1 Laboratory of Virology, Wageningen University, Binnenhaven 11,6709 PD Wageningen, The Netherlands and 2 International Livestock Research Institute, PO Box 30709, Nairobi, Kenya 3 Present address: The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850, USA
4 To whom correspondence should be addressed.E-mail: monique.vanoers{at}wur.nl
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Abstract |
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Keywords: baculovirus surface display/East Coast fever/protein folding/sporozoite surface antigen/Theileria parva
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Introduction |
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Immune responses against the infecting sporozoite and pathogenic schizont stages play a major role in mediating protection and immunity to ECF (McKeever et al., 1999). Cattle that recover from infection mount immunity to homologous strains. This immunity is thought to be dependent on a cellular immune response mediated by class I MHC-restricted CD8+ cytotoxic T lymphocytes, and directed against schizont-infected lymphoblasts (Morrison et al., 1995
; McKeever et al., 1999
). On a single exposure, animals do not mount a detectable anti-sporozoite response, but on repeated challenge with infected ticks cattle generate high levels of antibodies that neutralize sporozoites in vitro (Musoke et al., 1984
). These antibodies recognize p67, the major surface protein of sporozoites. A recombinant form of this antigen produced in bacteria has shown to provide protection in cattle (Musoke et al., 1992
). Because p67 is invariant in parasites isolated from cattle and exhibits 7696% sequence identity with stocks from buffalo, the antigen has a clear potential for development of a subunit vaccine against ECF. Pepscan analysis revealed that both murine and bovine epitopes cluster in the N- and C-terminal regions. Linear bovine B cell epitopes mapped within residues 25296, and 577591, while residues 105221 contained three epitopes defined by neutralizing monoclonal antibodies. A further two epitopes were located in the C-terminal fragment between residues 617631 (Nene et al., 1999
).
Expression of p67 in E.coli as a fusion protein to influenza NS1, resulted in an insoluble protein (Musoke et al., 1992). In insect cells infected with a conventional recombinant baculovirus, p67 was expressed at low levels and mainly in a non-native conformation, and was not transported to the cell surface (Nene et al., 1995
). The protection achieved by these two recombinant proteins was only partial and relatively high doses of antigen were needed for induction of protective immunity. We believe that incorporating conformational epitopes would enhance the efficacy of the vaccine.
In this paper, we have expressed domains of p67 on the surface of budded baculovirus particles (BVs) in an attempt to achieve a more native folding of immunodominant epitopes. The baculovirus surface display method (Boublik et al., 1995; Grabherr et al., 2001
) is based on the expression of foreign proteins fused to the baculovirus GP64 protein. This major BV envelope protein is responsible for fusion of the viral envelope with endosomes of the insect host and for virus budding from infected insect cells (Monsma et al., 1996
; Oomens and Blissard, 1999
). Baculovirus surface display has been used previously for the expression of viral surface antigens, such as HIV GP41 and GP120, rubella virus spike proteins and FMDV structural proteins (Boublik et al., 1995
; Grabherr et al., 1997
; Mottershead et al., 1997
; Tami et al., 2000
). Indications for a near-native folding of recombinant proteins produced in this system have been given by the display of functional scFv fragments (Mottershead et al., 2000
).
To allow easy generation and screening of recombinant viruses, we combined the existing surface display system with bacmid technology (Luckow et al., 1993), enabling the generation of recombinant viral genomes in bacterial cells. This is the first report of the expression of a parasite antigen on the surface of baculovirus particles and may open new avenues to develop vaccines against parasitic diseases.
