Methods to produce bunyavirus replicon particles

Abstract

The invention relates to methods of producing infectious bunyavirus replicon particles. These bunyavirus replicon particles are safe and can be used outside a biosafety containment. The invention further relates to recombinant bunyavirus replicon particles and uses of these recombinant bunyavirus replicon particles.

Claims

1. A non-spreading recombinant bunyavirus replicon particle, comprising: a bunyavirus L genome segment a bunyavirus S genome segment or part of a bunyavirus S genome segment comprising at least the N gene and the 3 and 5 UTRs; and, optionally, when a bunyavirus M genome segment is to be present, a bunyavirus M genome segment from which the GnGc coding region has been functionally inactivated, resulting in a non-spreading bunyavirus replicon particle.

2. The recombinant bunyavirus replicon particle according to claim 1, comprising a M genome segment from which the GnGc coding region has been deleted.

3. The recombinant bunyavirus replicon particle according to claim 1, in which a M genome segment is absent.

4. The recombinant bunyavirus replicon particle according to claim 1, wherein the bunyavirus L genome segment and/or the S genome segment and/or, when present, the M genome segment, comprises a foreign gene.

5. The recombinant bunyavirus replicon particle according to claim 1, comprising a M, L, or S minigenome.

6. The recombinant bunyavirus replicon particle according to claim 5, wherein a foreign gene is present on said M, L or S-minigenome.

7. The recombinant bunyavirus replicon particle according to claim 1, wherein the bunyavirus is Rift Valley fever virus.

8. An isolated eukaryotic cell comprising the recombinant bunyavirus replicon particle according to claim 1.

9. A medicament comprising the recombinant bunyavirus replicon particle according to claim 1.

10. A vaccine comprising the recombinant bunyavirus replicon particle according to claim 1.

Description

FIGURE LEGENDS

(1) FIG. 1. Expression of the N protein from the RVFV S genome segment. BSR-T7/5 cells (A) or FP-T7-infected BHK-21 cells (B) were transfected with plasmid pUC57-S, encoding the RVFV S genome segment in the antigenomic-sense orientation. Expression of the RVFV N protein was detected using a N protein-specific mAb and HRP-conjugated anti-mouse IgG antibodies.

(2) FIG. 2. Production of Rift Valley fever virus replicon particles (RRPs) containing three genome segments. BHK cells were infected with FP-T7 and subsequently transfected with a plasmid encoding the M-eGFP minigenome (M-eGFP), or in combination with plasmids encoding the RVFV L and S genome segments (M-eGFP/L/S), or in combination with the aforementioned plasmids and pCAGGS-NSmGnGc (M-eGFP/L/S+NSmGnGc). The number of eGFP-positive producer cells, recipient cells or recipient cells previously transfected with helperplasmids (+HP) pCIneo-RVFV-L and pCAGGS-N were determined by flow cytometry.

(3) FIG. 3. Production of RRPs containing two genome segments. BHK cells were infected with FP-T7 and subsequently transfected with a plasmid encoding the S-eGFP minigenome (S-eGFP), or in combination with plasmids encoding the RVFV L genome segments (S-eGFP/L), or in combination with the aforementioned plasmids and pCAGGS-NSmGnGc (S-eGFP/L+NSmGnGc). The number of eGFP-positive producer cells, recipient cells or recipient cells previously transfected with helperplasmids (+HP) pCIneo-RVFV-L and pCAGGS-N were determined by flow cytometry.

(4) FIG. 4. Expression of GnGc in BHK-GnGc cells. Detection of GnGc expression in BHK-21 cells (negative control, A) and BHK-GnGc cells (B) by IPMA using polyclonal antibodies directed against the Gn and Gc proteins and detection of Gc in cell lysates on a Western blot (C) using a peptide antiserum.

(5) FIG. 5. Expression of eGFP in BHK-GnGc cells persistently infected with NDV-GnGc maintaining the RVFV L genome segment and S-eGFP minigenome at cell passage 18.

(6) FIG. 6. RRP production by BHK-GnGc cells after introduction of pCAGGS-GnGc or pCAGGS-NSmGnGc. At 16 h after transfection, the culture medium was replaced by fresh medium. RRP titers were determined on BHK-21 cells using the Spearman-Krber method (Krber 1931. Arch. Exp. Path. Pharmak 162, 480-483; Spearman 1908 Br. J. Psychol 2, 227-242).

(7) FIG. 7. A) Partial sequence pUC57-L (SEQ ID NO:20); Plasmid encodes the RVFV strain 35/75 L genome segment in antigenomic-sense orientation. cDNA of the RVFV strain 35/74 L genome segment, flanked by a T7 promoter and cDNA encoding a HDV ribozyme was synthesized by the GenScript Corporation and cloned in the pUC57 vector using KpnI/HindIII. The T7 promoter, HDV ribozyme sequence and T7 terminator sequences, respectively, are underlined. The UTRs are indicated in italics, the open reading frame encoding the L protein is indicated in bold.

(8) (B) Partial sequence of plasmid pUC57-M (SEQ ID NO:21); Plasmid encodes the RVFV strain 35/74 M genome segment in antigenomic-sense orientation. cDNA encoding the antigenomic-sense RNA of the RVFV strain 35/74 M genome segment, flanked by a T7 promoter and cDNA encoding a HDV ribozyme was synthesized by the GenScript Corporation and cloned in the pUC57 vector using EcoRI/SalI. The T7 promoter, HDV ribozyme sequence and T7 terminator sequences, respectively, are underlined. The UTRs are indicated in italics, the open reading frame encoding the NSmGnGc proteins is indicated in bold.

(9) (C) Partial sequence of plasmid pUC57-S()(SEQ ID NO:22): Plasmid encodes the RVFV strain 35/74 S genome segment in genomic-sense orientation. cDNA encoding the genomic-sense RNA of the RVFV strain 35/74 S genome segment, flanked by a T7 promoter and cDNA encoding a HDV ribozyme was synthesized by the GenScript Corporation and cloned in the pUC57 vector using BamHI/XbaI. The T7 promoter, HDV ribozyme sequence and T7 terminator sequences, respectively, are underlined. The UTRs and intergenic region are indicated in italics, the open reading frames encoding the N and NSs proteins are indicated in bold.

(10) (D) Partial sequence of plasmid pUC57-SNSs (SEQ ID NO:23): Plasmid encodes the RVFV strain 35/74 S genome segment in antigenomic-sense orientation with a major deletion in NSs.

(11) The synthetic DNA was cloned between XbaI and ApaI of pUC57. The T7 promoter, HDV ribozyme sequence and T7 terminator sequences, respectively, are underlined. The UTRs and intergenic region are indicated in italics, the open reading frame encoding the N and the open reading frame encoding part of the NSs protein are indicated in bold.

(12) (E) Partial sequence of plasmid pUC57-S (SEQ ID NO:24): Plasmid encodes the RVFV strain 35/74 M genome segment in antigenomic-sense orientation. To construct cDNA encoding the complete S genome segment in antigenomic-sense orientation, the sequence between NcoI, EcoRV was isolated from the pUC57-S() construct and used to replace the sequence between NcoI and EcoRV of plasmid pUC57-SNSs, yielding pUC57-S. The T7 promoter and HDV ribozyme and T7 terminator sequences, respectively are underlined. The UTRs and intergenic region are indicated in italics, the open reading frames encoding the N and NSs proteins are indicated in bold.

(13) (F) Partial sequence of plasmid pUC57-S-eGFP (SEQ ID NO:25): Plasmid encodes the RVFV strain 35/74 M genome segment in antigenomic-sense orientation, where the NSs gene is replaced for the gene encoding enhanced green-fluorescent protein (eGFP).

(14) The T7 promoter and HDV ribozyme and T7 terminator sequences, respectively are underlined. The UTRs and intergenic region are indicated in italics, the open reading frames encoding the N and eGFP proteins are indicated in bold. The sequence was cloned between KpnI and SalI of pUC57. A silent C.fwdarw.T mutation is underlined.

(15) (G) Partial sequence of plasmid pUC57-Mv (SEQ ID NO:26): Plasmid encodes the RVFV strain 35/74 M genome segment in genomic-sense orientation, where the complete NSmGnGc ORF is deleted and NcoI and XbaI sites are introduced.

(16) The T7 promoter and HDV ribozyme and T7 terminator sequences, respectively are underlined. The UTRs are indicated in italics, the sequence between the UTRs is indicated in bold. Cloned between EcoRI and PstI of pUC57.

(17) (H) Partial sequence of plasmid pUC57-Mv-eGFP (SEQ ID NO:27): Plasmid encodes the RVFV strain 35/74 M genome segment in genomic-sense orientation, where the complete NSmGnGc ORF is deleted and eGFP is introduced between the NcoI and XbaI sites. The T7 promoter and HDV ribozyme sequences are underlined.

(18) (I) Partial sequence of plasmid pUC57-GnGc (SEQ ID NO:28): Plasmid encodes a codon-optimized version of the open reading frame of the RVFV strain 35/74 M genome segment starting at the fourth methionine codon. The gene was synthesized and cloned between EcoRI and HindIII. This gene was used to construct pCIneo-GnGc and pCAGGS-GnGc Using EcoRI and NotI (underlined). The GnGc open reading frame is indicated in bold.

(19) (J) Plasmids pCIneo-NSmGnGc and pCAGGS-NSmGnGc encode the open reading frame of the RVFV strain 35/74 M genome segment starting at the first methionine codon (SEQ ID NO:29). The gene was amplified from 35/74 cDNA and cloned in pCIneo and pCAGGS using EcoRI and NotI (underlined). The NSmGnGc open reading frame is indicated in bold.

(20) (K) Plasmid pUC57-N encodes a codon-optimized version of the N open reading frame of the RVFV strain 35/74 (SEQ ID NO:30). The gene was introduced into pCAGGS and pCIneo using EcoRI and NotI (underlined). The open reading frame of the N protein is indicated in bold.

(21) (L) Plasmid pCIneo-L contains the open reading frame of the L gene of RVFV strain 35/75 (SEQ ID NO:31). The gene was introduced into using XhoI and NotI (underlined). The transition mutation (T5912C), resulting in the substitution of isoleucin-1971 for threonine is also underlined. The open reading frame of the L protein is indicated in bold.

