to vector/host systems which can direct the expression of the recombinant polyhedrin genes 3C . in different hosts, including but not limited to, cultured cells, larvae, or microorganisms. -8- contains those epitopes which are capable of eliciting neutralizing antibodies; these include the glycoproteins of
- Vaccines are often administered in conjunction with various adjuvants.
- the adjuvants aid in attaining a more durable and higher level of immunity using smaller amounts o antigen in fewer doses than if the immunogen were administer alone.
- the mechanism of adjuvant action is complex and not completely understood. However, it may involve the stimulation of phagocytosis and other activities of the reticuloendothelial system as well as a delayed release and degradation of the antigen.
- adjuvants include
- VACCINES FOR VIRAL INFECTIONS A number of methods are currently in- use for the prevention and treatment of viral infections. These include vaccines which elicit an active immune response, treatment with chemotherapeutic agents and interferon treatment.
- Attenuation refers to the production of virus strains which have essentially lost their disease producing ability.
- One way to accomplish this is to subject the virus to unusual growth conditions and/or frequent passage in cell culture.
- subunit vaccines An alternative to the above methods is the use of subunit vaccines. This involves immunization only with those proteins which contain the relevant immunological material.
- the virally encoded glycoprotein -6- Recombinant baculoviruses have been produced by cotransfection of cells with recombinant bacterial pla ⁇ mids containing the foreign gene, together with baculovirus DNA.
- the foreign gene is inserted into or replaces the nonessenti polyhedrin gene of the viral genome through homologous recombination within the infected cell.
- the resulting recombinant plaques can be screened visually for lack of occlusion bodies resulting from the loss of the functional polyhedrin gene.
- the infected cells can also be screened using immunological techniques, DNA plaque hybridization, or genetic selection for recombinant viruses which subsequently can be isolated. These baculovirus recombinants retain thei essential functions and infectivity.
- Foreign gene expression can be detected by enzymatic immunological assays (for example, immunoprecipitation, radioimmunoassay, or immunoblotting) .
- High expression level can be obtained by using strong promoters or by cloning multiple copies of a single gene.
- Plasmid vectors also called insertion vectors, have been constructed to insert chimeric genes into AcNPV.
- an insertion vector is composed of: (a) an AcNPV promoter with the transcriptional initiation site; (b) severa unique restriction endonuclease recognition sites located downstream from the transcriptional start site, which can be used for the insertion of foreign DNA fragments; (c) AcNPV DN sequences (such as the polyhedrin gene) , which flank the U promoter and cloning sites, and which direct insertion of the chimeric gene into the homologous nonessential region of the virus genome; and (d) a bacterial origin of replication and antibiotic resistance marker for replication and selection in
- the isolation and characterization of monoclonal antibodies to intact OBs or to recrystallized polyhedrin will 5 identify regions of the polyhedrin protein that are exposed o the surface of the crystal. Once these regions are identifie it will be possible to test whether these domains can be altered without interfering with the integrity of the crystalline lattice. 0
- the occlusion bodies of NPVs are " surrounded by a carbohydrate envelope (Minion, F.C., et al., 1979, J. Invert. Pathol. 34:303) that may affect the immunogenicity of the OB. Therefore, in order to avoid such interference, monoclonal antibodies can be prepared to recrystallized polyhedrin, in 5 addition to using the purified OBs.
- An 11S-13S polyhedrin aggregate can be purified from alkali solubilized occlusion bodies and recrystallized by standard techniques (Shigematsu, H. , and Suzuki, S., 1971, J. Invert. Pathol. 17:375).
- OBs can be purified from a number of host cells by kno techniques (for example, see Section 7.1.4. infra, and Tweete
- such cell lines include, but are not limited to. Spodoptera frugiperda IPLB-SF-21AE cells, Heliothis zea IPLB
- HZ1075 cells Estigmene acrea BTI-EAA cells, Trichoplusia ni
- solubilized polyhedrin can be stored at -20°c
- BALB 5 mouse monoclonal antibodies can be prepared by use of a fusi protocol utilizing the BALB/c myeloma cell line, NS-1, and t fusagent, polyethyleneglycol 1000, as described by Ploplis e al. (Ploplis, V.A. , et al., 1982, Biochemistry 21:5891).
- Hybridomas producing antibodies to OBs can be identified preferably by use of a solid-phase immunoassay with a labelle ligand, such as an enzyme-linked immunosorbent assay (ELISA)
- ELISA enzyme-linked immunosorbent assay
- an epitope on the monomeric polyhedrin molecule that can be recognized by a monoclonal antibody generated to the OB or to recrystallized polyhedrin, Q can then be detected by use of a second antibody, directed against the monoclonal antibody, conjugated to a label such a an enzyme or radioisotope.
- a label such as an enzyme or radioisotope.
- horseradish peroxida can be used, in which case visualization of the antigen- antibody complex can be facilitated by using the enzyme
- protease digestion of polyhedrin can be accomplished by use of V8 protease (Brown, M. , et al., 1980, J. Gen. Virol. 50:309), although any protease known in the art such as trypsin, 5 chymotrypsin, papain, or pepsin, among others, can be used.
- Peptides generated by protease digestion are preferably isolated by HPLC utilizing reverse phase chromatography, although any standard techniques which result in the purification of the peptides can be used.
- the peptides can
- Monoclonal antibodies directed toward an OB antigenic determinant can be analyzed for crossreactivity to other polyhedra species. This can be most easily accomplished by an ELISA method although other immunoassays including, but not limited to, Western blotting, immunoprecipitation, and radioimmunoassays are within the sco
- Species-specific epitopes exposed on the surface of the crystal represent potentially modifiable regio of the polyhedrin protein.
- Some such species-specific monoclonal antibodies to OBs have been described (Huang, Y. S et al., 1985, Virology, 143:380), suggesting that there are Q epitopes which can be varied on the occlusion body surface.
- Another method for mapping epitopes on the polyhedri protein is by comparing proteolytic digests of polyhedrin in the presence and absence of the monoclonal antibody. It has been shown that epitopes are protected from proteolysis in th 5 presence of their respective antibodies (Jemmerson, R. , and Paterson, Y. , 1986, BioTechniques 4:18). Protease-digested fragments can be generated by known methods in the art, including, but not limited to, the use of proteases such as trypsin, chymotrypsin, V8 protease, papain, pepsin, etc.
- Protease-generated peptides can be identified by various techniques, including, but not limited to, reverse-phase chromatography and two-dimensional gel electrophoresis.
- tryptic peptides of polyhedrin can be identified using reverse-phase
- the digestion patterns of a nonspecific immunoglobulin molecule, digested in the presence of polyhedrin, can also be determined, in order to identify thos peptides which are derived from the immunoglobulin molecule that have retention times, electrophoretic migrations, or oth
- the thus identified polyhedrin-specific an immunoglobulin-specific digestion patterns can be compared to the digestion patterns obtained from the polyhedrin- antipolyhedrin complex.
- the antibody will protec 3 some proteolytic cleavage sites and reduce the recovery of peptides containing the epitope.
- These peptides, putatively comprising the epitope can be isolated and characterized by sequence analysis. Due to probable steric hindrance of the antibody on overall proteolysis of the protein, this proceedu could potentially identify some peptides which are not invol in antibody binding. Therefore, a variety of other protease can be used.
- proteases include, but are not limited to trypsin, chymotrypsin, V8 protease, papain, and pepsin.
- the generation of recombinant OBs which contain one or more foreign antigenic determinants, for use in vaccine formulations or immunoassay requires the identification and characterization of specific antigenic determinants which may be used in constructing recombinants.
- a peptide or protein should be identified which encodes an im unopotent sequence of a pathogenic microorganism.
- the peptide should be capable of eliciting an immune response against a pathogen.
- molecules which are hapten i.e.
- antigenic, but not immunogenic may also be used, sinc the polyhedra functions as a carrier molecule in conferring immunogenicity on the hapten.
- Peptides or proteins which are known to encode antige determinants can be incorporated into recombinant polyhedra. If specific antigens are unknown, identification and characterization of immunoreactive sequences should be carri out.
- One way in which to accomplish this is through the use monoclonal antibodies generated to the surface molecules of t pathogen. Such a technique has been used to help identify an characterize the major epitopes of myoglobin (Berzofsky, J.A. et al., 1982, J. Biol. Chem. 257:3189), lysozyme (Smith-Gill,
- the peptide sequences capable of being recognized by the antibodi are defined epitopes. These peptide sequences can be identified, for example, by virtue of the ability of small synthetic peptides containing such sequences, to compete with the intact protein for binding of monospecific antibodies.
- small synthetic peptides conjugated to carrier molecules can be tested for generation of monoclonal antibodi that bind to these sites, encoded by the peptide, on the inta molecule.
- Such an approach has been used for the recognition of an immunodominant peptide determinant in the influenza hemagglutin protein (Wilson, I. ., et al., 1984, Cell 37:767)
- epitopes are identifie by their protection from proteolysis in the presence of thei respective antibodies.
- cohesi termini are generated by restriction endonuclease digestion further modification of DNA before ligation may be needed. however, cohesive termini of the polyhedrin DNA are not available for generation by restriction endonuclease digest or different sites other than those available are preferred any of numerous techniques known in the art may be used to accomplish ligation of the heterologous DNA at the desired sites. For example, cleavage with a restriction enzyme can followed by modification to create blunt ends by digesting or filling in single-stranded DNA termini before ligation.
- the cleaved ends of the polyhedrin or heterologous DNA can be "chewed back" using a nuclease such nuclease Bal 31, exonuclease III, lambda exonuclease, mung nuclease, or T4 DNA polymerase exonuclease activity, to nam but a. few, in order to remove portions of the sequence.
- a nuclease such nuclease Bal 31, exonuclease III, lambda exonuclease, mung nuclease, or T4 DNA polymerase exonuclease activity, to nam but a. few, in order to remove portions of the sequence.
