BACULOVIRUS EXPRESSION SYSTEM

Abstract

The invention relates to a method for producing a recombinant baculovirus comprising n exogenous genes in an insect cell, by means of homologous recombination of a replication-deficient baculovirus genome and n transfer vectors, each comprising one of the n exogenous genes, n being an integer at least equal to 2.

Claims

1. A process for preparing, in an insect cell, a recombinant baculovirus comprising n exogenous genes, by homologous recombination between: a) a replication-deficient baculovirus genome in which n genes essential for viral replication are non-functional; and b) n transfer vectors each comprising: i) a nucleotide sequence that restores the function of one of the n non-functional genes essential for viral replication, ii) one of the n exogenous genes, the set of nucleotide sequences i) of the n transfer vectors being able to restore replication of the replication-deficient baculovirus genome; n being an integer at least equal to 2.

2. The process according to claim 1, wherein the genes essential for viral replication are selected from 1629 (ORF9), Pk1 (ORF10), lef-1 (ORF14), ORF34, lef-11 (ORF37), p47 (ORF40), lef8 (ORF50), DNAJ domain (ORF51), ORF53, vp1054 (ORF54), Lef-9 (ORF62), DNA Pol (ORF65), lef-3 (ORF67), ORF73, ORF75, ORF81, p95 (ORF83), vp39 (ORF89), lef-4 (ORF90), p33 (ORF92), helicase (ORF95), vp80 (ORF104), ORF106-107, odv-ec43 (ORF109), gp64/67 (ORF128), ORF132, ORF133, odv-ec27 (ORF144), ORF146, ie1 (ORF147), lef-2 (ORF6).

3. The process according to claim 1, wherein the n non-functional genes essential for viral replication are each adjacent to a gene non-essential for viral replication.

4. The process according to claim 3, wherein the gene non-essential for viral replication is selected from Ph (ORF 8), ORF11, ORF13, egt (ORF15), v-ubiquitin (ORF35), 39K (ORF36), ORF38, p43 (ORF39), lef-12 (ORF41), pcna (ORF49), ORF52, ORF55, Fp (ORF61), ORF63, gp37 (ORF64), ORF68, ORF72, ORF74, ORF82, cg30 (ORF88), ORF91, pif-4 (ORF96), he65 (ORF105), ORF108, ORF110, cathepsin (ORF127), p24 (ORF129), pp34 (ORF131), ORF134, ORF145, odv-e56 (ORF148), and ORF5.

5. The process according to claim 3, wherein the n exogenous genes each recombine at the level of a gene non-essential for viral replication adjacent to a non-functional gene essential for viral replication.

6. The process according to claim 1, wherein the transfer vectors comprise, on either side of the exogenous gene expression cassette, flanking sequences homologous to the replication-deficient baculovirus genome.

7. The process according to claim 6, wherein the flanking sequences have a length ranging from 10 bp (i.e. 10 base pairs) to 10 kb (i.e. 10 000 base pairs).

8. The process according to claim 1, wherein the insect cell is selected from the group consisting of: Sf9, Sf21, Tn5-b14, baculovirus AcMNPV-sensitive lepidopteran cell lines, and Sf21 lines.

9. The process according to claim 1, wherein the replication-deficient baculovirus genome is obtained from a baculovirus genome selected among or derived from the group of the baculovirus genomes consisting of BmNPV, AcMNPV, ApNPV, BsSNPV, CfMNPV, EoSNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SIMNPV, SeMNPV and TeNPV.

10. The process according to claim 1, wherein n is an integer ranging from 2 to 30.

11. A recombinant baculovirus comprising n nucleotide sequences of formula (I):
[exogenous gene]—[spacer nucleotide sequence]—[functional gene essential for viral replication]  (I), said spacer nucleic acid sequence consists of 0 to 600 bp, said functional gene essential for viral replication is selected from the group consisting of: 1629 (ORF9), Pk1 (ORF10), lef-1 (ORF14), ORF34, lef-11 (ORF37), p47 (ORF40), lef8 (ORF50), DNAJ domain (ORF51), ORF53, vp1054 (ORF54), Lef-9 (ORF62), DNA Pol (ORF65), lef-3 (ORF67), ORF73, ORF75, ORF81, p95 (ORF83), vp39 (ORF89), lef-4 (ORF90), p33 (ORF92), helicase (ORF95), Vp80 (ORF104), ORF106-107, odv-ec43 (ORF109), gp64/67 (ORF128), ORF132, ORF133, odv-ec27 (ORF144), ORF146, ie1 (ORF147), and lef-2 (ORF6); n being an integer at least equal to 2, said recombinant baculovirus does not comprise a nucleic acid sequence allowing it to replicate within a bacterial cell.