(22) FIG. 8. RRPs are incapable of autonomous spread. BHK cells were infected with RRPs at an m.o.i. of 1. After two days, eGFP expression was observed in infected cells (left panel). Fresh BHK cells were incubated with the collected supernatant and monitored for eGFP expression after three days (right panel).

(23) FIG. 9. RRP production kinetics. BHK-Rep cells were grown in GMEM supplemented with 5% serum and were either left untreated (GP) or transfected with pCAGGS-NSmGnGc (+GP). RRP titers were determined at different time points on BHK cells using eGFP expression as the readout parameter. Titers were determined using the Spearman-Krber method.

(24) FIG. 10. Western blot analysis of RRP proteins. Culture medium of BHK-Rep cells (GP) or of BHK-Rep cells transfected with pCAGGS-NSmGnGc (+GP) was ultracentrifuged at 100 000g for 2 h. The proteins present in the pellets were separated in 4-12% NuPAGE Bis-Tris gels and subsequently transferred to nitrocellulose blots. Specific proteins were detected by an anti-Gn (-Gn) or anti-Gc (-Gc) peptide antiserum or a mAb specific for the N protein (N). The positions of the NSm, Gn, Gc and N protein are indicated by arrows. Molecular weight standard proteins are indicated to the right in kilodaltons.

(25) FIG. 11. RRP infection of mammalian and insect cells. Cells were infected with RRPs and the number of positive cells was determined by flow-cytometry at 42 (BHK and HEK293T) or 72 hours post infection (S2 and C6/36). (A) Representative result from flow cytometry of RRP-infected mammalian and insect cells. Counts of non-infected control cells and infected cells are depicted in grey and green respectively. (B) Histograms showing averaged results of three independent measurements with S.D.

(26) FIG. 12. Vaccine efficacy of RRPs. Mice were either non-vaccinated (n=9; Mock) or vaccinated (n=10) either once (1) or twice (2) via the intramuscular route (IM) or subcutaneous route (SC) with 106 TCID50 of RRPs. Mice were challenged with a known lethal dose of RVFV strain 35/74 via the intraperitoneal route. The mortality rates were determined until 21 days post challenge (d.p.c.).

(27) FIG. 13. Injection site reactions observed at different time points after vaccination. Score 0: No aberrations noted; 1: Swelling observed; 2: Round swelling of maximal 5 cm in diameter; 3: Major swelling/abscess chance of rupture. PM, post mortem. Results are depicted as averages (n=6) with SD.

(28) FIG. 14. Rectal temperatures of vaccinated and unvaccinated (Mock) lambs before and after challenge with RVFV. Rectal body temperatures ( C.) were determined daily. Fever was defined as a body temperature above 41 C. (interrupted line). Results are depicted as averages (n=6) with SD.

(29) FIG. 15. Detection of viral RNA in plasma samples of vaccinated and unvaccinated lambs obtained at different time points after challenge infection with RVFV. The number of positive samples differs at each time point. Results are depicted as averages (n=6) with SD.

(30) FIG. 16. Body weight of lambs vaccinated once with one of the indicated vaccines. Results are depicted as averages (n=6) with SD.

(31) FIG. 17. Biochemical analysis of serum samples. ALT, ALP, TP, Creatinine and BUN concentrations are depicted as averages (n=6) with SD. The upper and lower reference values are indicated by dashed lines.

(32) FIG. 18. Schematic representation of the method used to create replicon cell lines producing the sHA.sub.3 and sNA.sub.4 proteins. GOI, gene of interest (in this work either the eGFP, sHA.sub.3 or sNA.sub.4, gene).

(33) FIG. 19. Flow cytometry demonstrating the percentage of replicon cell lines that produce the N protein at cell passage 8. N protein expression is dependent on the presence of the S and L genome segment, of which the former contains the foreign gene of interest (GOI). Cells expressing L and S-eGFP genome segments (A); cells expressing L and the S-CD5-HA-GCN4-ST (i.e. S-.sub.sHa.sub.3) segment (B); cells expressing L and the S-CD5-OS-GCN4-NA segment (i.e. S-.sub.s NA.sub.4) (C); control cells expressing GnGc are depicted in (D), (E) and (F).

(34) FIG. 20. Purified sHA.sub.3 and sNA.sub.4 were analyzed on Silver-stained polyacrylamide gels (A) and Western blots using Strep-Tacting-HRP for detection (B). The negative control sample (eGFP) was provided by following the purification procedure using medium collected from replicon cells expressing eGFP. The positions of molecular weight standard proteins are indicated to the left in kDa.

EXAMPLES

Example 1

Materials and Methods

(35) Cells and Growth Conditions.

(36) BSR-T7/5 were kindly provided by Prof. Dr. K. Conzelmann (Max von Pettenkofer-Institut, Mnchen, Germany). BHK-GnGc are BHK-21 cells that contain a genome-integrated plasmid pCIneo-GnGc described hereinbelow. BSR-T7/5 and BHK-GnGc cells were grown in Glasgow Minimum Essential Medium (GMEM; Invitrogen, Carlsbad, Calif., USA) supplemented with 4% tryptose phosphate broth (Invitrogen), 1% non-essential amino acids (Invitrogen), 10% fetal bovine serum (FBS; Pam Biotech, Aidenbach, Germany) and penicillin/streptomycin (Invitrogen) at a concentration of 100 U/ml and 100 g/ml, respectively. For maintenance of stable cell lines, geneticin (G-418; Invitrogen) was used at a concentration of 1 mg/ml. Cells were grown at 37 C. and 5% CO2.

(37) Plasmids and Viruses.

(38) Plasmids pCIneo-GnGc and pCAGGS-GnGc contain the open reading frame (ORF) of the M segment of RVFV strain 35/74, starting at the fourth methionine codon (FIG. 7I). Plasmid pCAGGS-N contains the ORF encoding the N gene (FIG. 7K). The N and GnGc-encoding sequences were codon-optimized for optimal expression in mammalian and insect cells and synthesized by the GenScript Corporation (Piscataway, N.J., USA). Plasmid pCAGGS-NSmGnGc contains the authentic ORF of the M segment starting at the first methionine codon (FIG. 7J). pCIneo-RVFV-L encodes the authentic ORF encoding the viral polymerase of RVFV strain 35/74 (FIG. 7L). This gene contains a transition mutation (T5912C), resulting in the substitution of isoleucin-1971 for threonine. The effect of this mutation was not studied.

(39) Expression of RVFV genes from pCIneo plasmids is controlled by a cytomegalovirus (CMV) immediate-early enhancer/promoter, whereas the expression of genes from pGAGGS plasmids is controlled by a CMV immediate enhancer/-actin (CAG) promoter (Niwa et al. 1991. Gene 108: 193-199). RVFV strain 35/74 was isolated at the Agricultural Research Council-Onderstepoort Veterinary Institute (ARC-OVI) from the liver of a sheep that died during a RVFV outbreak in the Free State province of South Africa in 1974 (Barnard 1979. J S Afr Vet Assoc 50: 155). The virus was passaged four times in suckling mice by intra-cerebral injection and three times on BHK-21 cells. Amplification of the genome segments was performed by one-step RT-PCR with SBS Genetech AMV-RT, TaKaRa Ex Taq HS and the primers described by Bird et al. (Bird et al. 2007. J Virol 81: 2805-2816). PCR products were purified with the Qiagen Gel extraction kit after separation on agarose gel and mixed in appropriate equal amounts before GS FLX sequencing at Inqaba Biotec (Pretoria, South Africa). Sequencing and sequence assembly was performed essentially as described for dsRNA virus genomes (Potgieter et al. 2009. J Gen Virol 90: 1423-1432). Consensus sequences corresponding to each genome segment were synthesized and cloned in pUC57, a standard cloning vector of GenScript Corporation (Piscataway, N.J., USA). pUC57-L (FIG. 7A), pUC57-M (FIG. 7B) and pUC57-S (FIG. 7E) encode the RVFV L, M and S genome segment in antigenomic (i.e. positive) sense orientation, respectively. These transcription plasmids each contain a complete copy of the viral RNA segments and are flanked by a T7 promoter and a HDV ribozyme sequence. In pUC57-S-eGFP() (FIG. 7F), the NSs gene is replaced by the gene encoding enhanced green fluorescent protein (eGFP). Of note, transcription of this plasmid results in a S-eGFP minigenome in which the eGFP gene is in the genomic (i.e. negative-sense) orientation. In plasmid pUC57-Mv-eGFP() (FIG. 7H), the complete ORF of the M segment is replaced by the eGFP gene. The M-eGFP minigenome produced from this plasmid is in the genomic-sense orientation as well.

(40) A recombinant Newcastle disease virus (NDV) that produces the RVFV structural glycoproteins Gn and Gc (i.e. NDFL-GnGc), from hereon referred to as NDV-GnGc, was previously described (Kortekaas et al. 2010. Vaccine 28: 4394-4401).

(41) A recombinant fowlpox virus that produces T7 polymerase, named fpEFLT7pol (Das et al. 2000. J Virol Meth 89: 119-127), from hereon referred to as FP-T7, was kindly provided by the Institute for Animal Health (IAH, Compton, UK). Virus titers were determined as 50% tissue culture infective dose (TCID50) on BHK-21 cells using the Spearman-Krber method (Krber 1931. Arch. Exp. Path. Pharmak 162, 480-483; Spearman 1908 Br. J. Psychol 2, 227-242),

(42) Rescue of Recombinant RVFV Strain 35/74.

(43) BSR-T7/5 cells were seeded in 6-well plates and were co-transfected with 1 g of plasmids pUC57-L, pUC57-M and pUC57-S using jetPEI transfection reagent according to the instructions of the manufacturers (Polyplus-transfection SA, Illkirch, France). After 6 days of incubation, medium was collected. For the detection of infectious virus, BHK-21 cells were incubated with the collected supernatant. When clear cytopathic effect was observed, the cells were fixed in 4% paraformaldehyde/PBS for 40 min. Plates were subsequently submerged completely in 80% ethanol/4% acetic acid to inactivate the virus and washed with PBS. Immunoperoxidase monolayer assays (IPMAs) were performed as described hereinbelow.