- An oligonucleotide sequence which encodes one or more restrict sites that are unique to the polyhedrin gene sequence and/oi the baculoviral genome itself can be inserted in a region o the polyhedrin gene that is nonessential for crystallizatio
- poly(ethylene glycol) linker (hereinafter this oligonucleotide linker will be referred t a polylinker) .
- the polylinker can be inserted into the polyhedrin sequence by in vitro techniques such as those discussed supra.
- the resulting recombinant gene is akin to
- cassette vector into which any heterologous gene can be inserted using appropriate restriction enzymes.
- heterologous gene into the cloning site located within the region of the polyhedrin gene sequence that is non-essential for crystallization, so that both sequences are in the corre translational reading frame uninterrupted by translational s signals, will result in a construct that directs the product of a fusion polyhedrin protein that will crystallize and for recombinant occlusion bodies.
- a polylinker may also be used generate suitable sites in the heterologous gene sequence.
- polyhedrin or heterologous gene sequences can b mutated _in vitro or in vivo in order to form new restriction endonuclease sites or destroy preexisting ones, to facilitate in vitro ligation procedures.
- Any technique for mutagenesis known in the art can be used, including but not limited to, i vitro site-directed mutagenesis (Kunkel, 1985, Proc. Natl.
- One specific embodiment of the invention is a strategy for replacing the region of the AcMNPV polyhedrin gene encodi amino acids 37-49 with oligonucleotides encoding an epitope o influenza hemagglutinin.
- a deletion strain can be constructed by cleavage with BamHI followed by digestion with exonuclease Bal 31, and ligation to a synthetic BamHI polylinker. In this way, we constructed a deletion strain in which DNA sequences encoding amino acids 35 through the BamHI site were replaced with a synthetic BamHI linker.
- the plasmid containing this gene can then be cut at the BamHI site, "blunt-ended" with either SI o mung bean nuclease, and ligated to the termini of the followi synthetic oligonucleotide:
- Nru I Bglll Xba I The synthetic oligonucleotide is thus inserted into the polyhedrin gene, and, by virtue of its encoded unique restriction sites, makes more cleavage sites available for recombination purposes.
- the clone thus generated contains the AcMNPV polyhedrin gene from the amino terminus to amino acid 37, the oligonucleotide with unique Nrul, Bglll, and
- Nrul and Xbal sites would generate a gene fusion in which the influenza hemagglutinin epitope extending from amino acid 98-106 would be inserted in frame between amino acids 38 and 50 of the Autographa polyhedrin gene.
- Alternative strategies for inserting the foreign oligonucleotide include, but are not limited to, insertion of a Bglll site at amino acid 43 by changing nucleotide 127 of the Autographa polyhedrin gene from a G to a T by in vitro mutagenesis (Kunkel, 1985, Proc. Natl. Acad. Sci. 82:488-492; Hutchinson, C. , et al., 1978, J. Biol. Chem. 253:6551). Synthetic oligonucleotides can then be inserted between the unique Bglll and BamHI sites.
- a DNA synthesizer e.g. , Applied Biosystems Model 380A
- Si ilar strategies may be used to recombine other regions of the polyhedrin gene. In vitro mutagenesis followed by insertion of synthetic polylinkers can enable manipulation of most r if not all, of the regions of the polyhedrin gene.
- an M13 phage, a phagemid which contains the polyhedrin DNA inserted within its genome.
- Specific cleavage at a particular restriction site within the DNA is accomplished by annealing a complementary synthetic oligonucleotide (oligo-1) to the single-stranded DNA, before restriction digestion. This annealing creates the requisite double-stranded region for recognition and cleavage by the restriction endonuclease. After cleavage, the single- stranded linear DNA can then be isolated by known techniques (e.g. heat denaturation and column chromatography) .
- An oligonucleotide with a sequence encoding a foreign epitope can also be synthesized (termed hereinafter oligo 2) .
- oligo 3 Another oligonucleotide can then be synthesized (termed hereinafter oligo 3) which is complementary to oligo 2 and which, in addition, has 5' and 3' termini which extend beyond oligo 2 which are complementary to the single- stranded termini of the polyhedrin DNA. Oligo 2 and oligo 3 can then be annealed together, followed by ligation of the duplex to the single-stranded polyhedrin DNA. Transfor ation of a suitable vector host such as E. coli will produce a recombinant transfer vector which contains the DNA encoding a foreign peptide inserted at a specific restriction site within the polyhedrin gene.
- DNA can be cotransfected into cells susceptible to infection, where in vivo recombination will then take place, producing the recombinant virus of the invention.
- the transfeetions can be accomplished by any procedures known in
- calciu - 25 phosphate precipitation method see, for example, Smith, G. , et al., 1983, J. Virol. 46:584), treatment with polybrene and dimethyl sulfoxide (Kawai, S. and Nishizawa, M. , 1984, Mol. Cell. Biol. 4:1172), or electroporation (e.g. , Kuta,
- a cassette vector can be constructed which comprises a polylinker sequence inserted within a region of the polyhedrin gene that is nonessential for
- This recombinant polyhedrin gene can then be transferred to a baculovirus by in vivo recombination within baculovirus-infected cells, producing a "cassette- expression" virus.
- the genome of this cassette-expression virus can be isolated for in vitro recombination purposes, in which insertion or replacement of polyhedrin regions which are nonessential for crystallization, by a heterologous sequence, is facilitated by virtue of the polylinker.
- baculoviruses that produce recombinant occlusion bodies may be constructed via recombination by replacing or interrupting regions of the polyhedrin gene sequence, that are nonessential for crystallization, .with the heterologous gene sequence so that the sequences are not interrupted by translational stop signals. Since the gene products of these recombinants will be expressed as recombinant occlusion bodies, it is preferred to use an OB- parent baculovirus strain, in order to select OB+ virus plagues against an OB- background. Viruses generating OBs can be detected in plaque assays among the large number of parental viruses which fail to make OBs, since 0B+ viruses form more refractile plaques than OB- viruses.
- an OB+ background would be preferred where the recombinant OBS form plaques that are less refractile than those formed by wild type viruses.
- the recombinant OBs expressed by InHem-43 and InHem-50 detailed in the Examples, infra are morphologically very different from wild type occlusion bodies. These recombinants were found to produce cuboidal occlusion bodies that express the foreign epitope.
- the cuboidal recombinant OBs formed placques which were less refractile than those produced by wild type virus.
- Selection can also be done on the basis of physical, i•mmunological, or functional properties of the inserted heterologous gene product.
- an enzyme-linked - I- immunosorbent assay ELISA
- ELISA enzyme-linked - I- immunosorbent assay
- selection may be done on the basis of enzymatic activity. Staining techniques based on chemical reactivity of the foreign peptide may be used. Many other techniques known in the art can be used, depending on the foreign sequence expressed, and are within the scope of the invention.
- a second gene encoding a selectable marker
- the selectable marker can also be introduced into a region of the baculovirus genome which is not essential for crystallization.
- the selectable marker Prior to transfer to the baculovirus genome, the selectable marker may exist as a totally distinct DNA fragment or, preferably may be contained in adjacent sequences of the transfer vector containing the recombinant polyhedrin gene. The selectable marker should be cotransfected with the recombinant polyhedrin gene into the baculovirus where ⁇ n vivo recombination will occur.
- Another method for selection is to screen for presence of the heterologous DNA sequence inserted into the polyhedrin gene. This can be accomplished by techniques known in the art, such as nucleic acid hybridization to replica plaques (Benton, W.D. and Davis, R.W. , 1977, Science 196:180), and variations thereof.
- parental baculoviruses can be constructed which contain a selectable marker flanked by sequences homologous to those surrounding the recombinant polyhedrin.
- a parental baculovirus can be constructed containing a beta-galactosidase gene downstream of the polyhedrin promoter. In vivo recombination between the recombinant polyhedrin gene and the constructed parental strain will result in insertion of the recombinant polyhedrin gene by virtue of its homologies with the parental polyhedrin sequences surrounding the beta- galactosidase gene.
- the recombination which results in insertion of the recombinant polyhedrin gene and inactivation or replacement of the beta-galactosidase gene may be selected for by the lack of beta-galactosidase activity by known methods (Messing, J. , et al., 1977, Proc. Natl. Acad. Sci. U.S.A. e 74:3642). This selection can be accomplished against an OB- or OB+ background as previously described.
- a control selection experiment which may be done is to cotransfect with wild-type viral DNA (OB+) in order to detect, among the wild-type progeny, recombinants failing to make OBs.
- OB+ wild-type viral DNA
- the identification and characterization of the recombinant generated in this type of cotransfection represents a negative result, it will provide valuable information regarding what modifications of the polyhedrin gene interfere with lattice formation and are therefore unsuitable for the practice of the present invention.
- OBs can be purified by any standard technique (for example, see Section 7.1.4., infra, and Tweeten, K.A. , et al., 1981, Microbiol. Rev. 45:379-408).
- monoclonal antibodies directed against the recombinant polyhedrin protein can be used as an effective means of purifying the polyhedrin protein.
- isolated preparations of recombinant OBs can be solubilized, the recombinant polyhedrin protein purified by immunoaffinity chromatography (Goding, J.W. , 1983, Monoclonal Antibodies:
- the foreign gene product can be analyzed by assays based on physical, immunological, or functional properties of the product. Immunological analysis is especially important where the ultimate goal is to use the recombinant OBs that express the product in vaccine formulations and/or as antigens in diagnostic immunoassays.
- Antibodies to the peptide preferably monoclonal, can be tested for their ability to interact with the crystalline recombinant polyhedrin. This can be accomplished by various techniques known in the art including, but not limited to, an enzyme- linked immunosorbent assay (ELISA) method (for example, a solid-phase binding assay on polyvinyl chloride plates) , or a radioimmunoassay. Methods known in the art such as western blotting or immunoprecipitation procedures can be used to determine the presence of the peptide on the polyhedrin monomer.