12. The recombinant baculovirus according to claim 11, not comprising n genes non-essential for viral replication selected from the group consisting of: Ph (ORF 8), ORF11, ORF13, egt (ORF15), v-ubiquitin (ORF35), 39K (ORF36), ORF38, p43 (ORF39), lef-12 (ORF41), pcna (ORF49), ORF52, ORF55, Fp (ORF61), ORF63, gp37(ORF64), ORF68, ORF72, ORF74, ORF82, cg30 (ORF88), ORF91, pif-4 (ORF96), he65 (ORF105), ORF108, ORF110, cathepsin (ORF127), p24 (ORF129), pp34 (ORF131), ORF134, ORF145, odv-e56 (ORF148), and ORF5.

13. A set of homologous recombination elements comprising: a) a replication-deficient baculovirus genome in which n genes essential for viral replication are non-functional; b) n transfer vectors each comprising: i) a nucleic acid sequence that restores the function of one of the n non-functional genes essential for viral replication, ii) optionally an exogenous gene, n being an integer at least equal to 2.

14. A cell comprising a recombinant baculovirus according to claim 11.

15. A process for the production of n exogenous polypeptides encoded by n exogenous genes comprising the use of a recombinant baculovirus according to claim 11.

16. A cell comprising a set of homologous recombination elements according to claim 13.

17. A process for the production of n exogenous polypeptides encoded by n exogenous genes comprising the use of a cell according to claim 14.

Description

FIGURES

[0117] FIG. 1 is a diagram illustrating the steps for the preparation of BacMid 1.

[0118] Legend:

[0119] ORF: open reading frame

[0120] Polyhedrin or PH: baculovirus gene encoding polyhedrin: gene non-essential for viral replication.

[0121] ORF603: baculovirus gene encoding the 603 protein, non-essential gene.

[0122] ORF1629: baculovirus gene encoding the 1629 protein: gene essential for viral replication

[0123] pVT: transfer vector plasmid.

[0124] Recombination fragment: DNA fragment containing the expression cassette to be integrated into the target DNA. This fragment has flanking regions on either side of the expression cassette to specifically target the region that will undergo homologous recombination in the bacterium via Red recombinase.

[0125] Mini-F: bacterial origin of replication.

[0126] Kan.sup.R: bacterial expression cassette expressing the kanamycin resistance gene.

[0127] AmpR: bacterial expression cassette expressing the ampicillin resistance gene.

[0128] FIG. 2 is a diagram illustrating the step of partial deletion of the gene encoding the viral DNA polymerase, the DNAPol gene, for the preparation of BacMid 2.

[0129] Legend:

[0130] gp37: baculovirus gene encoding glycoprotein gp37, gene non-essential for viral replication.

[0131] DNAPol: baculovirus gene encoding the viral DNA polymerase, gene essential for viral replication.

[0132] DNAPol.Δ466 aa: DNAPol gene from which the region encoding the 466 C-terminal amino acids has been deleted

[0133] Recombination fragment: DNA fragment containing the expression cassette to be integrated into the target DNA. This fragment has flanking regions on either side of the expression cassette to specifically target the region that will undergo homologous recombination in the bacterium via Red recombinase.

[0134] Hygro.sup.R: bacterial expression cassette expressing the hygromycin resistance gene.

[0135] FIG. 3 is a diagram illustrating the step of partial deletion of the gene encoding gp64 for the preparation of BacMid 3.

[0136] Legend:

[0137] Chit: baculovirus gene encoding chitinase, gene non-essential for viral replication.

[0138] Cath: baculovirus gene encoding viral cathepsin, gene non-essential for viral replication.

[0139] gp64: baculovirus gene encoding viral glycoprotein gp64, gene essential for viral replication.

[0140] gp64.Δ188 aa: gp64 gene from which the region encoding the 188 C-terminal amino acids has been deleted.