(44) Alternatively, BHK-21 cells were infected with FP-T7. Approximately 10E6 cells in each well of a six-well plate were inoculated with 1.5 ml of culture medium containing 10E5 TCID50 of FP-T7 (multiplicity of infection [m.o.i.] of 0.1). After incubation with FP-T7 for 1 h and recovery for another hour, the cells were treated in a similar way as described for BSR-T7/5 cells.

(45) Rescue of RVFV BRPs (RRPs).

(46) BHK-21 or BHK-GnGc cells were seeded in 6-well plates and incubated with FP-T7 for 1 h at 37 C. Medium was refreshed and cells were allowed to recover for 1 h. For the production of RRPs containing three genome segments, the cells were subsequently transfected with 600 ng of plasmids pUC57-L, pUC57-S, pUC57-Mv-eGFP() and pCAGGS-NSmGnGc. For the production of RRPs containing two genome segments, cells were transfected with pUC57-L, pUC57-S-eGFP() and pCAGGS-NSmGnGc. The medium was refreshed the next day. Alternatively, when NDV was used to provide Gn and Gc, NDV-GnGc infection was performed together with FP-T7. NDV-GnGc was used at an m.o.i. of 0.05. Supernatants were harvested after 72 h, pre-cleared at 5 000 rpm for 5 min at RT and stored at 4 C. until further use.

(47) NuPAGE and Western Blotting

(48) NuPAGE and Western blotting was performed as described (Kortekaas et al. 2010. Vaccine 28: 2271-2276). Briefly, proteins were diluted in 3 Laemmli sample buffer (0.5 M Tris pH 6.8, 6% (w/w) SDS, 26% (v/v) glycerol, 15% (v/v) 2-mercaptoethanol and 0.002% (w/w) bromophenol blue) and heated at 95 C. for 5 minutes, before loading onto 4-12% NuPAGE Bis-Tris gels. Proteins were subsequently transferred to nitrocellulose blots. To visualize Gc, rabbit polyclonal antibodies were used that were previously raised against a Gc-derived peptide (residues 975-VFERGSLPQTRNDKTFAASK-994), respectively (De Boer et al. 2010. Vaccine 28:2330-2339). Goat anti-rabbit IgG antibodies conjugated to horseradish peroxidase (HRP) (Dako, Heverlee, Belgium) were used as the secondary antibodies. Peroxidase activity was detected using the Amersham ECL Western blotting detection reagents (GE Healthcare, Diegem, Belgium). For the detection of the N protein, monoclonal antibody F1D11 (kindly provided by Dr. Alejandro Brun, CISA-INIA, Madrid, Spain) was used as the primary antibody and rabbit anti-mouse IgG (DAKO) as the secondary antibody.

(49) Immunoperoxidase Monolayer Assays (IPMA).

(50) IPMAs were performed as described previously (deBoer et al. 2010. Vaccine 28: 2330-2339). As the primary antibody, either a polyclonal antiserum was used that was previously obtained from a sheep that was vaccinated with NDFL-GnGc (Kortekaas et al. 2010. Vaccine 28: 4394-4401). For detection of the RVFV N protein, mAb F1D11 was used, which was previously kindly provided by Dr. Alejandro Brun (CISA-INIA, Spain). As the secondary antibody HRP-conjugated rabbit anti-mouse IgG (Dako, Heverlee, Belgium) was used.

(51) Flow Cytometry.

(52) For flow cytometry, cells of a six-well plate were washed with 3 ml PBS and then trypsinized with 0.3 ml 0.5% trypsine-EDTA (Invitrogen). After incubation for 2-3 min at 37 C. and resuspension, the cells were diluted in 1 ml culture medium. Cells were pelleted by centrifugation, resuspended in 0.5 ml PBS and pelleted again. Cells were fixed by adding 0.1 ml 4% PFA in PBS for 15-30 min and subsequently diluted in 0.5 ml PBS. Each sample contained all the cells from one well. The samples were stored at 4 C. until analysis. All measurements were performed at the day of harvesting the cells. Flow cytometry was performed using a CyAn ADP flow cytometer (Beckman, Woerden, The Netherlands), equipped with a 488 nm wavelength laser. Data analysis was performed with the Summit v4.3 software.

(53) Results

(54) Rescue of recombinant RVFV strain 35/74 from cDNA using BSR-T7/5 cells. Recombinant RVFV strain 35/74 (rec35/74) was rescued using three plasmids encoding the three viral RNA segments L, M and S in antigenomic-sense orientation. Transfection of the three plasmids into BSR-T7/5 cells resulted in cytopathic effect after 3-4 days. BHK-21 cells were inoculated with the collected supernatant. A titer of 10E9 TCID50/ml was obtained after two passages of the virus. No silent mutations were introduced in the genome of recombinant RVFV strain 35/74 to confirm rescue and exclude contamination with non-recombinant wildtype virus, since no wildtype virus was ever present in our laboratory before rescue of rec35/74.

(55) Production of VLPs Containing a Reporter Minigenome Using Transient Expression of NSmGnGc from Plasmid.

(56) As a first step towards the establishment of an Rift Valley fever virus replicon particle (RRP) production system, a minireplicon system was developed. A plasmid was designed in which the gene encoding eGFP is placed between M segment untranslated regions (UTRs). This plasmid was named pUC57-Mv-eGFP() and encodes the M-eGFP minigenome in genomic-sense orientation. Like the full-length constructs, the cDNA encoding this minigenome is flanked by a T7 polymerase promoter and cDNA encoding a HDV ribozyme sequence. Transfection of pUC57-Mv-eGFP() together with plasmids pUC57-L and pUC57-S resulted in eGFP expression in only a few cells (data not shown). In an attempt to improve reporter minigenome expression, the two helper plasmids pCIneo-RVFV-L and pCAGGS-N were added to the transfection mixture. This resulted in a slight increase in the number of positive cells (data not shown). Despite the very low number of eGFP-positive cells, an attempt was made to package the genome segments into VLPs by co-transfecting the cells with pCAGGS-NSmGnGc. If indeed VLPs were produced, we reasoned that these VLPs could contain either one, two or three genome segments. To be able to detect also VLPs that contain only the reporter minigenome, the supernatant was not only added to untreated BHK-21 cells, but also to BHK-21 cells that were previously transfected with helper plasmids pCIneo-RVFV-L and pCAGGS-N. Very few BHK-21 cells that were inoculated with the collected supernatant were shown to express eGFP after 18-24 hrs of incubation and expression was only observed in cells that were previously transfected with helper plasmids (data not shown). Thus, although VLPs were produced, none of these VLPs apparently contained all three genome segments.

(57) Production of VLPs Containing a Reporter Minigenome Using NDV to Provide Gn and Gc.

(58) Although we were previously able to produce large amounts of VLPs by expressing GnGc in insect cells (de Boer et al. 2010. Vaccine 28: 2330-2339), production of GnGc from pol-II promoters in mammalian cells was extremely poor in previous experiments, yielding no detectable VLPs in the culture supernatant (unpublished results). We also previously reported the production of an NDV recombinant (i.e. NDFL-GnGc, from hereon referred to as NDV-GnGc) that produces the GnGc glycoproteins. Interestingly, infection of BHK-21 cells with this recombinant virus did result in the production of detectable amounts of Gn and Gc in the supernatant (Kortekaas et al. 2010. Vaccine 28: 4394-4401). In the present work, we wondered if NDV-GnGc could be used to provide the Gn and Gc proteins for the packaging of RVFV genome segments. Cells were first transfected with pUC57-L, pUC57-S and pUC57-Mv-eGFP() and infected with NDV-GnGc, 24 hrs later. Expression of eGFP was observed in a small percentage of producer cells and in very few recipient cells (data not shown). Expression of eGFP in recipient cells was again dependent on a previous transfection with the helper plasmids pCIneo-RVFV-L and pCAGGS-N. These experiments demonstrated that we were able to package a reporter minigenome into VLPs using NDV-GnGc as a source of the glycoproteins, but that particles capable of autonomous replication were not obtained.

(59) Improved Rescue of RVFV from cDNA Using a Recombinant Fowlpox Virus as a Source of T7 Polymerase and Successful Production of RRPs.

(60) Virus recovery from BSR-T7/5 cells was not reproducible. As an alternative for BSR-T7/5 cells, we decided to use a recombinant fowlpox virus that expresses T7 polymerase (i.e. FP-T7) (Das et al. 2000. J Virol Methods 89: 119-127). In experiments where the rescue efficiency of RVFV using BSR-T7/5 cells and FP-T7-infected BHK-21 cells were compared, use of FP-T7 resulted in RVFV rescue in 5/5 attempts, whereas rescue using BSR-T7/5 cells was unsuccessful. We anticipated that a higher level of T7 polymerase expression by FP-T7 could result in higher production levels of the N and L proteins from the antigenomic (i.e. positive-sense) genome segments, facilitating initiation of replication. To test this hypothesis, the pUC57-S segment was transfected into BSR-T7/5 cells and into BHK-21 cells that were previously infected with FP-T7. Transcription of the pUC57-S plasmid by T7 polymerase results in antigenomic sense viral RNA of which the N gene is in sense orientation. Whereas the N protein could not be detected in BSR-T7/5 cells transfected with pUC57-S (FIG. 1A), FP-T7-infected BHK-21 cells that were transfected with this plasmid stained intensely with anti-N antibodies (FIG. 1B).

(61) We then proceeded with co-transfection of pUC57-S, pUC57-L and pUC57-Mv-eGFP() in FP-T7-infected BHK-21 cells. Whereas in previous experiments using BSR-T7/5 cells, only a few eGFP-positive cells were observed, in these experiments, FACS analysis showed that 1.3% of the cells were positive for eGFP (FIG. 2). A similar experiment was performed where cells were co-transfected with pCAGGS-NSmGnGc. In this experiment, 2.5% of the cells were positive for eGFP (FIG. 2). Interestingly, the observation of clusters of eGFP-positive cells suggested local spread of VLPs containing the reporter minigenome (data not shown). After three days, the culture supernatant was collected and incubated with BHK-21 cells that were either untreated or transfected with the helper plasmids pCIneo-RVFV-L and pCAGGS-N. In cells that were transfected with helperplasmids, 1.3% was positive for eGFP expression (FIG. 2). Moreover, 0.9% of the cells that were not previously transfected with helperplasmids was positive for eGFP expression, demonstrating that we were successful in producing replicon particles that contain all three genome segments (FIG. 2).