- ELISA enzyme- linked immunosorbent assay
- Any baculovirus may be used as the parent for construction of the recombinant baculoviruses of the present invention.
- These vectors include but are not limited to
- NPVs and GVs are examples of NPVs which may be used in accordance with the present invention.
- NPVs which may be used in accordance with the present invention include but are not limited to AcMNPV, HzSNPV, Heliothis virescens NPV, S. littoralis NPV, Rachoplusia ou MNPV, Galleria mellonella MNPV, Lymantria dispar MNPV, Bombyx mori SNPV, orygia pseudotsugata SNPV and MNPV, Orygia leucostigma NPV,
- N. sertifer SNPV T. paludosa SNPV
- Trichoplusia ni MNPV Trichoplusia ni MNPV
- Spodoptera frugiperda MNPV Vlak, J.M. and Rohrmann
- GVs which may be used in accordance with the present invention include but are not limited to P. brassicae GV, Estig ene acrea GV, Choristoneura vindis GV,
- Mamestra oleracea GV Pseudaletia unipuncta GV, Pygera anastomosis GV, S_. frugiperda GV, Zeiraphera diniana GV, and
- a plaque-purified isolate with a homogeneous genotype should be used as the parent baculovirus.
- a recombinant baculovirus can be constructed from parent viruses which possess particularly advantageous properties with respect to the host systems used in accordance with the present invention. For example, viruses which demonstrate high infectivity and high virus titers in the host system are preferred.
- Melanization is a normal response to viral infection which comprises the production of melanin, a pigment which is incorporated into the insect's cuticle, and appears to involve the polymerization of indol ring compounds derived by oxidation of tyrosine (Wigglesworth, V.B., 1974, in The Principles of Insect Physiology, Chapman and Hall, London, p. 610) .
- the tyrosinase which is involved in the melanization process appears to be abundant in the hemolymph of the insect and can react fairly non-specifically with available proteins.
- the tyrosinase activity in an insect carrying the recombinant baculoviruses of the invention may non- specifically metabolize the recombinant polyhedrin protein, interfering with and decreasing the yield and purity of the recombinant product.
- Melanization of occlusion bodies can cause subsequent chemical alteration of virion proteins and nucleic acids. Melanization can also severely reduce infectious extracellular virus titers in collected hemolymph, as well as poison cultured cells following inoculation. Thus non- elanizing or slow-melanizing host strains are preferred in order to avoid these problems.
- Heliothis zea cell lines Heliothis zea cell lines. It should be noted that this discussion is for descriptive purposes only and the scope of the invention includes many other baculoviruses, such as GVs and other NPVs.
- Heliothis zea SNPV may be used as the parent virus strain. Restriction digestion patterns of eight different geographic isolates of HzSNPV suggest each is a separate population of viruses having a slightly different predominant genotype, but none represents a totally unique virus species (Gettig and McCarthy, 1982, Virology 117:245-252) . Seven of the eight geographical isolates examined have similar major occluded virus structural protein profiles in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Monroe and McCarthy, 1984, J. Invert. Path. 43:32-40). Even though the eight Heliothis SNPV isolates are genetically and biochemically similar, several isolates exhibit significant differences in virulence towards H. zea larvae (Gettig and McCarthy, 1982, Virology 117:245-252).
- plaque purified strains derived from the ElcarTM isolate of HzSNPV originally isolated by Dr. J.J. Hamm, U.S.D.A., Tifton, GA.
- This region includes Hindlll fragments G, H, M, and N (see FIG. 6) . At least 15 of the twenty plaque-purified strains diverge from the wild-type strain in this region, with alterations in one or more of these Hindlll fragments.
- the genotype map of the HzS-15 strain differs from the wild-type map (Knell and Summers, 1984, J. Gen. Virol. 65:445-450) in the hypervariable region.
- the divergence in this strain is evident with the enzymes EcoRI, Hindlll, and SstI.
- Comparison of the maps of HzS-15 with those of the wild-type isolate shows that the divergence is caused by changes in the relative positions of several restriction sites, rather than in overall genome size or apparent organization.
- the variability between isolates is not limited to the genotype, but is also reflected in the structural proteins of the virions (see FIG. 9) .
- HOSTS USED IN THE VECTOR/HOST SYSTEMS
- the recombinant baculoviruses of the present invention can be used to direct the expression of the heterologous gene product in a number of host systems including but not limited to cell lines and larvae in which the virus can be propagated. Some useful cell lines and larval systems which can be used in accordance with the invention are described in the subsections below.
- Such cell lines include but are not limited to IPLB-SF-21AE (Spodoptera frugiperda cells); TN-368, BTI-
- TN4BI Trichoplusia ni cells; Granados, R.R., et al., 1986, Virology 152:472-476;
- ILPB-HZ1075 Heliothis zea cells; Goodwin, R.H. , et al.,
- the IPLB-HZ1075 cell line consists of a heterogeneous population of cells, and this heterogeneity seems to account in part for the inability to obtain 100% infection upon inoculation with HzSNPV.
- To obtain a more homogeneous response to infection through cloning of individual cell strains we subcloned and characterized twelve strains from IPLB-HZ1075 using dilution plating. All of the subcloned cell lines were similar to the parental cell line in that none of them were capable of 100% infection upon inoculation with the HzSNPV isolate HzS-15 under our culture conditions. Each cell strain exhibited slightly different growth kinetics (see FIG. 8) , predominant cell morphology, and ability to replicate virus (see Table VI infra) .
- Subcloned cell strains of TN-368 were distinguishable from the parental cell line with respect to cell doubling time (Faulkner, P., et al., supra) and ability to plaque Autographa californica MNPV (AcMNPV) (Volkman, L.E. and Summers, M.D., supra) , but none of the TN-368 subcloned cell strains replicated AcMNPV better than the uncloned parental cell line.
- the IPLB-HZ1075 UND-K cell line may be preferred for use in the expression vector/host systems of the present invention because of its ability to grow Heliothis virus quickly and at high titers which plaque, thus enabling identification.
- infectious cell culture supernatant can be stabilized by the addition of liquid agarose to a final concentration of 0.1%.
- virions can be isolated from the OBs according to procedures known in the art such as the technique described by Smith and Summers,
- Baculoviruses expressing the recombinant polyhedrin genes of the present invention can be propagated and/or mass-produced by infection of various host insect larvae.
- the propagation and isolation of baculoviruses using laboratory larval populations has been previously described (e.g. , Wood, H.A. , et al., 1981, J. Invertebr. Pathol. 38:236-241; Ignoffo, CM. and Garcia, C, 1979, Environ. Entomol. 8:1102-1104).
- Larva hosts which may be used in the propagation and production of viruses expessing recombinant polyhedrin genes include but are not limited to those species listed in Table I, infra. TABLE I
- Heliothis zea (Boddie) Trichoplusia ni (Huber) Galleria mellonella Spodoptera frugiperda Estigmene acrea Aedes aegypti Choristoneura fumiferana Heliothis virescens Autographa californica S. littoralis Rachoplusia ou Lymantria dispar Bombyx ori Orygia pseudotsugata Pseudohazis eglanterina N. sertifer T. paludosa P. brassicae Orygia leucostigma Choristoneura vindis Plodia interpunctella Choristoneura murinana Cirphis unipuncta L.
- JH juvenile hormone
- the production of recombinant polyhedrin crystals in bacterial cells has a number of attractive advantages. It would eliminate the need to transfer gene fusions into a baculovirus and to identify and characterize the resulting recombinant virus. In addition, it is cheaper and easier to grow large quantities of bacterial cells than to culture insect cells. Many different strains of bacteria and types of plasmids known in the art can be used in this embodiment of the present invention, as long as the host allows for appropriate expression of the recombinant polyhedrin gene of the vector.
- a "polyhedrin polylinker” sequence is created, a type of cassette vector, which can be ligated to the remainder of the parental polyhedrin gene, and which can be utilized to insert sequences encoding foreign epitopes at its unique restriction sites.
- a gene construction provides the potential to easily engineer a large number of changes into the polyhedrin gene.
- the gene construction can be designed with flanking restriction sites suitable for insertion into E. coli expression vectors. Insertion of the polyhedrin polylinker sequence into an E. coli expression vector will produce a cassette-expression vector which can greatly facilitate construction and expression, in bacteria, of a recombinant polyhedrin gene of the present invention.
- Such a construction is not restricted to use in E. coli; it can also be engineered for use in the baculovirus or other systems.
- One example of a polyhedrin polylinker is shown in
- Figure 4 shows a gene segment encoding the amino terminus to amino acid 58 (at the BamHI site) of the
- the unique restriction sites can facilitate the replacement of small regions in the 5' section of the gene with synthetic oligonucleotides encoding new antigenic determinants.
- replacing the segment between the Xbal site and either the BamHI or Bell site enables the insertion of new determinants in a putatively modifiable region between amino acids 37-49.
- the SphI site can be used to add determinants to the amino terminus of the protein.
- the unique EcoRI site immediately 5' to the synthetic gene permits cloning the fusion gene into an EcoRI site of an E. coli expression vector.
- a vector which may be used is the E. coli expression vector PK223-3
- PK223-3 plasmid construction would permit efficient regulated expression of genes inserted at its cloning site.
- Numerous other plasmids with suitable cloning sites and signals for expression may also be used.
- Construction and expression in a suitable vector/host system will determine whether the recombinant polyhedrin expressed in such a system will crystallize. If the protein will not crystallize in vivo, the solubilized polyhedrin 5 protein can be purified and crystallized in vitro
- Immunopotency of the foreign epitope expressed on or within recombinant occlusion bodies can be determined by monitorin the immune response of test animals following immunization with the recombinant OB.