[0141] Recombination fragment: DNA fragment containing the expression cassette to be integrated into the target DNA. This fragment has flanking regions on either side of the expression cassette to specifically target the region that will undergo homologous recombination in the bacterium via Red recombinase.

[0142] Zeo.sup.R: bacterial expression cassette expressing the zeocin resistance gene.

[0143] FIG. 4 is a diagram illustrating transfer vector pVT/gp37 and its use for the expression of a gene X (where X is a gene different from the gene encoding the heavy chain of an antibody).

[0144] Legend:

[0145] pVT: transfer vector plasmid.

[0146] gp37: baculovirus gene encoding glycoprotein gp37, gene non-essential for viral replication.

[0147] DNAPol: baculovirus gene encoding the viral DNA polymerase, gene essential for viral replication.

[0148] DNAPol.Δ466 aa: DNAPol gene from which the region encoding the 466 C-terminal amino acids has been deleted.

[0149] Exogenous gene: gene of interest.

[0150] Pr: viral or cellular promoter controlling expression of the exogenous gene.

[0151] Hygro.sup.R: bacterial expression cassette expressing the hygromycin resistance gene.

[0152] FIG. 5 is a diagram illustrating the construction and use of PH transfer vector pVT/PH for the expression of an exogenous gene X (where X is an exogenous gene different from the gene encoding a light chain of an antibody).

[0153] Legend:

[0154] pVT/PH: polyhedrin transfer vector plasmid.

[0155] PH: all or part of the baculovirus gene encoding polyhedrin, gene non-essential for viral replication.

[0156] ORF603: baculovirus gene encoding the 603 protein.

[0157] ORF1629: baculovirus gene encoding the 1629 protein, gene essential for viral replication. Exogenous gene: gene of interest.

[0158] Pr: viral or cellular promoter controlling expression of the exogenous gene.

[0159] Kan.sup.R: bacterial expression cassette expressing the kanamycin resistance gene.

[0160] Mini-F: bacterial origin of replication.

[0161] FIG. 6 is a diagram illustrating the construction and use of transfer vector pVT/gp37C.sub.γ. for the expression of the heavy chain of an antibody.

[0162] Legend:

[0163] pVT: transfer vector plasmid.

[0164] pVT/gp37-Cγ1: transfer vector plasmid specific for the heavy chain of an immunoglobulin.

[0165] Cγ1: cDNA encoding the γ1 constant domain of a human immunoglobulin.

[0166] VH: cDNA encoding the variable domain of the heavy chain of an immunoglobulin.

[0167] DNAPol: baculovirus gene encoding the viral DNA polymerase, gene essential for viral replication.

[0168] DNAPolΔ466 aa: DNAPol gene from which the region encoding the 466 C-terminal amino acids has been deleted.

[0169] Exogenous gene: gene of interest.

[0170] Pr: viral or cellular promoter controlling expression of the exogenous gene.

[0171] Hygro.sup.R: bacterial expression cassette expressing the hygromycin resistance gene.

[0172] PS: cDNA encoding a signal sequence (heavy chain secretion).

[0173] FIG. 7 is a diagram illustrating the construction and use of transfer vector pVT/PHCK for the expression of the light chain of an antibody.

[0174] Legend:

[0175] ORF603: baculovirus gene encoding the 603 protein.

[0176] ORF1629: baculovirus gene encoding the 1629 protein: gene essential for viral replication.

[0177] Mini-F: bacterial origin of replication.

[0178] pVT: transfer vector plasmid.

[0179] pVT/PH-Cκ: transfer vector plasmid specific for the light chain of an immunoglobulin.

[0180] Cκ: cDNA encoding the K constant domain of a human immunoglobulin.

[0181] VL: cDNA encoding the variable domain of the light chain of an immunoglobulin.

[0182] Pr: viral or cellular promoter controlling expression of the exogenous gene.

[0183] Kan.sup.R: bacterial expression cassette expressing the kanamycin resistance gene.

[0184] PS: cDNA encoding a signal sequence (light chain secretion).

[0185] FIG. 8 is a diagram illustrating the construction of vector pVT/Chit-Cath and its use for the production of an exogenous gene.

[0186] pVT: Transfer vector plasmid

[0187] pVT Chit/cath: Transfer vector plasmid specifically targeting the Chit/Cath region.