(62) Production of RRPs Containing Two Genome Segments.

(63) To facilitate further optimization of the system, we aimed to produce RRPs that contain only two genome segments. To this end, a reporter minigenome was produced in which the gene encoding the NSs gene of the S segment was exchanged for the gene encoding eGFP (i.e. S-eGFP). Previous work demonstrated that the NSs gene is not essential for growth in tissue culture (Muller et al. 1995. Am J Trop Med Hyg 53: 405-411) and other studies demonstrated that a virus containing this deletion is viable (Ikegami et al. 2006. J Virol 80: 2933-2940). Co-transfection of the resulting plasmid, pUC57-S-eGFP() with pUC57-L resulted in expression of eGFP in a small percentage of cells. However, when pCAGGS-NSmGnGc was added to the transfection mixture, 21.5% of the cells were positive for eGFP, as determined by FACS analysis (FIG. 3). Incubation of BHK-21 cells with the collected supernatant resulted in 4.7% of positive cells when helper plasmids were not provided, whereas the number of cells increased to 28.7% when helper plasmids were included (FIG. 3).

(64) Production of Stable BHK-21 Cell Lines that Produce the Gn and Gc Glycoproteins.

(65) With the aim to produce a system for the continuous production of RRPs, stable cell lines were produced that constitutively produce the Gn and Gc proteins. Briefly, BHK-21 cells were transfected with pCIneo-GnGc and clones with integrated plasmids were grown in the presence of geneticin G-418. A number of clones were tested for Gn/Gc expression by IPMA. A highly positive antiserum from a naturally infected sheep did not reveal any Gn/Gc positive cells (data not shown). An antiserum derived from a sheep that was previously vaccinated with NDV-GnGc was, however, successfully used to identify positive clones. One of the clones clearly displayed the highest Gn and Gc expression level as revealed by IPMA, although the expression levels appeared low (FIG. 4). This clone was tested for expression of Gc by Western blotting of proteins from cell lysates. Using a previously described polyclonal antiserum specific for a Gc-derived peptide, Gc expression was clearly detected (FIG. 4C). This cell line was designated BHK-GnGc. To determine if VLPs were produced by these cells, supernatants were ultracentrifuged and the proteins present in the collected pellets were analyzed by Western blotting. The Gc protein was not detected in the pellet fractions, suggesting that either no VLPs were produced, or that glycoprotein production was too low to allow detection.

(66) Use of a Cell Line that Maintains the RVFV L and S-eGFP Segments is Essential for the Efficient Production of RRPs.

(67) Production of RRPs by infection of BHK-21 cells with FP-T7 and subsequent introduction of plasmids providing the L segment, S-eGFP segment and pCAGGS-NSmGnGc resulted in a maximum of RRP titers of 10E3 to 10E4 TCID50/ml (data not shown). Although the BHK-GnGc cell line appeared not suitable for the constitutive large scale production of RRPs, it was striking to find that introduction of FP-T7 and subsequent introduction of plasmids providing the L segment, S-eGFP segment and pCAGGS-NSmGnGc, resulted in clusters of positive cells that were larger in both number and size than those obtained with normal BHK-21 cells.

(68) A cell line maintaining the RVFV L and S-eGFP segments could be highly valuable for several applications, including high-throughput screens of antiviral agents outside biosafety containment facilities. We therefore aimed to produce a cell culture of which each cell contains the RVFV L and S-eGFP segment. To this end, the FP-T7 virus was introduced, followed by introduction of the plasmids providing the L segment and the S-eGFP segment. To facilitate spread of the L and S-eGFP segment, pCAGGS-NSmGnGc was introduced several times after passage of the cells. Using this method, cell lines of both wildtype BHK-21 cells as well as BHK-GnGc were obtained of which most, if not all, cells expressed the eGFP reporter. However, whereas repetitive passage of the eGFP-expressing BHK-21 cells resulted in loss of eGFP, passage of the eGFP-expressing BHK-GnGc cells did not result in any loss of eGFP expression. For at least 50 cell passages, eGFP expression in these cells remained unchanged. When not transfected, BHK-GnGc cells containing the RVFV replicons were found to produce very small amounts of RRPs, with a maximum yield of 10E2 TCID50/ml. Importantly, introduction of pCAGGS-NSmGnGc or pCAGGS-GnGc in these cells resulted in RRP titers of maximally 10E6.8 or 10E6.4 TCID50/ml, respectively (FIG. 5).

(69) Efficient Production of RRPs Using NDV-GnGc.

(70) Considering the superior production of GnGc by NDV when compared to expression from plasmid, we tested if RRPs could be produced by infection of the cells with NDV-GnGc. BHK-GnGc cells were co-infected with FP-T7 and NDV-GnGc and, after recovery, were transfected with pUC57-L and pUC57-S-eGFP(). After 72 hrs of incubation, extremely large comets of positive cells were observed in the flasks (data not shown). This result suggested that large amounts of RRPs are produced when NDV-GnGc is used to provide the glycoproteins. We subsequently split the culture into two flasks. One was left untreated, the other was again infected with NDV-GnGc. The supernatants of these cells were collected after 48 h and the TCID50 RRP titers were determined on BHK-21 cells. The supernatant of the cells that were infected with NDV-GnGc twice, contained a titer of 10E7 TCID50/ml, whereas the supernatant of the cells that were infected only once contained a titer of 10E4.5 TCID50/ml. The latter cell line was passaged 18 times, after which the RRP titer was again determined. Surprisingly, this revealed a titer of 10E6 TCID50/ml. Moreover, visual examination of this cell line by fluorescence microscopy revealed that most, if not all, cells expressed eGFP (FIG. 6). This result led us to suggest that the cells were persistently infected with NDFL-GnGc, thereby providing a continuous source of Gn and Gc. Indeed, IPMAs using a mAb specific for the NDV F protein, revealed the presence of the virus in a subset of the cells. It is important to note, however, that the virus present in the supernatant of these cells was shown to be non-infectious. This was expected however, since lentogenic NDV strains such as the recombinant LaSota strain used in the current work, requires cleavage of the F protein by trypsin-like proteases for infectivity. In conclusion, by virtue of the persistent infection of the cells with NDFL-GnGc, RRPs can continuously be produced to titers of up to 10E7 TCID50/ml.

(71) To demonstrate that RRPs are incapable of autonomous spread, BHK cells were infected with RRPs at a multiplicity of infection (m.o.i.) of 1. After two days, eGFP expression was observed by fluorescence microscopy (FIG. 8, left panel). BHK cells were incubated with collected pre-cleared supernatant and after three days, cells were monitored for eGFP expression. No eGFP expression was observed, demonstrating that no progeny infectious particles were produced by the RRP-infected BHK cells (FIG. 8, right panel).

(72) To establish the kinetics of RRP production, BHK-rep cells were transfected with pCAGGS-NSmGnGc and the culture medium was collected at different time points post transfection. RRPs were titrated on BHK cells using eGFP expression as the readout parameter. This experiment demonstrated that a titer close to 106 TCID50/ml was obtained already after 22 h (FIG. 9).

(73) To visualize RRP proteins, RRPs were pelleted by ultracentrifugation. The proteins were separated in NuPAGE gels, transferred to nitrocellulose membranes and detected using peptide antisera specific for the Gn and Gc protein or a monoclonal antibody specific for the N protein. Analysis of the supernatant obtained from non-transfected BHK-Rep cells revealed only the N protein (FIG. 10). Interestingly, this result suggests that the RVFV N protein is released from cells, presumably in the form of ribonucleoprotein core particles, resembling results previously described from studies on CCHFV (Bergeron et al., 2007. J Virol 81: 13271-13276). Analysis of supernatant from BHK-Rep cells transfected with pCAGGS-NSmGnGc (pCAGGS-M) revealed the Gn protein, the NSm protein, the Gc protein and the N protein (FIG. 10).

Example 2

Production of Bunyavirus Replicon Particles (BRPs) of Crimean-Congo Hemorrhagic Fever Virus Strain IbAr10200

(74) cDNA encoding anti virus-sense full-length RNA of the L (GenBank: AY389508.2), M (GenBank: AF467768.2) and S (GenBank: U88410.1) genome segments CCHFV strain IbAr10200 are synthesized and flanked by a T7 polymerase promoter and cDNA encoding a HDV ribozyme sequence. A T7 transcription termination sequence is preferably positioned downstream of the ribozyme cDNA. These sequences are cloned into pUC57 vectors essentially as exemplified in Example 1, resulting in pUC57-CCHFV-L, pUC57-CCHFV-M and pUC57-CCHFV-S.

(75) To facilitate cloning into pUC57 of the cDNA encoding the CCHFV L genome segment, a KpnI restriction site is introduced immediately upstream of the T7 promoter sequence and immediately downstream of the T7 transcription termination sequence.

(76) To facilitate cloning of the cDNA encoding the CCHFV M genome segment, a KpnI restriction site is introduced immediately upstream of the T7 promoter sequence and a SalI restriction site is introduced immediately downstream of the T7 transcription termination sequence.

(77) To facilitate cloning of the cDNA encoding the CCHFV S genome segment, an EcoRI restriction site is introduced immediately upstream of the T7 promoter sequence and a BamHI restriction site is introduced immediately downstream of the T7 transcription termination sequence.

(78) The complete open reading frame of the M genome segment (M-ORF, nucleotides 93-5147 of GenBank sequence AF467768.2) is introduced downstream of the CMV promoter of pCIneo. Stable cell lines are produced by transfecting pCIneo-M-ORF into BHK-21 cells and cells with integrated plasmids are selected by culturing in the presence of G-418.

(79) As an alternative, BHK-21 cells are provided with the CCHFV M-ORF encoded proteins by infecting the eukaryotic cell with a recombinant viral vector that transduces the CCHFV M-ORF-encoded polyprotein, followed by selecting a cell in which the recombinant viral vector is persistently present without causing overt cytopathogenic effect.