- Occlusion bodies for immunization purposes can be obtained by purification from insects or insect cell cultures (for example, by the procedures of
- Test animals may include but are not limited to mice, rabbits, chimpanzees, and eventually human subjects.
- Methods of introduction of the immunogen may include oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, or any other standard route of immunization.
- the immune response of the test subjects can be analyzed by three approaches: (a) the reactivity of the resultant immune serum to the authentic pathogenic molecule or a fragment thereof containing the desired epitope, or to the isolated naturally occurring pathogenic microorganism, as assayed by known techniques, e.g. enzyme linked immunosorbant assay
- ELISA immunoblots, radioimmunoprecipitations, etc.
- b the ability of the immune serum to neutralize infectivity of the pathogen in vitro
- c protection from infection and/or attenuation of infectious symptoms in immunized animals.
- rabbits can be inoculated by a variety of protocols with recombinant
- mice can be inoculated intraperitoneally with the recombinant OBs, subsequently challenged by intranasal inoculation of virulent virus, and monitored for the onset of disease symptoms.
- VACCINES Recombinant OBs expressing epitopes of pathogenic microorganisms are particularly useful in the formulation of vaccines.
- the foreign epitope is exposed on the surface of the crystal. Since the crystalline lattice of the occlusion body is composed predominantly of the polyhedrin molecule, foreign epitopes within this molecule are presented a large number of times 5 on the surface of the OB. Recombinant OBs can be used in vaccine formulations even if the foreign epitope is not presented on the surface of the crystal but is internal, since alterations in crystallization properties (e.g.
- recombinant OBs may be especially advantageous when the heterologous peptide or protein to be Q used in a vaccine formulation is a hapten (i.e. , a molecule that is antigenic but not immunogenic) which ordinarily must be coupled to a carrier molecule that confers immunogeni- city.
- a hapten i.e. , a molecule that is antigenic but not immunogenic
- the production of recombinant OBs carrying the heterologous hapten on their surface using the expression 5 vector/host systems of the present invention would render the molecule immunogenic and eliminate coupling reactions.
- heterologous protein can be solubilized and recrystallized.
- the resulting OBs would bear each of the heterologous proteins and would be particularly useful as a multivalent vaccine.
- multivalent vaccines can be produced by engineering multiple epitopes into the
- a heterologous sequence encoding an amphipathic alpha-helical structure can be inserted into or replace portions of the polyhedrin gene which are nonessential for crystallization and which encode alpha- helical regions, which may be hydrophobic.
- Any protein epitope of a pathogenic microorganism which is capable of inducing an immune response specific to the microorganism can potentially be used in a recombinant OB vaccine formulation.
- Demonstration of the production of recombinant OBs which express the foreign epitope in an immunopotent state, as provided for by the present invention, is necessary prior to formulation as a vaccine.
- Potentially useful antigens for recombinant OB vaccine formulations can be identified by various criteria, such as the antigen's involvement in neutralization of the pathogen's infectivity (Norrby, E. , 1985, Summary, In
- the antigen's Q encoded epitope should preferably display a small or no degree of antigenic variation in time.
- the gene sequence encoding the epitope to be expressed on or within recombinant OBs may be obtained by techniques known in the art including but not limited to purification from genomic 5 DNA of the microorganism, by cDNA synthesis from RNA of the microorganism, by recombinant DNA techniques, or by chemical synthesis.
- Recombinant OBs have potential uses as vaccines for diseases and disorders of viral, parasitic, and bacterial Q origins.
- Many viral-specific antigens are known and can potentially be incorporated into the recombinant OB vaccine formulations of the invention.
- antigens, and/or portions thereof which encode the epitope(s) which may be used include but are not limited to influenza A 5 hemagglutinin; Hepatitis A virus VP1; Hepatitis B surface, core, or e antigens; retroviral envelope glycoproteins or capsid proteins; poliovirus capsid protein VP1; rabies virus glycoprotein; foot and mouth disease virus VP1; Herpes simplex virus glycoprotein D; Epstein-Barr virus 0 glycoprotein; pseudorabies virus glycoprotein; vesicular stomatitis virus glycoprotein, etc.
- the recombinant OBs of the present invention can comprise an epitope of the AIDS virus (HTLV-III/LAV/HIV) glycoprotein and/or capsid proteins.
- AIDS virus HTLV-III/LAV/HIV
- capsid proteins Such an embodiment may 25 be particularly useful in vaccinating against AIDS without concomitant induction of detrimental effects caused by the presence of the active AIDS virus glycoprotein such as the induction of T lymphocyte cell fusion and death.
- Vaccines85 Lerner, R.A., R.M. Chanock, and F. Brown (eds.), Cold Spring Harbor Laboratory, New York, pp. 1-5) , cholera toxin, diptheria toxin, and gonococci antigens.
- microbial genes which have been successfully cloned and may be used in recombinant OB vaccine formulations include but are not limited to, enterotoxin genes of E. coli, the toxin and filamentous hemagglutinin genes of Bordetella pertussis, and the circumsporozoite (CS) antigen of the malaria parasite Plasmodium falciparum (Norrby, E. , 1985, In Vaccines85, supra, pp. 387-394; interpreted, J.B., et al., 1985, In Vaccines85, supra, pp. 7-11) .
- the antibodies generated against pathogenic microorganisms by immunization with the recombinant OBs of the present invention also have potential uses in diagnostic immunoassays, passive immunotherapy , and generation of antiidiotypic antibodies.
- any immunoassay system known in the art may be used for this purpose including but not limited to competitive and noncompetitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assays) , "sandwich” immunoassays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement- fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays and immunoelectrophoresis assays, to name but a few.
- the vaccine formulations of the present invention can also be used to produce antibodies for use in passive immunotherapy, in which short-term protection of a host is achieved by the administration of pre-formed antibody directed against a pathogenic microorganism.
- Passive immunization could be used on an emergency basis for immediate protection of unimmunized individuals who have been exposed to a pathogenic microorganism, for instance, in hospitals and other health-care facilities.
- Human immunoglobulin is preferred for use in humans since a heterologous immunoglobulin will induce an immune response directed against its foreign immunogenic components.
- the antibodies generated by the vaccine formulations -o£ the present invention can also be used in the production of antiidiotypic antibody.
- the antiidiotypic antibody can then in turn be used for immunization, in order to produce a subpopulation of antibodies that bind the initial antigen of the pathogenic microorganism (Jerne, N.K. , 1974, Ann. rmmunol. (Paris) 125c:373; Jerne, N.K. , et al., 1982, EMBO 1:234) .
- BIOLOGICAL INSECTICIDES Baculoviruses are major pathogens of a large number of agricultural pests (Vlak, J.M. and Rohrmann, G.F., supra; Tweeten, K.A. , et al. , 1981, Microbiol. Rev. 45:379-408).
- one baculovirus host the corn earworm Heliothis zea, in many areas routinely damages 90-100% of the ears of sweet corn (Kirk-Othmer, Encyclopedia of
- the OB is the infectious particle responsible for transmission of the virus from organism to organism in the wild.
- the production of recombinant occlusion bodies in accordance with the present 0 invention thus provides for horizontal transmission of infection with concomitant expression of the foreign gene.
- Manipulation of the polyhedrin protein to incorporate enzymatic activities, toxic peptides, or any molecule with insecticidal activity can increase the lethality of the OB 5 to host agricultural pests.
- the recombinant OBs of the present invention have valuable applications as biological insecticides.
- Neuropeptides which are toxic or which induce detrimental behavioral modifications may be encoded within the polyhedrin gene.
- Sex pheromones which act as chemattractants may be used to increase spread of the baculovirus infection throughout the insect population.
- a chitinase incorporated into the OB may increase the infectivity of the virus.
- An endotoxin of another insect pathogen, such as the Bacillus thuringiensis endotoxin may be expressed in order to increase pathogenicity.
- the recombinant form of the insecticidal molecule is functionally active within the physiological environment of the infected insect.
- Metabolic precursors to insecticidal molecules may also be encoded by the recombinant polyhedrin gene, provided that the metabolic machinery to convert the peptide to a biologically active form is available and functional at the site of infection within the host insect.
- Any standard method can be used to assay lethality of the recombinant baculovirus. Such methods include but are not limited to the diet-surface technique and container, to bioassay OB activity (Ignoffo, CM., 1966, J. Invert. Pathol. 8:531-536; Ignoffo, CM. and Boening, O.P., 1970, J. Econ. Entomol. 63:1696-1697).
- the recombinant viruses which form occlusion bodies expressing heterologous peptides can be used generally as expression vector systems for the production of the foreign peptide(s) which they encode.
- the recombinant baculoviruses which express the foreign peptide under control of the polyhedrin promoter are used to infect an appropriate host cell in order to obtain the desired quantities of the heterologous peptide.
- the foreign peptide (which is a fusion polyhedrin protein) may be purified from the occluded virions, isolated occlusion bodies, cell culture media, infected larvae, etc.
- the recombinant OBs of the present invention may be used as antigens in immunoassays for the detection of antibodies to the epitope(s).
- the recombinant OBs may also be used to detect the same or related epitope(s) by competition assays.
- the recombinant OBs, or the foreign epitope(s) expressed by them may be used in any immunoassay system known in the art including but not limited to competitive and noncompetitive assay systems using techniques such as radioimmunoassays,
- ELISA enzyme-linked immunosorbent assay
- "sandwich” immunoassays precipitin reactions
- gel diffusion precipitin reactions immunodiffusion assays
- agglutination assays complement-fixation assays
- immunoradiometric assays fluorescent immunoassays
- protein A immunoassays protein A immunoassays
- i munoelectrophoresis assays to name but a few.
- the recombinant OBs of the invention are capable of capturing an precipitating antibodies specific for the foreign epitope(s) presented on the recombinant OBs.