[0188] Chit: baculovirus gene encoding chitinase non-essential for viral replication

[0189] Cath: baculovirus gene encoding cathepsin non-essential for viral replication

[0190] Exogenous gene: gene of interest.

[0191] Pr: viral or cellular promoter controlling expression of the exogenous gene.

[0192] gp64: baculovirus gene encoding gp64, gene essential for viral replication

[0193] gp64Δ188aa: gene encoding gp64 from which 566 bp have been deleted from the 3′ end, leading to the production of a non-functional gp64 with a deletion of the 188 C-terminal amino acids.

[0194] Zeo.sup.R: bacterial expression cassette expressing the zeocin resistance gene.

[0195] FIG. 9 is A) Southern blot analysis of the genome of 3 purified independent recombinant baculoviruses generated during the same transfection, and B) analysis of the recombinant antibody after purification on protein A.

[0196] Legend:

[0197] A: Analysis of the organization of the genomes of 3 independent recombinant baculoviruses expressing antibody 1313811. These baculoviruses were isolated from a single transfection experiment. The hybridizations carried out respectively with a probe specific for the constant region of the kappa light chain: CK probe, and a probe specific for the constant region of the gamma 1 heavy chain: Cγ1 probe, demonstrate correct and identical organization of the 3 recombinant viruses.

[0198] B. Analysis by polyacrylamide gel electrophoresis (SDS, 2-mercaptoethanol) and silver staining of the purified recombinant antibody. The antibody secreted in the culture medium of cells infected with the recombinant baculovirus was purified on protein A Sepharose column. PC1: Control plasmid, plasmid containing the kappa light chain gene, PC2: Control plasmid, plasmid containing the γ1 heavy chain gene, R1-3, recombinant baculovirus 1, 2 and 3, MW: size marker.

[0199] FIG. 10 is the Southern blot analysis of the genome of a triple recombinant virus expressing influenza virus M, HA and NA proteins.

[0200] Legend:

[0201] M: influenza virus gene encoding the matrix protein.

[0202] HA: influenza virus gene encoding hemagglutinin.

[0203] NA: influenza virus gene encoding neuraminidase.

[0204] bp: DNA fragment size expressed in base pairs.

[0205] FIG. 11 is a diagram illustrating in A the structure of the bispecific antibody and in B the polyacrylamide gel electrophoresis analysis of the purified bispecific antibody on protein A Sepharose column.

[0206] Legend:

[0207] A: Schematic representation of the structure of the bispecific antibody. L1: antibody 1 light chain; L2, antibody 2 light chain.

[0208] B: Purified bispecific antibody, analysed by polyacrylamide gel electrophoresis. Proteins are revealed by silver staining. (1) electrophoresis under reducing conditions (SDS, 2-mercaptoethanol). (2) electrophoresis under non-reducing conditions. H: antibody heavy chain, L: antibody light chain, H2L4: composition of the bispecific antibody: 2 fused heavy chains linked by 4 disulphide bridges at 2 hinge regions+4 light chains (2 L1 chains+2 L2 chains) specifically matched to the corresponding VH1-CH1 and VH2-CH1 regions.

EXAMPLES

Example 1: Construction of a Replication-Deficient Baculovirus Genome in which 1 Gene Essential for Viral Replication is Non-Functional (BacMid 1)

[0209] BacMid 1 has the deletion of a gene essential for viral replication, the 1629 gene.

1.1. Integration of the Bacterial Origin of Replication in a Baculovirus Genome

[0210] This operation is performed in the insect cell.

[0211] The bacterial Mini-F origin of replication was introduced into the polyhedrin locus of the baculovirus AcMNPV genome by homologous recombination in Sf9 (Spodoptera frugiperda) insect cells. To that end, the cells were transfected with (i) a PH transfer vector (pVT/Mini-F Kan.sup.R) in which the ph gene sequence was replaced by a DNA fragment carrying the Mini-F+ a bacterial expression cassette conferring Kanamycin resistance (Kan.sup.R), and (ii) a baculovirus AcMNPV genome (baculovirus isolated from the lepidoptera Autographa californica). The generated baculoviruses were purified by the lysis plaque technique and then characterized to confirm that they had in fact integrated the Mini-F and the Kan.sup.R expression cassette. A baculovirus was selected and then transferred to E. coli EL350 bacteria, thus generating a first BacMid (BacMid 0, not deficient for viral replication in insect cells).