(80) The cells that (conditionally or constitutively) express the CCHFV M-ORF-encoded proteins are infected with FP-T7 and, after recovery, transfected with plasmids pUC57-CCHFV-L and pUC57-CCHFV-S. When required for efficient production, a construct encoding the CCHFV structural glycoproteins under the control of a suitable Polymerase-II promoter (such as the CAG promoter in the pCAGGS plasmid) is also transfected or infected to provide the CCHFV structural glycoproteins. This procedure results in the production of CCHFV replicon particles that do not contain a CCHFV M genome segment.

(81) TABLE-US-00001 The T7 promoter that is used has the nucleotide sequence: 5-TAATACGACTCACTATAG-3 The HDV ribozyme sequence that is used has the nucleotide sequence 5-GGGTCGGCATGGCATCTCC-3. The T7 terminator sequence that is used has the nucleotide sequence: 5-TAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTT TG-3

Example 3

Production of Bunyavirus Replicon Particles (BRPs) of Dobrava-Belgrade Virus (DOBV) Strain DOBV/Ano-Poroia/Afl9/1999

(82) cDNA encoding anti virus-sense full-length RNA of the L (GenBank: AJ410617.1), M (GenBank: AJ410616.1) and S (GenBank: AJ410615.1) genome segments DOBV strain DOBV/Ano-Poroia/Afl9/1999 is synthesized and flanked by a T7 polymerase promoter and cDNA encoding a HDV ribozyme sequence. A T7 transcription termination sequence is preferably present downstream of the introduced ribozyme cDNA. These sequences are cloned into pUC57 vectors essentially as exemplified in Example 1, resulting in pUC57-DOBV-L, pUC57-DOBV-M and pUC57-DOBV-S.

(83) To facilitate cloning into pUC57 of the cDNA encoding the DOBV L genome segment, a BamHI restriction site is introduced immediately upstream of the T7 promoter sequence and immediately downstream of the T7 transcription termination sequence.

(84) To facilitate cloning of the cDNA encoding the DOBV M genome segment, a KpnI restriction site is introduced immediately upstream of the T7 promoter sequence and a SalI restriction site is introduced immediately downstream of the T7 transcription termination sequence.

(85) To facilitate cloning of the cDNA encoding the DOBV S genome segment, a KpnI restriction site is introduced immediately upstream of the T7 promoter sequence and a SalI restriction site is introduced immediately downstream of the T7 transcription termination sequence.

(86) The complete open reading frame of the M genome segment (MDOBV-ORF, nucleotides 41-3448 of GenBank sequence AJ410616.1) is introduced downstream of the CMV promoter of pCIneo. Stable cell lines are produced by transfecting pCIneo-MDOBV-ORF into BHK-21 cells and selecting cells with integrated plasmids by culturing in the presence of G-418.

(87) Alternatively, the eukaryotic cell is provided with MDOBV-ORF-encoded proteins by infecting the eukaryotic cell with a recombinant viral vector that produces the MDOBV proteins, followed by selecting a cell in which the recombinant viral vector is persistently present without causing overt cytopathogenic effect.

(88) The cells that (conditionally or constitutively) express the MDOBV-ORF-encoded proteins are infected with FP-T7 and, after recovery, transfected with plasmids pUC57-DOBV-L and pUC57-DOBV-S. When required for efficient production, a construct encoding the DOBV structural glycoproteins under the control of a suitable Polymerase-II promoter (such as the CAG promoter in the pCAGGS plasmid) is also transfected or infected to provide the DOBV structural glycoproteins. This procedure results in the production of DOBV replicon particles.

(89) TABLE-US-00002 The T7 promoter that is used has the nucleotide sequence: 5-TAATACGACTCACTATAG-3 The HDV ribozyme sequence that is used has the nucleotide sequence 5-GGGTCGGCATGGCATCTCC-3. The T7 terminator sequence that is used has the nucleotide sequence: 5-TAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTT TG-3

Example 4

Tet-Off System for the Inducible Expression of RVFV NSm, Gn and Gc Proteins

(90) BHK-21 cells are transfected with the pTet-Off Advanced plasmid (Clontech, CA, USA) according to the instructions of the manufacturers. After selection with G-418, resistant clones are selected, yielding a BHK-Tet-Off Advanced cell line.

(91) The complete open reading frames of the M segment of a RVFV strain 35/74, (nucleotides 21-3614) are synthesized with flanked KpnI (5 end) and NotI and SalI (3 end) restriction sites and cloned into pUC57 using KpnI and SalI restriction sites (GenScript Corporation). The inserts are released from the pUC57 plasmids by KpnI/NotI digestion and cloned into the KpnI/NotI-digested pTREtight vector (Clontech).

(92) The BHK-Tet-Off Advanced cells are transfected with pTREtight-NSmGnGc and a linear marker that facilitates the selection of transfected cells by hygromycin or puromycin. Clones that produce the proteins of interest are selected by growing the clones in the absence of doxycycline (DOX).

(93) After selection of suitable clones, the cells are grown in the presence of DOX and infected with FP-T7. After recovery, the cells are transfected with pUC57-L and pUC57-S-eGFP. After recovery, the culture medium of the cells is replaced by medium without DOX, resulting in expression of NSm, Gn and Gc and the formation of RVFV replicon particles.

Example 5

RVFV Replicon Particles that Produce Recombinant Soluble Multimeric HA of Pandemic Swine-Origin 2009 A (H1N1) Influenza Virus

(94) Oligo MSC-1 and Oligo MSC-2 are synthesized. Annealing of Oligo MCS-1 and Oligo MCS-2 results in a double-stranded DNA molecule containing NcoI and XbaI overhangs and additional SpeI, XhoI, BglI, NotI restriction sites.

(95) TABLE-US-00003 Oligo MCS 1: 5-CATGGACTAGTCTCGAGGCTAGCAGATCTGCGGCCGCT-3 Oligo MCS 2: 5-CTAGAGCGGCCGCAGATCTGCTAGCCTCGAGACTAGTC-3

(96) The eGFP gene is removed from pUC57-S-eGFP (FIG. 7F) by NcoI/XbaI digestion and the MCS linker is ligated into this vector, yielding pUC57-S-MCS.

(97) The sequence listed below (Seq CD5-HA-GCN4-ST) encodes a human codon-optimized soluble hemagglutinin ectodomain (sHA, amino acids 17 to 522) of influenza virus A/California/04/2009 (H1N1). This sequence is synthesized at the GenScript Corporation. The HA gene is preceded by a sequence encoding an N-terminal CD5 signal peptide and followed by sequences encoding a C-terminal artificial GCN4 trimerization domain (GCN4-pII, Harbury et al. 1993. Science 262: 1401-1407) and a Streptavidin-tag (Strep) for affinity purification. This construct is described in Bosch et al. 2010. J. Virol. 84: 10366-10374).

(98) To generate a vector comprising CD5-sHA-GCN4-pII, pUC57-S-MCS is digested with NheI/XbaI and NheI/XbaI digested HA-GCN4-ST sequence is cloned into this plasmid, yielding pUC57-S-HA-GCN4-ST. The CD5 sequence is subsequently introduced by annealing of the following oligo's:

(99) TABLE-US-00004 Oligo CD5-1: 5-CTAGTATGCCCATGGGGTCTCTGCAACCGCTGGCCACCTTGTACCTG CTGGGGATGCTGGTCGCTTCCGTG-3 Oligo CD5-2: 5-CTAGCACGGAAGCGACCAGCATCCCCAGCAGGTACAAGGTGGCCAGC GGTTGCAGAGACCCCATGGGCATA-3

(100) The CD5 linker is ligated into SpeI/NheI-digested pUC57-S-HA-GCN4-ST, yielding pUC57-S-CD5-HA-GCN4-ST.

(101) BHK-GnGc cells are infected with FP-T7. After recovery, the cells are transfected with pUC57-L, pUC57-S-CD5-HA-GCN4-ST and pCAGGS-NSmGnGc, essentially as exemplified in Example 1. After cell passage, the transfection is repeated. This sequence of events is repeated until all the cells of the monolayer express the CD5-HA-GCN4-ST protein, as is determined by IPMA. The quantity of HA-GCN4-ST in the culture medium of these cells is determined. To establish high levels of NSmGnGc proteins, plasmid pCAGGS-NSmGnGc is again introduced in the cell line, yielding replicon particles containing the S-CD5-HA-GCN4-ST gene. The replicon particles are used for vaccination/challenge experiments in mice to establish the protective efficacy against a lethal H1N1 challenge.