- This is an attractive feature of the recombinant OBs which makes them particularly useful for the detection of antibodies in sample fluids, especially where the antibodies are presented at low concentrations.
- the recombinant OBs bound to their captured antibodies could be immobilized by anti- polyhedrin antibodies.
- the presence of the captured antibod can be detected using appropriate anti-immunoglobulin antibodies.
- a "sandwich" type of immunoassay for detecting different antibodies in sample fluids can be accomplished using "universal" capture (e.g. , antipolyhedrin) and detection antibodies (e.g. , anti-human Ig) .
- 5 binding region of protein G or A could be produced by cloning appropriate regions of those genes into the polyhedrin gene within the modifiable domains described herein. These recombinant OBs can be used to bind any antibody. The resulting recombinant OB/antibody complexes can then be used ⁇ n in immunoassays to bind and capture antigens in sample fluids. These could similarly be used in the "sandwich" type of assay system described above to detect antigens in sample fluids.
- the recombinant OBs of the present invention which lb express the active site of an enzyme on their surface can be used in a variety of procedures which require immobilized enzymes.
- the recombinant enzymatic OBs may be packed into a column on which reactions catalyzed by the enzyme can be carried out. The resulting products can easily be separated from the mixture of reactants and enzyme.
- Plasmid DNAs were digested with restriction 35 endonucleases Hindlll, EcoRI, PstI, Xbal, BamHI, Salll Xhol,
- Tris-borate 90 mM boric acid, 2 mM EDTA (pH 8.0), and 0.1 ug/ml of ethidium bromide. DNA bands were visualized with a ultraviolet transilluminator and photographed. Analyses of single and multiple digestions were used to construct the restriction maps.
- Filters were rinsed with distilled water and incubated at 37*C overnight with fresh prehybridization solution plus denatured labelled radioactive probes. Filters were rinsed in 0.2 X SSC and washed for one hour in prehybridization solution without Denhardts. Rinses and washes were repeated four times. Filters were dried and autoradiographed with Kodak X-Omat AR5 film.
- DNA sequences were determined using the dideoxy chain termination method with M13 subclones (Sanger, F. , Nicklen,
- sequencing primers Bethesda Research Laboratories or Pharmacia
- Primer extensions in the presence of the dideoxy nucleotides were initiated by adding 3 ul of the annealing mix to tubes containing the appropriate mix of dideoxy (dd) and deoxy " nucleotides.
- a reaction 1 ul of 0.5 mM ddATP and 1 ul 125 uM dCTP, dGTP and dTTP.
- G reaction 1 ul of 0.625 mM ddGTP and 1 ul 8 uM dGTP, 170 uM dCTP and 170 uM dTTP.
- Heliothis zea DNA was obtained from virus isolated from Heliothis zea infected larvae. Viral Hindlll and Xhol fragments were cloned into the Hindlll and Sail site, respectively, of pUC12. Two plasmids were characterized: pHH5 which contains a 3.1 kb Hindlll virus fragment and pHX which contains a 6.5 kb Xhol virus fragment. Both inserts cross-hybridized to a DNA fragment encoding part of the Autographa polyhedrin gene. Restriction maps of pHH5 and pHX12 demonstrated that the pHH5 insert is contained within pHX12.
- Fragment # Fragment size Starts at Ends at
- Fragments shown are those expected from the DNA sequence of the Heliothis polyhedrin gene shown in Figure 1, after digestion with Hindlll, Nrul, Hindi, and Accl.
- the DNA sequence of the subclones was compared with that of the Autographa polyhedrin gene in order to identify the coding sequence of the Heliothis polyhedrin gene.
- DNA sequence shown in FIG. 1 reveals an open reading frame of 753 nucleotides.
- the 7th codon of the open reading frame encodes a methionine which is followed by a sequence encoding
- Heliothis and Bombyx sequences are somewhat more divergent, sharing only 77% sequence homology.
- 52 amino acid substitutions there are two single amino acid insertions as well as two deletions in the
- MNPV Heliothis
- SNPV Heliothis
- Bombyx MNPV
- the pattern of hydrophilicity is very similar for the Autographa and Heliothis proteins (FIG. 3) .
- the region of highest hydrophilicity of the polyhedrin proteins is the region of greatest sequence divergence. 5
- sequence homology between the Autographa and Heliothis polyhedrins in the region between amino acids 38 and 50 of the sequence.
- the Autographa and Bombyx sequences share only 31% sequence homology, while the Heliothis and Bombyx sequences are 39% homologous in this Q region.
- these hydrophilic regions identify a site involved in some species specific interaction with other viral or cellular components.
- Small peptides generated from this region perhaps may be used 5 to raise monoclonal antibodies that could discriminate among different baculoviruses.
- the plasmids pHH5 and pHX12 were used to construct a transfer vector, termed pHE2.6, which allows for the insertion of foreign genes within the polyhedrin gene sequence so that recombinant Hz viruses containing the foreign genes can be produced via in vivo recombination.
- the preparation of pHH5 and pHX12 is described above in Section 6.2.
- the pHH5 plasmid contains a 3.1 kb Hindlll ou fragment of the Hz virus DNA (starting from nucleotide residue number 281 of the polyhedrin gene sequence depicted in FIG. 1) in the Hindlll site_of pUC12 (Vieira, J. and Messing, J. , 1982, Gene 19:259) (FIG. 5A) .
- the Hindlll Hz DNA insert of pHH5 encodes approximately two-thirds of the polyhedrin gene comprising the carboxy-coding region (i.e. , approximately one-third of the polyhedrin coding sequence comprising the a ino-coding region is missing) .
- the polyhedrin gene sequence is oriented so that the polylinker of the pUC12 parent plasmid is located upstream or 5' to the polyhedrin gene sequence (FIG. 5A) .
- the Xhol Hz DNA insert of the pHXl2 plasmid contains the entire polyhedrin gene sequence inserted into the Sail site of pUC12.
- the Hz polyhedrin gene sequence in pHX12 is oriented in the opposite direction with respect to the pUC12 polylinker as compared with the Hz polyhedrin coding sequenc
- pHH5 that is, the EcoRI site of the polylinker of pUC12 is located 3' to the polyhedrin gene sequence in pHX12 (FIG. 5A) .
- the pHX12 plasmid was cleaved with EcoRI and Nrul, and an approximately 1125 bp EcoRI-Nrul fragment, containing the promoter and amino-terminal portion of the Heliothis polyhedrin gene, was isolated. This 1125 bp fragment was
- a transfer vector termed pHE2.61ac, was constructed to contain the E. coli beta-galactosidase (B-gal) gene inserted within an MCS flanked by Heliothis polyhedrin 0 sequences (FIG. 5B) .
- B-gal E. coli beta-galactosidase
- FIG. 5B A 3 kb fragment of plasmid pMC1871
- Plasmid pHE2.6 was cleaved with Bglll, treated with bacterial alkaline phosphatase, and ligated (T4 DNA ligase)
- the resulting plasmids contained the B-gal gene in both orientations (5' to 3', and
- Plasmid pHE2.61ac has been used for transfections into Heliothis virus-infected cells in order to transfer the B-gal gene fusion into Heliothis virus. Once a Heliothis virus expressing beta-galactosidase is obtained, the virus can be used as the parental virus for further manipulations involving insertions and deletions of the polyhedrin gene, through transfection of parental virus-infected cells with transfer vectors such as plasmid pHE2.6. Selection of the appropriate recombinant viruses would be greatly facilitated by detection of white plaques amidst a background of blue plaques.
- Plasmid pHE2.6 was digested with EcoRI and PstI.
- the double-stranded replicative form of the resulting M13 derivative was digested with Xbal and Kpnl, which cut within the MCS, to generate a single-stranded 5' overhang at the Xbal cleavage site and a single-stranded 3' overhang at the Kpnl cleavage site.
- the resulting DNA was treated with Exonuclease III (exo III) which digested a single strand of the double-stranded DNA for a variable length in the 3' to 5' direction starting from the Xbal- cleaved end.
- the KpnI-cleaved end which has a 3' overhang is resistant to exo III digestion.
- the DNA was then digested with Mung Bean nuclease, which digests single- stranded DNA, to generate blunt ends by removing the single-stranded DNA left after exo III digestion.
- the blunt ends were ligated together with DNA ligase, resulting in transfer vectors that contain deletions of various length within the N-terminal portions of the Heliothis polyhedrin gene.
- the transfer vectors thus derived contain the Heliothis polyhedrin promoter, 5' polyhedrin regions of various length, an abbreviated MCS, and 3' polyhedrin sequences. Thus far, several N-terminal deletion transfer vectors have been obtained in this fashion.
- Transfer vector 1 has a 282 base pair (bp) deletion spanning nucleotide number 63 of Figure 1 through the Kpnl site.
- Transfer vector 2 has a 274 bp deletion spanning nucleotide number 71 of Figure 1 through the Kpnl site. Additional deletion mutations are being generated. Similar manipulations can be done at other suitable restriction sites in order to obtain deletions of regions that are nonessential for OB formation.
- Twenty plaque-purified strains of HzSNPV Elcar were characterized based on their restriction endonuclease digestion patterns of viral DNA and structural protein profiles. Each of the twenty strains had a unique genotype which was distinguishable by digestion with restriction endonucleases BamHI, EcoRI, Hindlll, or PstI. Most of the genomic heterogeneity between strains was located between map units 23.5 and 43.3. Differences were evident in the occluded virus structural protein profiles of all the plaque-purified strains relative to the wild-type isolate.
- HzS-15 The genotype of the weakly melanizing strain, HzS-15, was extensively characterized relative to the wild population genotype, using numerous restriction enzymes. A genomic map was constructed for HzS-15 using the enzymes BamHI, PstI, and SstI.