1.2. Deletion of the Essential 1629 Gene

[0212] A bacterial expression cassette conferring ampicillin resistance (AmpR) and having at 5′ and 3′ the MauBI restriction site—a site absent from the baculovirus AcMNPV genome—was integrated downstream of the Kan.sup.R bacterial expression cassette by homologous recombination in E. coli EL350 bacteria. During this recombination, a DNA fragment encoding the 27 C-terminal amino acids of the 1629 protein was deleted, making the 1629 protein non-functional. The ampicillin resistance gene was then removed after MauBI digestion and re-ligation, thus generating BacMid 1. The baculovirus genome (i.e. BacMid 1) is thus replication-deficient in insect cells because a gene essential for viral replication (i.e. the gene encoding the 1629 protein) is non-functional. Bacteria containing BacMid 1 are hereinafter referred to as “E. coli EL350/BacMid1 bacteria”.
FIG. 1 illustrates the steps for the preparation of BacMid 1.

Example 2: Construction of a Replication-Deficient Baculovirus Genome in which 2 Genes Essential for Viral Replication are Non-Functional (BacMid 2)

[0213] BacMid 2 has the deletion of 2 genes essential for viral replication, the 1629 gene and the gene encoding the viral DNA polymerase (DNAPol). From BacMid 1, deletion of the DNAPol gene was performed in E. coli EL350/BacMid1 bacteria after electroporation of a 4222 bp recombination fragment in which part of the genes encoding gp37 (240 amino acids) and DNAPol (466 C-terminal amino acids) has been deleted and replaced by a bacterial expression cassette for producing hygromycin B phosphotransferase (Hygro.sup.R), thus conferring hygromycin resistance (Hygro.sup.R). The Hygro.sup.R gene was placed under the control of the bacterial EM7 promoter (from the commercial vector pSelect-Hygro-mcs, Invitrogen), the glms terminator was introduced downstream of the Hygro.sup.R gene (Gay N.J. et al. Biochem J., 1986, 234, 111-117). Bacteria containing BacMid2 (E. coli EL350/BacMid2) were selected for their resistance to hygromycin. The baculovirus genome (i.e. BacMid 2) is replication-deficient in insect cells because two genes essential for viral replication (i.e. the gene encoding the 1689 protein and the gene encoding DNAPol) are non-functional.
FIG. 2 is a diagram illustrating the step of deletion of DNAPol for the preparation of BacMid 2.
Note: BacMid 2 can be used to produce a single protein (see Example 4). It is sufficient to have two transfer vectors, one providing the exogenous gene to be produced and all or part of the deleted gene 1 and the other providing the wild-type gene corresponding to the deleted gene 2. Both deleted genes are repaired during homologous recombination.

Example 3: Construction of a Replication-Deficient Baculovirus Genome in which 3 Genes Essential for Viral Replication are Non-Functional (BacMid 3)

[0214] BacMid 3 has the deletion of 3 essential genes, 1629, DNAPol and gp64.
From BacMid 2, deletion of the gp64 gene was performed in E. coli EL350/BacMid2 bacteria after electroporation of a 3260 bp recombination fragment in which the entire cathepsin gene plus 779 bp of the sequence encoding 259 aa of chitinase and part of the gp64 gene, deletion of 566 bp encoding 188 amino acids, was replaced by a bacterial expression cassette conferring zeocin resistance (Drocourt et al., Nudeic Acids Research, vol. 18 no. 13, 1990). The Zeocin® gene derived from the commercial plasmid pCR®—Blunt (Invitrogen) was placed under the control of the bacterial T5N25 promoter, derived from phage T5 (Gentz and Bujard, J. Bacteriology, vol. 164 no. 1, 1985) and followed by the rrnBT1 transcription terminator (E. coli ribosomal RNA operon T1 terminator) (Kwon et al., J Biol. Chem., vol. 274 no. 41, 1999). Bacteria containing BacMid3 (E. coli EL350/BacMid3) were selected for their resistance to Zeocin. The baculovirus genome (i.e. BacMid 3) is replication-deficient in insect cells because three genes essential for viral replication (i.e. the gene encoding the 1689 protein, the gene encoding DNAPol and the gene encoding gp64) are non-functional.
FIG. 3 is a diagram illustrating the step of deletion of gp64 for the preparation of BacMid 3.