(102) Seq. CD5-HA-GCN4-ST

(103) Nucleotides 1-69 (underlined) encode the CD5 sequence

(104) Nucleotides 70-1590 encode the HA ectodomain

(105) Nucleotides 1591-1713 (underlined) encode the GCN4 domain

(106) Nucleotides 1714-1737 encode the Strep tag

(107) TABLE-US-00005 atgcccatggggtctctgcaaccgctggccaccttgtacctgctggggatgctggtcgct MPMGSLQPLATLYLLGMLVA tccgtgctagcagacaccctgtgcatcggctaccacgccaacaacagcaccgacaccgtg SVLADTLCIGYHANNSTDTV gacaccgtgctggagaagaacgtgaccgtgacccacagcgtgaacctgctggaggacaag DTVLEKNVTVTHSVNLLEDK cacaacggcaagctgtgcaagctgcgcggcgtggctccactgcacctgggcaagtgcaac HNGKLCKLRGVAPLHLGKCN attgctggatggatcctgggaaacccagagtgcgagagcctgagcaccgccagcagctgg IAGWILGNPECESLSTASSW agctacatcgtggagacccccagcagcgacaacggcacctgctaccccggcgacttcatc SYIVETPSSDNGTCYPGDFI gactacgaggagctgcgcgagcagctgagcagcgtgagcagcttcgagcgcttcgagatc DYEELREQLSSVSSFERFEI ttccccaagaccagcagctggccaaaccacgacagcaacaagggagtgaccgctgcttgc FPKTSSWPNHDSNKGVTAAC ccacacgctggagccaagagcttctacaagaacctgatctggctggtgaagaagggcaac PHAGAKSFYKNLIWLVKKGN agctaccccaagctgagcaagagctacatcaacgacaagggcaaggaggtgctggtgctg SYPKLSKSYINDKGKEVLVL tggggcatccaccaccccagcaccagcgccgaccagcagagcctgtaccagaacgccgac WGIHHPSTSADQQSLYQNAD acctacgtgttcgtgggcagcagccgctacagcaagaagttcaagcccgagatcgccatc TYVFVGSSRYSKKFKPEIAI cgccccaaggtgcgcgaccaggagggccgcatgaactactactggaccctggtggagccc RPKVRDQEGRMNYYWTLVEP ggcgacaagatcacctttgaggctaccggaaacctggtggtgccacgctacgcttttgct GDKITFEATGNLVVPRYAFA atggagaggaatgctggcagcggcatcatcatcagcgacacccccgtgcacgactgcaac MERNAGSGIIISDTPVHDCN accacctgccagacccccaagggcgccatcaacaccagcctgcccttccagaacatccac TTCQTPKGAINTSLPFQNIH cccatcaccatcggcaagtgccccaagtacgtgaagagcaccaagctgcgcctggccacc PITIGKCPKYVKSTKLRLAT ggactgaggaacatcccaagcatccagagccgcggcctgtttggagctattgctggattc GLRNIPSIQSRGLFGAIAGF attgagggcggctggaccggaatggtggatggatggtacggctaccaccaccagaacgag IEGGWTGMVDGWYGYHHQNE cagggcagcggctacgccgccgacctgaagagcacccagaacgccatcgacgagatcacc QGSGYAADLKSTQNAIDEIT aacaaggtgaacagcgtgatcgagaagatgaacacccagttcaccgccgtgggcaaggag NKVNSVIEKMNTQFTAVGKE ttcaaccacctggagaagcgcatcgagaacctgaacaagaaggtggacgacggcttcctg FNHLEKRIENLNKKVDDGFL gacatctggacctacaacgccgagctgctggtgctgctggagaacgagcgcaccctggac DIWTYNAELLVLLENERTLD taccacgacagcaacgtgaagaacctgtacgagaaggtgcgcagccagctgaagaacaac YHDSNVKNLYEKVRSQLKNN gccaaggagatcggcaacggctgcttcgagttctaccacaagtgcgacaacacctgcatg AKEIGNGCFEFYHKCDNTCM gagagcgtgaagaacggcacctacgactaccccaagtacagcgaggaggccaagctgaac ESVKNGTYDYPKYSEEAKLN Cgcgaggagatcgacggcgtgaagctcgagttaattaagcgcatgaagcagatcgaggac REEIDGVKLELIKRMKQIED aagatcgaagagatcgagtccaagcagaagaagatcgagaacgagatcgcccgcatcaag KIEEIESKQKKIENEIARIK Aagattaagctggtgccgcgcggcagcctcgagtggagccacccgcagttcgagaagtga KIKLVPRGSLEWSHPQFEK-

Example 6

RRP Infection of Mammalian and Insect Cells and Production of RRPs

(108) To determine if other mammalian and insect cells can be infected with RRPs, Human Embryonic Kidney 293 cells (HEK293T), Drosophila S2 cells and Aedes albopictus C6/36 cells were infected with RRPs at an m.o.i. of 1. Of note, the m.o.i. was calculated using the titer determined on BHK cells. This experiment demonstrated that both mammalian and insect cells can be readily infected with RRPs, although reporter gene expression in insect cells is considerably lower (FIG. 11A). Expression of eGFP in mammalian cells and insect cells was optimal at 42 or 72 hours post infection (hpi), respectively (FIG. 11).

(109) Next, we were interested in determining if HEK293T cells can be used for the production of RRPs. To this end, BHK cells and HEK293T cells were infected with RRPs at an m.o.i. of 3. After three days, the cells were seeded in a 6-well plate and transfected with pCAGGS-NSmGnGc. The supernatant was collected after three days and the RRP titers obtained were 10E7 (10.sup.7) or BHK cells and 10E6.5 (10.sup.6.5) TCID.sub.50/ml for HEK293T cells. This result demonstrates that wildtype BHK cells and HEK293T cells can be used for the production of RRPs by combining an RRP infection with a transfection of the pCAGGS-NSmGnGc plasmid

Example 7

Use of RRPs in a Virus Neutralization Test (VNT)

(110) Classical VNT and a novel VNT that uses RRPs instead of live virus (RaPid VNT) were performed with sera from lambs that were previously experimentally infected with the 35/74 virus. To confirm the presence of RVFV-specific antibodies, the sera were analyzed by the recN RVFV ELISA (BDSL, Ayrshire Scotland, UK) prior to analysis by VNT. The classical VNT was performed as described previously (de Boer et al., 2010. Vaccine 28: 2330-2339). For the RaPid VNT, serum dilutions were prepared in 96-well plates in 50 l GMEM supplemented with 5% FBS, 4% TPB, 1% MEM NEAA, 1% pen/strep. Growth medium containing 200 RRPs in a 50 l volume was added to the serum dilutions and incubated for 1.5 h at room temperature. Next, 50 l of growth medium containing 40 000 BHK cells was added to each well. Plates were incubated at 37 C. and 5% CO2. After 36-48 hrs the neutralization titer was calculated using the Spearman-Krber method (Karber (1931). Arch Exp Path Pharmak 162:480-483; Spearman (1908). Br J Psychol 2: 227-242).

(111) TABLE-US-00006 TABLE 1 Comparison of the classical VNT assay with the RaPid VNT. Sera from experimentally infected lambs were analysed by the recN ELISA (ELISA), classical VNT, and RaPid VNT. Neutralization titres are determined as .sup.10log 50% end-point titres. Lamb no: Classical VNT RaPid VNT ELISA 4308 3.56 3.94 POS 4309 4.09 4.16 POS 4310 0 0 NEG 4311 4.01 4.24 POS 4312 3.71 4.76 POS 4314 3.56 4.46 POS 4315 3.71 4.39 POS 4318 4.16 4.39 POS 4321 0 0 NEG 4324 4.24 4.31 POS 4328 4.01 4.69 POS

(112) This experiment revealed that the use of RRPs in the so-called RaPid VNT has an optimal readout between 36 and 48 hrs and is of equal, if not higher sensitivity than the classical VNT (Table 1).

Example 8

Vaccination and Challenge of Mice

(113) Female BALB/c mice (Charles River laboratories, Maastricht, The Netherlands) were housed in groups of five animals in type-III filter-top cages and kept under biosafety level-3 containment. Groups of 10 mice were vaccinated via the intramuscular or subcutaneous route either once on day 21 or two times on days 0 and 21 with 10E6 TCID50 of RRPs in 50 l PBS. One group of nine mice was left untreated (non-vaccinated). The body weights of the mice were monitored weekly. On day 42, all mice were challenged via the intraperitoneal route with 10E2.7 TCID50 of RVFV strain 35/74 in 0.5 ml culture medium. Challenged mice were monitored daily for visual signs of illness and mortality. This experiment was approved by the Ethics Committee for Animal Experiments of the Central Veterinary Institute of Wageningen University and Research Centre.

(114) To study the vaccine efficacy of RRPs, groups of 10 mice were immunized with 50 l of an inoculum containing 10E6 TCID50 RRPs, via either the subcutaneous or intramuscular route, either once or twice, with a three week interval. One group of 9 non-vaccinated mice was added as a control group. The mice were challenged on day 42 with a known lethal dose of RVFV strain 35/74. All non-vaccinated mice displayed overt clinical signs and weight loss and eight of a total of nine non-vaccinated mice succumbed to the infection within four days after challenge. One mouse survived for twelve days, but eventually died. The percentage of survival in the groups of mice vaccinated either once or twice via the subcutaneous route was 60% (FIG. 12). In contrast, 100% of the mice vaccinated via the intramuscular route, either once or twice, survived the challenge (FIG. 12). These mice did not show any clinical signs or weight loss throughout the experiment. This demonstrates that a single intramuscular vaccination with 10E6 RRPs can protect mice from a lethal dose of RVFV.

Example 9

Production of Bunyavirus Replicon Particles of Severe Fever with Thrombocytopenia Syndrome (SFTS) Virus Isolate HB29

(115) SFTS virus was first described by Yu et al. (Yu X J, et al. Fever with Thrombocytopenia Associated with a Novel Bunyavirus in China. N Engl J Med 2011 Mar. 16).

(116) cDNAs encoding full-length viral RNA corresponding to the L (GenBank: HM745930.1), M (GenBank: HM745931.1) and S (GenBank: HM745932.1) genome segments of SFTS virus isolate HB29 are synthesized and flanked by a T7 polymerase promoter and cDNA encoding a HDV ribozyme sequence. A T7 transcription termination sequence is preferably positioned downstream of the ribozyme cDNA. These sequences are cloned into pUC57 vectors essentially as exemplified in Example 1, resulting in pUC57-SFTS-L, pUC57-SFTS-M and pUC57-SFTS-S.

(117) To facilitate cloning into pUC57 of the cDNA encoding the SFTS L genome segment, a KpnI restriction site is introduced immediately upstream of the T7 promoter sequence and immediately downstream of the T7 transcription termination sequence.

(118) To facilitate cloning of the cDNA encoding the SFTS M genome segment, a KpnI restriction site is introduced immediately upstream of the T7 promoter and immediately downstream of the T7 transcription termination sequence.

(119) To facilitate cloning of the cDNA encoding the SFTS S genome segment, a KpnI restriction site is introduced immediately upstream of the T7 promoter sequence and immediately downstream of the T7 transcription termination sequence.

(120) An additional construct encoding an SFTS S genome-like segment in which the NSs gene (nucleotides 835-1716 of GenBank: HM745932.1) is replaced by a gene encoding a suitable reporter protein (i.e. eGFP or luciferase) is also developed. A plasmid encoding an S-like genome segment in which the NSs gene is replaced by the eGFP gene is named pUC57-SFTS-S-eGFP. Similar as was shown for RVFV, introduction of the SFTS-L and SFTS-S-eGFP genome segments in suitable cells (e.g. BHK-21 cells) results in viable cells maintaining these genome segments.