- Infectious extracellular virus was obtained from larvae five days post infection and before melanization. Hemolymph was collected by clipping a proleg and bleeding 10 larvae into 5 ml of TNM-FH medium (Hink,
- IPLB-HZ1075 cells (Goodwin, R.H. , et al., 1982, In Vitro
- Inoculation of cell cultures was accomplished by adding 5 ml of filtered inoculum to a 24 hour old monolayer of 1.0 x 10
- IPLB-HZ1075 cells in a tissue culture flask (25 cm ) . After one hour at 29"C, the inoculum was removed and the monolayer washed once with fresh media. Five ml of TNM-FH was added and the cultures were incubated at 29*C and monitored at 24 hour intervals for the presence of occlusion bodies.
- Plaque assays were performed on supernatants of cell cultures infected with the larval-isolated HzSNPV as previously described (Fraser, 1982, J. Tis. Cult. Meth. 7:43-46) . Twenty-four wild-type (MP type) plaques were picked and used as inoculum for 1 x 10 cells in each well of a 24 well plate. These once-plaque-purified isolates were re-assayed and individual plaques from the second assay were amplified first in 24-well plate cultures, and then in
- OBs Infectious occlusion bodies
- TE buffer 10 mM Tris-HCl, lmM EDTA, pH 7.6
- the larval ho ogenate was filtered through two layers of cheesecloth and centrifuged at 1,800 x g for 15 minutes. The supernatants were discarded and the OB pellet washed twice by resuspension in 20 ml of E buffer and centrifugation at 1,800 x g. The washed OBs were resuspended in a final volume of 10 ml TE buffer.
- the precipitated DNA was pelleted at 1,800 x g for 15 minutes and resuspended in sterile distilled water by heating at 65°C for 30 minutes.
- the concentration of DNA was determined by absorbance at 260 nm, and the DNA was stored at 4'C until use.
- Structural proteins of virions released from occlusion bodies by alkali treatment were compared by electrophoresis in discontinuous polyacrylamide slab gels according to the method of Lae mli (1970, Nature 227:680).
- Virion proteins were solubilized by boiling for 3 minutes in denaturation buffer (62.2 mM Tris-HCl, 2.0% SDS, 20% giycerol, 2.5% dithiothreitol, pH 6.8) at a concentration of 1 mg protein/ml. Electrophoresis was carried out at 30 milliamps for 4.5 hours in a 12% separating gel (10 cm long
- IPLB-HZ1075 insect cell line grew well in TNM-FH medium supplemented with 8% fetal calf serum. Cells remained susceptible to infection by HzSNPV, but infectivity was not 100% under these conditions. The highest levels observed were between 50 and 70% infected cells with maximal titers of 5 x 10 plaque forming units per ml. The best 35 infections were achieved when cells were allowed to grow at least 24 hours before inoculation with virus. We have since discovered that the addition of 1% bovine serum albumin
- plaque-purified strains were amplified in third 15 to fourth instar H. zea larvae. Larval propagation was necessary to rapidly expand the virus and reduce the probability of selecting in vitro passage mutants.
- Mutant selection is a phenomenon which occurs readily during ii vitro propagation of baculoviruses (Potter, K.N., et al., 1976, J. Virol. 18:1040-1050; Hink and Strauss, 1976, J. Invert. Path. 27:49-55; Fraser and Hink, 1982, Virology 117:366-378; Fraser and McCarthy, 1984, J. Invert. Path. 43:427-429), but is not observed during short term in vivo propagations of HzSNPV (Mclntosh, A.H. and Ignoffo, CM., 1986, Intervirol. 25:172-176).
- the inoculations were performed by placing a drop of a 1 x 10 OB/ml suspension directly on the head capsule of each larvae.
- Larval derived OBs were used for subsequent inoculations to characterize the relative virulence and degree of pathogenicity of each strain and the wild-type isolate.
- No Melanization is defined as less than 30% melanization by nine days after larval death.
- HzS-15 is highly virulent.
- Hindlll digests there were only three strains with identical fragment patterns (FIG. 4) . These three strains could be distinguished from the others upon digestion with BamHI. Comparisons of the several individual digests suggested that there is a hypervariable region of the HzSNPV genome between map units 23.5 and 43.3 (Knell and Summers, 1984, J. Gen. Virol. 65:445-450).
- Virions were liberated from larval-derived occlusion bodies by alkali treatment and purified by banding in linear sucrose gradients. Electrophoresis of occluded virion structural proteins of the wild-type isolate revealed 13 major polypeptides following staining with Coomassie blue 5
- VP 37.2, VP 41.1, VP 49.2, and VP 62.9 were found in the occluded virion protein profiles of all the plaque-purified strains.
- the remaining eight wild-type polypeptides varied in occurrence among the plaque-purified strains.
- the total number of major polypeptides in the plaque-purified strains varied from a low of 13 to a high of
- the HzS-15 strain exhibited most of the wild-type polypeptides except VP 33.8 and VP 66.1, and also exhibited many of the additional polypeptides found individually in several of the other strains.
- Several protein bands were apparently unique to HzS-15 including VP 51.0, and three bands above VP 69.0.
- the IPLB-HZ1075 cell line was obtained from Dr. J. 5 Vaughn (USDA Invertebrate Pathology Laboratory, Beltsville) and adapted to growth in TNM-FH medium over several passages. Cloning of cell strains was accomplished by diluting cells to an average density of 1 cell per 100 ul, and plating 100 ul in each well of a 96 well culture plate. Q The wells were examined after 12 hours and those containing only one cell each were marked.
- the growth medium for cell clones was composed of an equal mixture of. filter- sterilized, conditioned TNM-FH medium and fresh TNM-FH medium (50% conditioned medium) . The conditioned medium was
- the cells were allowed to attach and enter log phase growth for 24 hours after which an initial cell count was made.
- Each strain was inoculated at a density of 1.25 x 10 5 cells per well m separate wells of a 24 well cluster plate and the cells were allowed to attach for 24 hours.
- TNM-FH medium The cells were monitored for 10 days, after which both the media and cells were collected for quantitation of ECV and OBs.
- the cultures were collected and the cells and OBs were pelleted by centrifugation at 15,000 x g for 2 minutes.
- the ECV- containing supernatants were decanted and titered using the
- the OBs were released from the infected cells by resuspending the pellets from each well in 1 ml of TE buffer (0.01 M Tris-HCl, 0.001 M EDTA, pH 7.5) containing 0.1% SDS. The OBs were pelleted from the cell lysate at 15,000 x g for
- Isozymes were detected following electrophoresis of the cleared cell lysates in 5% polyacrylamide gels in either TBE buffer (81.2 mM Tris, 20 mM boric acid, 1.5 mM EDTA, pH
- FIG. 9 shows malate dehydrogenase (MDH, not shown) with thos of both the IPLB-HZ1075 parental cell line and larval tissues from the host of origin, H ⁇ zea.
- MDH malate dehydrogenase
- IPLB-HZ1075 cell line was compared to other lepidopteran and one dipteran cell lines maintained in our laboratory. Cell homogenates were prepared and electrophorese as described above, and stained for either LDH or MDH (FIG.
- IPLB-HZ1075 1.00 1.0 HELIOTHIS ZEA
- IPLB-SF-21AE 0.77 1.0 SPODOPTERA FURGIPERD
- IPLB-HZ1075 ce line from all but one (IPLB-SF21AE) of the lepidopteran cell lines, and from the single dipteran cell line originated from
- HzSNPV non- melanizing strains of HzSNPV, such as those described in Section 6, supra, are preferred for use in the larval expression systems of the present invention.
- Pinto beans in 90 ml water 18 g 9 g Agar (Sigma) 25 g 12.5 g
- Vitamin diet fortification 3.3 g 1.66 g mixture (ICN)
- the diet mix is prepared as follows: 1. The methyl paraben is dissolved in alcohol before the procedure is started.
- COLONY MAINTENANCE The diet prepared as described is dispensed so that each larva receives a minimum of approximately 10 ml.
- T. n_i or H. zea The rearing conditions for T. n_i or H. zea are: 28-30 ⁇ c, relative humidity of 65-70%, and photoperiod of 12 hours (each 24 hours) .
- the pupae Upon pupation, the pupae are combined into a flight cage of one cubic foot size, at 40 pupae/cage.
- the adult moths are allowed to emerge and feed on a mixture of 250 g sucrose, 25 ml honey, 5 g ascorbic acid, 5 g methyl paraben (dissolved initially in 5 ml 95% ethanol before addition) , plus 500 ml distilled H-O (components are dissolved with moderate heat) .
- the feeding mix is presented to the adult moths in a 15 ml conical centrifuge tube equipped with a cap through which a two inch long dental wick extends, so that the adults can feed on the dental wick.
- the walls of the flight cage are lined with sterilized paper towels, on which the adult moths lay their eggs.
- the paper towels containing the eggs are removed from the cage with aseptic precautions and transferred to a plastic box, termed a crisper, in which som of the agar-based diet mixture is present.
- the larvae emerg from the eggs in the crisper. Since the larvae are positively phototropic, a light is shone at the end of the crisper where the diet mix is located, so that the larvae move toward the diet mix and feed upon it.
- the feeding of the larvae on the diet mix increases yield of larvae, since the larvae are cannibalistic and would otherwise eat other larvae and unhatched eggs.
- the larvae are cannibalistic, they are segregated within a day after they emerge. This segregation is accomplished by hand, using an artist's brush which has been sterilized in 0.25% CloroxTM, and rinsed with double distilled H_0. The brush is used to gently lift the larvae and place each individual larva alone into a cup. The larvae are allowed to grow in the cups until pupation, at which time the pupae are placed into the flight cages.
- T. ni are not as cannibalistic as H. zea, a slightly different protocol is alternatively used.
- a one inch square piece of the paper towel is placed on the lid of a quart cup (J. Cup, Dart Container Corp., Mason, Michigan, Cat. No. 8SJ20) containing 20-30 ml of liquid agar-based diet mixture that has solidified.