Example 4: Use of BacMid 2

[0215] A transfer vector, pVT/gp37, was constructed to be able to generate recombinant baculoviruses expressing 2 exogenous genes. To that end, the EcoRI F fragment of the baculovirus AcMNPV genome containing the gp37 gene and the DNAPol gene was cloned into a bacterial plasmid, pUC, thus generating pUC/gp37.
This plasmid was then modified as follows: a large part of the gene encoding gp37 was deleted (724 bp), the ATG initiator was mutated and replaced by two unique restriction sites, XbaI and AvrII, allowing integration of an exogenous gene under the control of the natural gp37 promoter. These modifications thus led to transfer vector pVT/gp37.
Sf9 cells were transfected by lipofection with transfer vectors pVT/PH and pVT/gp37 loaded with the exogenous genes and the BacMid 2 DNA. The viruses generated after homologous recombination were cloned by the lysis plaque method. Production of the recombinant protein was verified by a suitable method (e.g. ELISA, Western blot, enzyme assay). The genome of the recombinant viruses was verified by Southern blot and the sequence of the exogenous gene integrated into the viral genome was verified by sequencing after PCR amplification.
FIG. 4 is a diagram illustrating transfer vector pVT/gp37 for the expression of a gene X (where X is a gene different from the gene encoding the heavy chain of an antibody).
The recombinant baculovirus genomes generated after homologous recombination between BacMid2 and the transfer vectors no longer express gp37 (protein non-essential for viral replication).
For the viral DNA to be repaired in both BacMid 2 loci, a second recombination must take place with a PH transfer vector optionally loaded with an exogenous gene. In all cases, the baculovirus genome DNA will be repaired and therefore infectious.
It will also be possible to use the pVT/PH containing a wild-type sequence, i.e. containing the wild-type (unmodified) expression cassette leading to the production of polyhedrin. The pVT/PH may also be “empty”, i.e. containing neither an exogenous gene nor the polyhedrin gene.
FIG. 5 is a diagram illustrating the construction and use of PH transfer vector pVT/PH for the expression of an exogenous gene X (where X is an exogenous gene different from the gene encoding a light chain of an antibody).

Expression of the Heavy Chain of an Antibody.

[0216] Construction of a specific pVT/gp37, pVT/gp37-Cγ1
This transfer vector contains the following expression cassette: [0217] Wild-type P10 viral promoter (SEQ ID NO: 1) [0218] DNA sequence encoding a human immunoglobulin signal sequence (secretion sequence) [0219] 2 unique restriction sites for phase cloning of the variable region (VH) of the antibody (region that gives the specificity of the antibody) [0220] DNA sequence that encodes an epsilon, mu, or alpha constant region of human IgG (γ1-4).
FIG. 6 is a diagram illustrating the construction and use of transfer vector pVT/gp37Cγ1 for the expression of the heavy chain of an antibody.

Expression of the Light Chain of an Antibody.

[0221] Construction of a specific pVT/PH-VL. This transfer vector contains the following expression cassette: [0222] P10 viral promoter P10S1B (SEQ ID NO: 2) [0223] DNA sequence encoding a human immunoglobulin signal sequence (secretion sequence) [0224] 2 unique restriction sites for phase cloning of the variable region (VL) of the antibody (region that gives the specificity of the antibody) [0225] DNA sequence that encodes a kappa (κ) or lambda (l) light chain constant region of human IgG.
FIG. 7 is a diagram illustrating the construction and use of transfer vector pVT/PHCK for the expression of the light chain of an antibody.

Example 5: Use of BacMid 3

[0226] A transfer vector, pVT/Chit-Cath, was constructed to be able to generate recombinant baculovirus genomes expressing 3 exogenous genes.

[0227] The BstXI-XbaI fragment from the EcoRI E and H regions of the baculovirus AcMNPV was cloned in a pUC plasmid. An EcoNI-EcoRI deletion of 1175 bp inactivates the genes encoding chitinase (chiA), non-essential in vitro, and cathepsin (v-cath), also non-essential. The addition of an XbaI site between the EcoNI and EcoRI sites makes it possible to integrate an exogenous gene. These modifications thus led to transfer vector pVT/Chit-Cath.