(121) The complete open reading frame of the M genome segment (M-ORF, nucleotides 19-3240 of GenBank sequence HM745931.1) is introduced downstream of the CMV promoter of pCIneo. Stable cell lines are produced by transfecting pCIneo-SFTS-M-ORF into BHK-21 cells and cells with integrated plasmids are selected by culturing in the presence of G-418.

(122) As an alternative, BHK-21 cells are provided with the SFTS-M-ORF encoded proteins by infecting the eukaryotic cell with a recombinant viral vector that transduces the SFTS M-ORF-encoded polyprotein and a cell line is selected in which the recombinant viral vector is persistently present without causing overt cytopathogenic effect.

(123) The cells expressing the SFTS M-ORF-encoded proteins are infected with FP-T7 and, after recovery, transfected with plasmids pUC57-SFTS-L and either pUC57-SFTS-S or pUC57-SFTS-S-eGFP. When required for efficient production, a construct encoding the SFTS structural glycoproteins under the control of a suitable Polymerase-II promoter (such as the CAG promoter in the pCAGGS plasmid) is also transfected or infected to provide the SFTS structural glycoproteins. This procedure results in the production of SFTS replicon particles of isolate HB29 that do not contain an SFTS M genome segment.

(124) TABLE-US-00007 The T7 promoter that is used has the nucleotide sequence: 5-TAATACGACTCACTATAG-3 The HDV ribozyme sequence that is used has the nucleotide sequence 5-GGGTCGGCATGGCATCTCC-3. The T7 terminator sequence that is used has the nucleotide sequence: 5-TAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTT TG-3

(125) A similar method can be used to produce replicon particles of other SFTS isolates.

Example 10

Rapid Immunity Against RVFV in Sheep after a Single Vaccination with the NSR Vaccine

(126) Materials and Methods

(127) Preparation of the Challenge Virus

(128) The RVFV virus that was used for challenge was rescued from cDNA (see Example 1 and Kortekaas et al. 2011. J Virol. Accepted for publication). The recombinant 35/74 (rec35/74) virus was titrated on BHK-21 cells and the titer was determined as tissue culture infective dose 50 (TCID.sub.50) using the Spearman-Krber algorithm (Example 1). The complete sequence of the L, M and S genome sequences of the 35/74 isolate can be found on GenBank under the accession numbers JF784386, JF784387 and JF784388, respectively.

2.2 Preparation of the Vaccines

(129) The ectodomain of the Gn protein (GneS3) was produced from the insect expression vector pMT/BiP/V5-HisA (Invitrogen, Carlsbad, Calif., USA). The sequence encoding the authentic Gn signal peptide was replaced by a sequence coding for the BiP signal peptide, specifying the junction sequence GLSLG-RSLRSLAEDPH, in which GLSLG denotes the BiP, RSLRSL denotes a linker sequence, and AEDPH the start of the Gn ectodomain. In the pMT-GneS3 plasmid, the Gn ectodomain sequence was fused to a sequence encoding a combined FLAG-tag/enterokinase (EK) cleavage site for easy detection and purification of the monomeric protein (DYKDDDDK) and three Strep tags (WSHPQFEK) separated by glycine linkers (GGGSGGGSGGGS), resulting in the following sequence: ( . . . YQCHTDPTGDYKDDDDKAGPGWSHPQFEK GGGSGGGSGGGSWSHPQFEKGGGSGGGSGGGSWSHPQFEK in which the sequences resulting from introduced restriction sites are indicated in bold. The enterokinase cleavage site was introduced to allow removal of the Strep-tag after purification.

(130) The secreted Gn ectodomain was purified from the cell culture supernatant by virtue of its C-terminal 3 Strep-tag using Strep-Tactin Sepharose according to the manufacturer's recommendations (IBA, Gttingen, Germany). The GneS3 protein was eluted from the washed beads with 4 mM D-Desthiobiotin (IBA) and concentrated using an Amicon Ultra-4 concentrator with a molecular mass cut-off of 30 kDa (Millipore, Billerica, Mass., USA). The protein, named GneS3, was formulated in Stimune water-in-oil adjuvant (Prionics, Lelystad, The Netherlands) to a final concentration of 20 g/ml.

(131) Production of the NDFL-GnGc was previously reported (Kortekaas et al. 2010. Vaccine 28: 4394-4401). OBP vaccine is a commercially available inactivated RVFV vaccine (Onderstepoort Biological Products [OBP], Onderstepoort, South Africa). This vaccine was purchased from OBP and administered according to the instructions of the manufacturer. The administered doses and routes of vaccination of the four indicated vaccines are depicted in Table 2.

(132) TABLE-US-00008 TABLE 2 Route and dose of vaccines Vaccine Route Dose Adjuvant OBP vaccine Subcutaneous According to Aluminium hydroxide protocol of gel manufacturer NDFL-GnGc Intramuscular 2.10.sup.7 TCID.sub.50 None NSR Intramuscular 10.sup.7 TCID.sub.50 None GneS3 Subcutaneous 20 g Stimune water-in-oil

2.3 Vaccination and Challenge

(133) Thirty conventional European breed lambs were purchased from a commercial sheep farm in The Netherlands and divided over five groups. Lambs were vaccinated once at the age of six weeks (day 0), as depicted in Table 2. On day 19 (days post challenge [DPC] 0), all lambs were challenged via the intraperitoneal route with 10.sup.5 TCID.sub.50 of RVFV rec35/74. EDTA blood samples were collected daily starting from day 19 (DPC 0) until day 26 (DPC 7) and again on days 28, 30, 33, 35, 37 and 40 (DPC 9, 11, 14, 15, 17 and 20). Serum samples were collected on days 7, 0, 7, 14, and daily from day 19 (day of challenge) to 26 (DPC 7) and finally on days 33 (DPC 14) and 40 (DPC 20).

(134) Body weights were determined weekly, on DPI 7, 1, 6, 13, 18, 25, 32 and 39. Rectal body temperatures were determined daily starting on day 17 (two days prior to challenge) until the end of the experiment.

2.4 Quantitative Real-Time PCR and ELISA

(135) Viral RNA was isolated from plasma samples using the QuickGene DNA tissue kit S (DT-S, Fuji Photo Film Europe GmbH, Dusseldorf, Germany) with the following modifications. Proteinase K solution (EDT, DT-S kit, 30 l) and 3 l polyadenylic acid A (polyA 5 g/l, Sigma, St. Louis, Mo., USA) were added to 250 l lysis buffer (LDT, DT-S kit). Of this mixture, 250 l was added to 300 l plasma. The mixture was heated at 72 C. for 10 min in a heating block and stored at 20 C. until further analysis. RNA isolation was subsequently performed using the QG-Mini80 Workflow (Fuji Film). The lysate was mixed with 350 l 99% ethanol before loading on the column. After three wash steps with 750 l wash buffer (WDT, DT-S kit) the RNA was eluted with 50 l elution buffer (CDT, DT-S kit). The material was stored at 70 C. until further analysis.

(136) RNA samples (5 l) were used for quantitative Taqman reverse-transcriptase real-time PCR (qRRT-PCR). The LightCycler RNA Amplification Kit HybProbe (Roche, Almere, The Netherlands) was used and primers, probes and cycling conditions were used as previously described (Drosten et al. 2002 J Clin Microbiol 40: 2323-2330).

2.5 Clinical Chemistry

(137) Clinical chemistry was performed with serum collected on the day of challenge (study day 19, DPC 0) and subsequently on days 20-25 (DPC 1-6), and on days 32 (DPC 14) and 38 (DPC 21). Enzyme analysis was performed using the Spotchem EZ SP-4430 analyser (Menarini Diagnostics, Valkenswaard, The Netherlands) using strips capable of detecting alkaline phosphatase (ALP), alanine transaminase (ALT), creatinine, glucose, total protein and urea.

(138) On day 0, blood samples (n=30) were collected and used to define the upper and lower limits of blood parameters. Limits were set at the average upper and lower averages plus two times their standard deviations.

2.6 Statistical Analysis

(139) Statistical analyses were performed with the one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison test using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, Calif., USA). Statistical differences with p-values <0.05 were considered significant.

(140) Results

(141) After acclimatization for one week, all lambs were vaccinated as depicted in Table 2. On different time points after vaccination, the injection sites were inspected for possible adverse reactions. These inspections revealed mild to moderate swelling in four and five out of six lambs vaccinated with the OBP and GneS3 vaccine, respectively. In the other groups, no injection site reactions were observed (FIG. 13). No adverse reactions at the injection site were observed in lambs vaccinated with the NDV-GnGc vaccine or the NSR vaccine.

(142) After challenge the rectal temperatures in the control group peaked at 2 DPC (FIG. 14). Peak rectal temperatures and the total days of fever (rectal body temperature 40.1 C.) were both significantly lower (p<0.0001) in all vaccinated groups, compared to the control group.

(143) The occurrence of viremia was determined by qRRT-PCR on RNA isolated from plasma samples. High levels of viral RNA were detected in all but one of the unvaccinated control animals, peaking on DPC 3 (FIG. 15). Viral RNA levels were significantly lower in all vaccinated groups (p<0.0001).

(144) The body weights of the lambs were determined weekly. The lambs in the unvaccinated control group all displayed weight loss in the first two weeks after challenge (between days 18-25). Some weight loss, at a later time point (between days 25-32), was noted in five lambs vaccinated with NDV-GnGc and two lambs vaccinated with the NSR vaccine (FIG. 16). Differences between body weights were not statistically significant.

(145) To investigate the occurrence of liver and renal damage, biochemical analyses were performed on serum samples using the Spotchem EZ dry chemistry analyzer. Hepatic dysfunction was assessed by sequential measurements of serum alkaline phosphatase (ALP), alanine transaminase (ALT) and total protein concentrations (TP) (FIG. 17). Total protein concentration is assumed to represent mostly albumin levels. In unvaccinated lambs, ALP and ALT levels were clearly increased when compared to vaccinated lambs. Statistical significance (p<0.05) was however only achieved when comparing ALP levels in plasma obtained from unvaccinated lambs (Mock) and NDV-GnGc-vaccinated lambs.