- the cup is inverted on top of the paper.
- the larvae hatch and migrate up toward the diet surface.
- the paper is then removed from the bottom and the cups are turned right-side up. The larvae are allowed to grow in the cups until pupation, at which time the pupae are placed into the flight cages.
- the temperature can range from 25 to 30*C.
- the relative humidity of ambient room conditions is suitable.
- the larva diet for G. melonella is composed of a mixture of 200 ml honey, 100 ml glycerine, and 1 box GerberTM's mixed cereal.
- the diet mixture is put into a quart Mason jar fitted with a wire-mesh screen at the top, into which the G. melonella eggs are placed. After the larvae emerge, more diet mix is added as necessary until the larvae form cocoons. Since G. melonella larvae are not as cannibalistic as H. zea larvae, it is not necessary to segregate the larvae.
- the larvae When the larvae are in the last instar stage and start forming cocoons, they are taken out of the jar by hand (with gloves) and placed together in a crisper- The insects pupate and the adults emerge within the crisper. The adult moths lay eggs in the cracks or crevices of the crisper without feeding. Eggs are thus laid at the interface of the lid and the box, so that when the lid is removed, the eggs adhere to the lid. The eggs are then stripped off the lid by using a razor blade, and placed in a Mason jar containing the diet mixture.
- a 2 kb Xhol to BamHI fragment was isolated and subcloned into the Sail and BamHI sites of M13mpl9, generati clone mpl9pEcoIXB.
- a DNA fragment was synthesized, corresponding to the Autographa polyhedrin sequence extendin from the EcoRV site in the promoter region to the transcripti initiation site, followed by a multiple cloning site (MCS) containing BamHI, EcoRI, Sail, and Kpnl restriction enzyme recognition sites.
- MCS multiple cloning site
- the synthetic fragment was clone between the EcoRV and Kpnl sites of mpl9pEcolXB, resulting in clone mpl9Ald.
- the Hindlll to Kpnl fragment of mpl9Ald was isolated. (The Xhol site was lost in the cloning into the Sail site of mpl9, and the Hindlll site of the mpl9 MCS is a convenient nearby site) .
- the Kpnl to BamHI fragment of pEcoRI-I was als isolated. These two fragments were cloned into the Hindlll a sstl sites of the MCS of pUC12 (FIG. 13).
- the ligation included a synthetic oligonucleotide, 5'-GATCAGCT-3' , in orde to permit the ligation of the BamHI end of the pEcoRI-I fragment into an SstI end of pUC12, and to remove the BamHI site probably by the mechanism shown below.
- the resulting clone, pAV1.5 included Autographa sequences extending from the Xhol site 5' of the polyhedrin gene to the transcription initiation site, a MCS, and Autographa sequences extending from the Kpnl site in the carboxy-coding end of the polyhedrin gene to a BamHI site 3' of the gene.
- the Xhol and BamHI sites were lost.
- Plasmid pAV1.5 and plasmid pHX12 were used as the parenta plasmids for the construction shown in Figure 14.
- a 2 kb Pstl-EcoRI fragment of pAV1.5 (containing the Autographa polyhedrin promoter) and a 4.2 kb Sail-PstI fragment of pAV1.5 (containing pUC12 sequences) were isolated.
- a 2.2 kb EcoRI- Sall fragment of pHX12 (containing the Heliothis polyhedrin promoter and coding sequences) was isolated, and ligated to th Pstl-EcoRI and Sall-PstI pAVl.5-derived fragments.
- pAVHp ⁇ contains the Heliothis polyhedrin promoter and coding sequences, flanked by Autograph polyhedrin sequences including the Autographa polyhedrin promoter.
- pAVHp6 can thus be used to transfer the Heliothis polyhedrin gene into AcNPV through _in vivo recombination, resulting in a recombinant virus that can comprise an expression system in accordance with the present invention.
- pAVHp ⁇ can also be used to create a recombinant AcNPV with two polyhedrin promoters. One thus has the potential to express two different heterologous genes within the same virus.
- foreign DNA is inserted and expressed under the control of the Autographa promoter in such a recombinant vir the parental Heliothis polyhedrin promoter and gene can presumably ensure the retention of occlusion body formation.
- Figure 15 depicts a strategy for cloning amino acids 98- 106 of the influenza hemagglutinin into the amino-terminal coding sequence of the Autographa polyhedrin gene.
- This strategy can be used to attempt to insert the influenza sequence into the Autographa polyhedrin sequence contained in the M13 derivative mpl9EcoIXB (described in Section 10., supr within the sequence encoding the second amino acid of the polyhedrin protein.
- An oligonucleotide (termed Rol-1) can be synthesized (Applied Biosystems Model 380A) , which is homologous to the region containing the Hpall cleavage site within the codon for amino acid 2.
- Rol-1 is annealed to mpl9EcoIXB single-stranded DNA, which is then cut with Hpall. Annealing of the oligonucleotide creates the requisite double stranded region for restriction endonuclease cleavage.
- the linear single-stranded DNA with Hp_alI-derived ends is isolate by heat denaturation and gel purification.
- An oligonucleotid corresponding to amino acids 98-106 of influenza hemagglutini (termed Rol-2) is synthesized.
- Rol-2 is then annealed to a third synthetic oligonucleotide (Rol-3) which is complementar to Rol-2.
- Rol-3 has 5' and 3' termini which extend beyond Rol-2 which are complementary to the Hpall- derived ends of the isolated single-stranded phage DNA.
- the annealed Rol-2/Rol-3 DNA can be ligated to the isolated single-stranded phage DNA, forming a circular DNA molecule.
- the desired transformant can be selected by hydridization to radiolabeled Rol-3 according to the procedu of Benton and Davis (1977, Science 196:180-182).
- additio Rol-3 encodes two restriction sites, Mlul and Nsil, which ar not found in the parental mpl9EcoIXB DNA.
- the identit of selected transformants can be confirmed by the presence o Mlul and Nsil restriction sites in the phage DNA isolated fr transformants.
- a similar strategy to that described supra may be used in order to cut the polyhedrin sequence contained within mpl9EcoIXB at the BamHI site within the sequence encoding amino acid 58.
- the subsections below describe manipulations of the polyhedrin gene of Autographa californica to form recombinan occlusion bodies that expose antigenic determinants of foreign organisms.
- the construction of 5 different recombinant polyhedrin genes containing a short DNA sequence encoding an influenza hemaglutinin epitope are described.
- the five recombinants are named InHem-1, InHem-2, InHem-43, InHem-50, and InHem-43/50, in which "InHem” signifies the influenza hemagglutinin epitope and the numbered suffix indicates the amino acid residue of the baculovirus polyhedrin sequence into which the hemagglutinin epitope was inserted.
- these antibodies also interact with non-denatured purified recombinant OBs.
- the ability of the recombinant OBs to precipitate or capture antibodies to the influenza hemagglutinin epitope suggests that the recombinant structures may be valuable as diagnosti reagents. Additionally, preliminary results with a limited number of animals indicates that one of the recombinants induces an immunogenic response to the hemagglutinin epitope.
- Alternations within the polyhedrin gene were introduced into the baculovirus genome by homologous recombinations iri vivo following cotransfection of susceptible cells with both viral DNA and transfer plasmids containing the altered gene.
- the transfer plasmids are bacterial plasmids containing the viral segment surrounding the polyhedrin gene.
- a series of transfer vectors that contain 2kb of baculovirus sequences 5' of the polyhedrin gene, the sequence of the altered polyhedrin gene, and approximately 1.5 kb of 3' flanking sequences were used.
- the long flanking sequences facilitated the transfer of the polyhedrin gene in the transfer plasmid into the viral genome by homologous recombination _in vivo.
- New restriction sites were introduced into the gene by in vitro mutagenesis using the procedures developed by
- the uracil containing plus strand is not efficiently used as a template in a ung dut strain.
- This change introduced a Bglll site into the wild type polyhedrin gene sequence.
- the alteration in the mpl9 subclone was transferred into the transfer vector by replacing the Pst/Bam fragment of the transfer vector with the corresponding fragment of the mutated mpl9 subclone (Crec5mpl9Xho/Bam) .
- the resulting transfer vector, pAV15 contained a new unique Bglll site at a position corresponding to amino acid residue number 43 and a naturally occurring BamHI site at a position corresponding to amino acid residue number 58.
- a synthetic oligonucleotide encoding the influenza epitope followed by the polyhedrin sequence from amino acid residue 50 to 58 was cloned into the Bglll/BamHI site of pAV15 (see FIG. 16A) .
- the oligonucleotide introduced an Xbal site at a position corresponding to amino acid residue number 50 of the polyhedrin sequence.
- the resulting transfer vector, pAV15Inhem contained an altered polyhedrin gene coding for a polyhedrin in which amino acid residues between 43 and 50 were replaced with the influenza epitope.
- pAV1.5 (FIG. 13) was used to construct three 5 intermediate plasmids, pAVll, pAV12 and pAV13; these, in turn, were used to construct pAV17 which has a unique SphI site located at the ATG of the polyhedrin gene (FIG. 17A) .
- pAV17 was converted to pAV17b (FIG. 0
- a cassette vector was constructed using pBR322 as the plasmid backbone.
- This vector called PBRX13, allows for the polyhedrin gene spanning the coding region for amino acid residue numbers 36 through 50.
- the entire polyhedrin gene sequence can be cut out of the pBRX13 vector and cloned into a transfer vector where it is flanked by baculoviral sequences that allow for in vivo recombination with virus.
- pBR322 was cut with EcoRI and PstI and the following oligonucleotide (HE50/HE51) was cloned into the pBR322 backbone so that the EcoRI, PstI and one XmnI site of pBR322 is eliminated and replaced with a unique Kpnl and Xhol site:
- pBRX13 contains the first 213 amino acids of the polyhedrin sequence except that
- the polyhedrin sequence of pBRX13 contains a unique XmnI, Bglll and Xbal site spanning the amino acid 36-50 region so that the coding sequence for any 5 epitope can be cloned into this region.