Sf9 cells are transfected by lipofection with transfer vectors pVT/PH, pVT/gp37 and pVT/chitCath loaded with the exogenous genes and the BacMid 3 DNA. The viruses generated during homologous recombination were cloned by the lysis plaque method. Production of the recombinant protein was verified by a suitable method (ELISA, Western blot, enzyme assay, etc.). The genome of the recombinant viruses was verified by Southern blot and the sequence of the exogenous gene was verified after PCR amplification.
FIG. 8 is a diagram illustrating the construction of vector pVT/Chit-Cath and its homologous recombination with BacMid 3.

Example 6: Production of an Anti-CD4 Monoclonal Antibody (13B8II) Using BacMid 2

[0228] cDNAs encoding the VH and VL regions of the antibody were integrated into transfer vectors pVT/PH-Cκ and pVT/gp37-Cγ1, respectively. Recombinant baculoviruses were generated after homologous recombination between the 2 pVTs and BacMid 2 DNA from Example 4:

[0229] cDNA encoding the VL region of the antibody was introduced into pVTPH/Ck which recombines with the PH/1629 region of BacMid2,

[0230] cDNA encoding the VH region of the antibody was cloned into pVT/gp37-Cγ1 which recombines with the gp37 region of BacMid2.

Sf9 cells were transfected by lipofection with BacMid 2 and the 2 transfer vectors obtained in Example 4 and incubated for 4 days at 28° C. Culture supernatants were collected and the recombinant baculoviruses generated, secreted in the culture medium, were cloned by the lysis plaque technique.
The organization of the genome of the recombinant baculoviruses was verified by Southern blot (see FIG. 9) and the integrated exogenous genes (i.e. VL and VH) were verified after PCR amplification, cloning and sequencing.

Example 7: Use of BacMid 3 for the Production of Virus-Like Particles (VLPs)

[0231] Production of Influenza VLPs

To produce these VLPs, the 3 genes of the influenza virus, M, HA and NA, were coexpressed.
These 3 genes were integrated into the three transfer vectors necessary to recombine with BacMid 3 from Example 5:

[0232] The M gene was introduced into transfer vector pVT/PH as described in FIG. 5.

[0233] The HA gene was introduced into transfer vector pVT/gp37 as described in FIG. 4.

[0234] The NA gene was introduced into transfer vector pVT/Chit/Cath as described in FIG. 8.

Sf9 cells were transfected by lipofection with BacMid 3 and the 3 transfer vectors obtained above, then incubated for 4 days at 28° C. The recombinant baculoviruses generated and secreted in the culture supernatant were cloned by the lysis plaque method.
The organization of the genomes of the recombinant baculoviruses was verified by Southern blot (see FIG. 10) and the integrated genes were verified after PCR amplification, cloning and sequencing. The Southern blot was performed on genomic DNA of the recombinant virus expressing the 3 influenza virus proteins HA, NA and M to detect cDNA encoding the integrated M, HA and NA proteins. The membranes were hybridized with probes specific to these 3 genes.

Example 8: Use of BacMid 3 for the Production of Bispecific Antibodies

[0235] The bispecific antibody constructed according to the patent (PCT/IB2012/053482) consists of a heavy chain composed of the VH+CH1+CH2+CH2+CH3 domains of an antibody 1, N-terminal fused to the VH+CH1 domains of an antibody 2. Mutations introduced at the interface of the CL and CH1 regions of antibody 1 promote correct matching between the VL1 and VL2 domains of light chains L1 and L2, which are produced separately, and the corresponding VH1 and VH2 domains. Production of this antibody requires simultaneous and equal production of 3 chains, the fused heavy chain, light chain L1 and light chain L2.

[0236] cDNA encoding light chain L1 was introduced into transfer vector pVT/PH as described in FIG. 5.

[0237] cDNA encoding light chain L2 was introduced into transfer vector pVT/gp37 as described in FIG. 4.

[0238] cDNA encoding the fused heavy chain was introduced into transfer vector pVT/Chit-Cath as described in FIG. 8.

FIG. 11 is a diagram illustrating in A the structure of the bispecific antibody and in B the polyacrylamide gel electrophoresis analysis of the bispecific antibody purified on protein A Sepharose column.