(146) The concentrations of blood urea nitrogen (BUN) and creatinine were measured to assess renal function. BUN levels in serum obtained from unvaccinated lambs were clearly on average higher than levels detected in the serum of vaccinated lambs (FIG. 17).

(147) Creatinine levels in serum from unvaccinated lambs were significantly higher when compared to levels detected in serum from vaccinated lambs (OBP vaccine, p<0.0005; GneS3, p<0.005; NSR, p<0.005; NDV-GnGc, p<0.05).

(148) One lamb in the Mock-vaccinated control group succumbed to the infection. In this lamb, creatinine levels peaked on DPC 8 to a level of 582 mol/l and BUN levels peaked to 30.6 mmol/l. This strongly suggests that this lamb died from severe kidney failure. Liver failure was revealed by a peak in ALT concentration of 20 U/l.

(149) Conclusions

(150) We here demonstrate that a single vaccination of sheep with the non-spreading NSR vaccine prevents mortality and morbidity and significantly reduces viremia, fever and clinical signs resulting from a RVFV challenge. The efficacy of the NSR vaccine did not significantly differ from the other vaccines evaluated in our study. The high immunogenicity of the NSR vaccine explains that the use of adjuvants is not required. The inability of the NSR vaccine to spread from the vaccinated animal provides optimal safety.

Example 11

RVFV Replicon Particles that Produce Recombinant Soluble Multimeric NA of Pandemic Swine-Origin 2009 A (H1N1) Influenza Virus

(151) Seq S-CD5-OS-GCN4-NA (see below) encodes a human codon-optimized soluble neuraminidase ectodomain (NA amino acids 75 to 469) of influenza virus A/California/04/2009 (H1N1). This sequence was synthesized at the GenScript Corporation. The NA gene is preceded by a sequence encoding an N-terminal CD5 signal peptide which is followed by sequences encoding a N-terminal OneSTrEP (OS) comprising a purification motif and a tetramerization motif (GCN4-pLI; Bosch et al. 2010. J. Virol. 84: 10366-10374).

(152) To generate a vector comprising CD5-OS-GCN4-NA, pUC57-S-MCS was digested with NheI and XbaI and NheI/XbaI digested OS-GCN4-NA sequence was cloned into this plasmid, yielding pUC57-S-GCN4-NA. The CD5 sequence was subsequently introduced by annealing of the following oligo's:

(153) TABLE-US-00009 Oligo CD5-3: 5-CATGCCCATGGGGTCTCTGCAACCGCTGGCCACCTTGTACCTGCTGG GGATGCTGGTCGCTTCCGTG-3 Oligo CD5-4: 5-CTAGCACGGAAGCGACCAGCATCCCCAGCAGGTACAAGGTGGCCAGC GGTTGCAGAGACCCCATGGG-3

(154) The CD5 linker is ligated into NcoI/NheI-digested pUC57-S-GCN4-NA, yielding pUC57-S-CD5-OS-GCN4-NA.

(155) BHK-GnGc cells were infected with FP-T7. After recovery, the cells were transfected with pUC57-L, pUC57-S-CD5-OS-GCN4-NA and pCAGGS-NSmGnGc, essentially as exemplified in Example 1. After cell passage, the transfection was repeated. This sequence of events was repeated until all the cells of the monolayer express the CD5-OS-GCN4-NA protein, as determined by IPMA. The quantity of GCN4-NA in the culture medium of these cells was determined. To establish high levels of NSmGnGc proteins, plasmid pCAGGS-NSmGnGc was again introduced in the cell line, yielding replicon particles containing the S-CD5-OS-GCN4-NA gene. The replicon particles are used for vaccination/challenge experiments in mice to establish the protective efficacy against a lethal H1N1 challenge.

(156) Seq. S-CD5-OS-GCN4-NA

(157) Nucleotides 1-69 (underlined) encode the CD5 sequence

(158) Nucleotides 73-156 encodes the OneSTrEP (OS) peptide

(159) Nucleotides 163-261 encodes the GCN4 domain

(160) Nucleotides 262-1449 encodes the NA ectodomain

(161) TABLE-US-00010 atgcccatggggtctctgcaaccgctggccaccttgtacctgctggggatgctggtcgct MPMGSLQPLATLYLLGMLVA tccgtgctagcgtggagccacccgcagttcgagaaaggtggaggttccggaggtggatcg SVLAWSHPQFEEKGGGSGGGS ggaggtggatcgtggagccacccgcagttcgaaaaaagatctatgaaacaaatcgaagac GGGSWSHPQFEKRSMKQIED aagctggaagaaatcctttcgaaactgtaccacatcgaaaacgagctggccaggatcaag KLEEILSKLYHIENELARIK aaactgctgggcgaaggatccgctgctggacagtccgtcgtgagcgtgaagctggccgga KLLGEGSAAGQSVVSVKLAG aacagcagcctgtgcccagtgagcggatgggccatctacagcaaggacaacagcgtgcgc NSSLCPVSGWAIYSKDNSVR atcggcagcaagggcgacgtgttcgtgatccgcgagcccttcatcagctgcagccccctg IGSKGDVFVIREPFISCSPL gagtgccgcaccttcttcctgacccagggcgccctgctgaacgacaagcacagcaacggc ECRTFFLTQGALLNDKHSNG accattaaggaccgcagcccatacaggaccctgatgagctgccccattggagaggtgcca TIKDRSPYRTLMSCPIGEVP agcccatacaacagcaggtttgagagcgtggcttggtccgccagcgcttgccacgatgga SPYNSRFESVAWSASACHDG atcaactggctgaccattggaatcagcggaccagacaacggcgccgtggccgtgctgaag INWLTIGISGPDNGAVAVLK tacaacggcatcatcaccgacaccatcaagagctggcgcaacaacatcctgcgcacccag YNGIITDTIKSWRNNILRTQ gagagcgagtgcgcctgcgtgaacggcagctgcttcaccgtgatgaccgacggccccagc ESECACVNGSCFTVMTDGPS aacggccaggccagctacaagattttccgcatcgagaagggcaagatcgtgaagagcgtg NGQASYKIFRIEKGKIVKSV gagatgaacgcccccaactaccactacgaggagtgcagctgctaccccgacagcagcgag EMNAPNYHYEECSCYPDSSE atcacctgcgtgtgccgcgacaactggcacggcagcaaccgcccctgggtcagcttcaac ITCVCRDNWHGSNRPWVSFN Cagaacctggagtaccagatcggctacatctgctccggaatctttggagacaatcccagg QNLEYQIGYICSGIFGDNPR ccaaatgacaagaccggcagctgcggaccagtgagcagcaatggagctaacggcgtgaag PNDKTGSCGPVSSNGANGVK ggcttcagcttcaagtacggcaacggcgtgtggatcggccgcaccaagagcatcagcagc GFSFKYGNGVWIGRTKSISS cgcaacggcttcgagatgatctgggaccccaacggctggaccggcaccgacaacaacttc RNGFEMIWDPNGWTGTDNNF agcatcaagcaggacatcgtgggcatcaacgagtggagcggatacagcggcagctttgtg SIKQDIVGINEWSGYSGSFV cagcacccagagctgaccggactggactgcatcaggccctgcttctgggtggagctgatc QHPELTGLDCIRPCFWVELI aggggaagacccaaggagaacaccatctggaccagcggcagcagcattagcttttgcgga RGRPKENTIWTSGSSISFCG gtgaacagcgacaccgtgggatggagctggccagatggagctgagctgcccttcaccatc VNSDTVGWSWPDGAELPFTI gacaagtga DK-

Example 12

Expression of Foreign Proteins from the NSR Genome

(162) Replicon cell lines expressing trimeric soluble hemagglutinin protein (sHA.sub.3) and tetrameric soluble neuraminidase protein (sNA.sub.4) from influenza virus H1N1 were produced essentially as described in Example 5 with the modifications schematically depicted in FIG. 18. A replicon cell line expressing the eGFP protein was also produced alongside in these experiments.

(163) Flow cytometry analysis using an antibody specific for the N protein was used to determine the percentage of cells containing both the L and S (S-eGFP, S-.sub.sHA.sub.3 or S-.sub.sNA.sub.4) genome segments. N protein expression is dependent on the presence of both L and S genome segments. Flow cytometry demonstrated that 96% of the cells in which the L and S-eGFP genome segments were introduced were positive for N protein expression at passage 8 (FIG. 19A, results obtained from analysis of control BHK-GnGc cells are depicted in FIGS. 19D, E and F). Flow cytometry analysis of cells containing L and the S-CD5-HA-GCN4-ST (i.e. S-.sub.sHA.sub.3) segment demonstrated that 98% of the cells were positive for N expression (FIG. 19B). Flow cytometry analysis of cells containing L and the S-CD5-OS-GCN4-NA segment (i.e. S-.sub.ssNA.sub.4) demonstrated that 98.5% of the cells were positive for N expression (FIG. 19C).

(164) Production of the sHA.sub.3 and sNA.sub.4 Proteins by the Replicon Cell Lines

(165) Cell culture medium was collected and pre-cleared by slow-speed centrifugation. Proteins were purified from the culture medium using Strep-Tactin Sepharose gravity-flow columns according to the instructions from the manufacturers (IBA, Gttingen, Germany). The eluted fractions were analyzed by Silver-stained polyacrylamide gels (FIG. 20A) and Western blot using Strep-Tactin conjugated to horseradish peroxidase (Strep-Tactin-HRP, IBA, FIG. 20B).

(166) The yield of both proteins was estimated at 1 mg/l of culture medium (BCA assay, Pierce, Thermo Scientific, Landsmeer, The Netherlands). The production yields of the sHA.sub.3 and sNA.sub.4 proteins were again determined after cell passage 20 and found unchanged.

(167) Transfection of the replicon cell lines with the pCAGGS-NSmGnGc plasmid resulted in NSR titers of 10.sup.7.3 TCID.sub.50/ml of NSR-sHA.sub.3 and 10.sup.6 TCID.sub.50/ml of NSR-.sub.sNA.sub.4. These particles will be used as vaccines for the control of influenza.