- the entire recombinant polyhedrin gene can be excised from pBRX-13 usin Kpnl and EcoRV. This fragment can then be cloned into a transfer vector so that the recombinant polyhedrin sequence Q is flanked by baculovirus sequences to allow for _in vivo recombination with virus.
- the transfer of the altered polyhedrin genes into the 5 baculovirus genome was accomplished by homologous recombination .in vivo between viral DNA and the transfer plasmids.
- the viral and plasmid DNAs were introduced into susceptible cells by cotransfection. Transfections involvin calcium phosphate precipitation of DNA yielded the most consistent results.
- Cells were preseeded on 60 mm culture 0 dishes in growth media. Calcium phosphate precipitated DNA was added to the media and the cells incubated for 12-18 hours. The media was then removed and fresh media added.
- the cells were then incubated for 4 or 5 days at which time most cells were infected with virus. Although only a small percentage of the cells are initially transfected, the rest of the cells are infected by the progeny of later rounds of infection.
- the progeny of the transfection were plaqued and the recombinant viruses were identified from the parental virus 0 on the basis of plaque morphology.
- Two types of cotransfections were set up to identify recombinant occlusion body formation.
- viral DNA was derived from a strain in which the polyhedrin gene had been replaced with the bacterial CAT gene. Since this virus has O no polyhedrin gene it fails to make OBs. If the recombinant polyhedrin gene encodes a protein that will form an occlusion body, the recombinant virus is detected in plaque assays among the large number of parental types which fail to make
- OBs Since viruses producing occlusion bodies form refractile plaques, these recombinants are easily detected against the OB negative background.
- the second cotransfection involved the use of wild type viral DNA. In this case, a recombinant failing to make OBs could be detected among the wild type progeny. In this case the rare recombinant forms a non-refractile plaque.
- influenza epitope replaces the polyhedrin sequence between amino acids 43 and 50.
- the virions embedded in the wild type OB act as impurities and interfere with regular lattice formation. By failing to embed virions these mutants may form large, regular lattices.
- OBs described above expose the influenza hemagglutinin epitope as analyzed by ELISA immunoassay, immunoprecipitatio and Western immunoblotting.
- preliminary result indicate that the recombinant OBs are immunogenic and capabl of eliciting an immune response specific for the hemagglutinin epitope.
- ACTR Autographa wild type virus
- OBs recombinant OBs isolated from one T75 flask in 2 ml of TE buffer (43-2B1, 43-2B1A, 50-11A1
- Immunoprecipitation data indicate that the anti- influenza hemagglutinin monoclonal antibody (MAb) also interacts with non-denatured recombinant polyhedrin.
- MAb anti- influenza hemagglutinin monoclonal antibody
- purified occlusion bodies from InHem-43, -I 15- InHem-50 or ACTR (wild type) infected cells were incubated with either BSA, mouse anti-influenza Mab, or mouse anti- plasminogen Mab (as the negative control) .
- the occlusion bodies were pelleted and washed repeatedly.
- the OBs were then incubated with alkaline phosphatase conjugated rabbit anti-mouse antibody.
- the OBs were pelleted, washed several times and then incubated with a chromogenic substrate.
- mice were inoculated with purified OBs from Inhem-43 infected cells. At various times the animals were bled and the sera tested for antibodies specific for the influenza hemagglutinin epitope in ELISA assays. In these
- OBs induced an immunogenic response to the influenza peptide.
- the measurements represent a single animal per time point.
- E. coli strains carrying the listed plasmid have been deposited with the Agricultural Research Culture Collection (NRRL) , Peoria, IL, and have been assigned the following accession numbers:
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US07/153,736 US4870023A (en) | 1987-03-16 | 1988-02-08 | Recombinant baculovirus occlusion bodies in vaccines and biological insecticides |
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EP19880904294 Withdrawn EP0349594A4 (en) | 1987-03-16 | 1988-03-02 | Recombinant baculovirus occlusion bodies in vaccines and biological insecticides |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0349594A4 (de) |
JP (1) | JPH02502876A (de) |
AU (1) | AU1717688A (de) |
WO (1) | WO1988007082A1 (de) |
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FR2631974B1 (fr) * | 1988-05-31 | 1992-12-11 | Agronomique Inst Nat Rech | Baculovirus modifie, son procede de preparation et son application en tant que vecteur d'expression de genes |
US5145775A (en) * | 1989-02-28 | 1992-09-08 | Research Association For Biotechnology Of Agricultural Chemicals | Polyhedrin gene and genetic engineering thereof |
JPH07500962A (ja) * | 1991-08-05 | 1995-02-02 | コモンウェルス サイエンティフィック アンド インダストリアル リサーチ オーガニゼーション | ウイルスのベクターに基づく殺虫剤及び発現系 |
EP2374894B1 (de) * | 1993-09-13 | 2014-09-17 | Protein Sciences Corporation | Verfahren zum Herstellen von hämaglutinin-multivalenten Influenza-Impfstoffen |
US5762939A (en) * | 1993-09-13 | 1998-06-09 | Mg-Pmc, Llc | Method for producing influenza hemagglutinin multivalent vaccines using baculovirus |
FR2761994B1 (fr) | 1997-04-11 | 1999-06-18 | Centre Nat Rech Scient | Preparation de recepteurs membranaires a partir de baculovirus extracellulaires |
KR100264953B1 (ko) * | 1998-04-03 | 2001-03-02 | 박현우 | 재조합 베큘로바이러스, 이의 제조방법 및 이를 포함하는 미생물 살충제 |
WO2002036785A1 (fr) * | 2000-10-30 | 2002-05-10 | Protein Crystal Co., Ltd. | Complexes de proteines et leur procede de production |
CA2553040A1 (en) | 2004-02-02 | 2005-08-18 | Ambrx, Inc. | Modified human four helical bundle polypeptides and their uses |
CN102603895B (zh) | 2004-06-18 | 2016-09-28 | Ambrx公司 | 新颖抗原结合多肽和其用途 |
NZ555386A (en) | 2004-12-22 | 2011-01-28 | Ambrx Inc | Formulations of human growth hormone comprising a non-naturally encoded amino acid |
KR101252835B1 (ko) | 2004-12-22 | 2013-04-10 | 암브룩스, 인코포레이티드 | 아미노아실-tRNA 합성효소의 조성물 및 그것의 용도 |
WO2006073846A2 (en) | 2004-12-22 | 2006-07-13 | Ambrx, Inc. | Methods for expression and purification of recombinant human growth hormone |
US7385028B2 (en) | 2004-12-22 | 2008-06-10 | Ambrx, Inc | Derivatization of non-natural amino acids and polypeptides |
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KR20080079643A (ko) | 2005-11-16 | 2008-09-01 | 암브룩스, 인코포레이티드 | 비-천연 아미노산을 포함하는 방법 및 조성물 |
EP2069396B1 (de) | 2006-09-08 | 2015-10-21 | Ambrx, Inc. | Modifiziertes polypeptid oder fc-gerüste aus humanem plasma und ihre verwendung |
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CN101939443B (zh) | 2008-02-08 | 2014-01-29 | Ambrx公司 | 经修饰瘦素多肽和其用途 |
PT2318029T (pt) | 2008-07-23 | 2018-01-10 | Ambrx Inc | Polipéptidos de g-csf bovino modificados e suas utilizações |
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KR20240024362A (ko) | 2014-10-24 | 2024-02-23 | 브리스톨-마이어스 스큅 컴퍼니 | 변형된 fgf-21 폴리펩티드 및 그의 용도 |
CN106701826B (zh) * | 2015-11-13 | 2021-04-20 | 中国科学院武汉病毒研究所 | 一种可用于包装大量外源蛋白的重组质粒及构建方法和应用 |
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Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0127839A2 (de) * | 1983-05-27 | 1984-12-12 | THE TEXAS A&M UNIVERSITY SYSTEM | Verfahren zur Herstellung eines rekombinanten Baculovirus-Expressionsvektors |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4745051A (en) * | 1983-05-27 | 1988-05-17 | The Texas A&M University System | Method for producing a recombinant baculovirus expression vector |
-
1988
- 1988-03-02 WO PCT/US1988/000631 patent/WO1988007082A1/en not_active Application Discontinuation
- 1988-03-02 JP JP63504028A patent/JPH02502876A/ja active Pending
- 1988-03-02 AU AU17176/88A patent/AU1717688A/en not_active Abandoned
- 1988-03-02 EP EP19880904294 patent/EP0349594A4/en not_active Withdrawn
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0127839A2 (de) * | 1983-05-27 | 1984-12-12 | THE TEXAS A&M UNIVERSITY SYSTEM | Verfahren zur Herstellung eines rekombinanten Baculovirus-Expressionsvektors |
Non-Patent Citations (3)
Title |
---|
EMBO JOURNAL, vol. 5, no. 6, 1986, pages 1359-1365, IRL Press Ltd, Oxford, GB; K. KURODA et al.: "Expression of the influenza virus haemagglutinin in insect cells by a baculovirus vector" * |
PROTEIN ENGINEERING, vol. 1, no. 4, August/September 1987, pages 359-366, IRL Press Ltd, Oxford, GB; V.C. EMERY et al.: "The development of multiple expression vectors for high level synthesis of eukaryotic proteins: expression of LCMV-N and AcNPV polyhedrin protein by a recombinant baculovirus" * |
See also references of WO8807082A1 * |
Also Published As
Publication number | Publication date |
---|---|
JPH02502876A (ja) | 1990-09-13 |
AU1717688A (en) | 1988-10-10 |
WO1988007082A1 (en) | 1988-09-22 |
EP0349594A4 (en) | 1990-09-26 |
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