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Materials and methods |
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The coding sequence for enhanced GFP (Cormack et al., 1996) was cloned as a NcoINsiI fragment downstream of the p10 promoter in the pFastBacDual vector (Invitrogen Breda, The Netherlands). The KpnI restriction site in the multiple cloning site (MCS) downstream of the p10 promoter was removed by digesting with NsiI and KpnI, and filling in with Klenow. After self-ligation, this resulted in the vector pFBD-GFP-
KpnI. In this way, we were able to use a unique KpnI cloning site between the gp64 signal peptide and major domain (see Figure 1
). A p67 N-terminal domain corresponding to amino acids 21225 was obtained by PCR from plasmid pMG1-p67 (Musoke et al., 1992
) with primers surf-3 and surf-4, generating KpnI sites (Table I
, Figure 1A
). The C-terminal domain of p67, encoding amino acids 572651, was amplified with primers surf-1 and surf-2 to introduce KpnI sites on both ends (Table I
, Figure 1A
). The PCR products were verified by sequencing. The C-terminal domain was cloned into the KpnI site of pBACSurf-1 (Novagen, Darmstadt, Germany) to give plasmid pBACSurf-p67C. With this plasmid as template, the p67 C-domain flanked by the gp64 leader sequence (86 bp) and the gp64 major domain (1511 bp) was amplified using primers surf-5 and surf-6 (Table I
) and the Expand Long Template PCR system (Roche Diagnostics, Almere, The Netherlands). The PCR product was cloned between the EcoRI and HindIII restriction sites in the MCS of pFBD-GFP-
KpnI, resulting in pFBD-GFP-gp64/p67C (Figure 1B
). The sequence was verified. Plasmid pFBD-GFP-gp64/p67N was generated by removing the p67 C-domain by KpnI digestion and replacing it with the N-domain. Self-ligation after KpnI digestion resulted in an empty vector, pFBD-GFP-gp64, which was used as a control in the experiments described below.
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Escherichia coli DH10BAC cells (Invitrogen) were transformed with the plasmids pFBD-GFP-gp64/p67N, pFBD-GFP-gp64/p67C or pFBD-GFP-gp64 to generate recombinant AcMNPV bacmids. Putative recombinant bacmids were analyzed by PCR using the M13 reverse and surf-5 primers. Isolated bacmid DNA was used to transfect Spodoptera frugiperda Sf21 cells (Vaughn et al., 1977) using Cellfectin (Invitrogen). This resulted in the recombinant viruses Ac-gp64/p67N, Ac-gp64/p67C and Ac-gp64. Recombinant viruses were grown to high titer stocks using standard procedures (King and Possee, 1992
).
Immunofluorescence studies with non-fixed cells
Sf21 cells were grown in Graces supplemented medium containing 10% FBS (Invitrogen). Sf21 cells were infected with Ac-gp64, Ac-gp64/p67N and Ac-gp64/p67C at a multiplicity of infection (m.o.i.) of 10 tissue culture infection dose 50 (TCID50) units/cell. The cells were harvested and collected in 2 ml of Graces supplemented medium with 10% FBS at 48 h post-infection (p.i.). Infected non-fixed cells were incubated in this medium with monoclonal antibodies ARIII 22.7, recognizing the N-domain of p67, or with ARIII 21.4, specific for the C-domain (Nene et al., 1999) at a dilution of 1:200 for 1 h at room temperature. Cells were washed three times with Graces supplemented medium containing 10% FBS. Cells were further incubated with goat anti-mouse IgG conjugated to rhodamine Red X (Molecular Probes Europe, Leiden, The Netherlands) for 1 h at a 1:200 dilution. Similar studies were also performed with monoclonal TpM12 at a 1:50 dilution. After extensive washing, the insect cells were viewed in a Zeiss LSM510 confocal laser scanning microscope. GFP fluorescence was observed through excitation with blue laser light at 488 nm and emission through a 505530 nm bandpass filter. Rhodamine was simultaneously visualized using green laser light at 545 nm for excitation and a 560 nm longpass filter for emission.
Analysis of budded virions
Sf21 cells were infected with the various recombinant viruses at a m.o.i. of 10 TCID50 units/cell and harvested 48 h p.i. Cell debris was removed by centrifugation at 3000 r.p.m. in a Labofuge with swing-out rotor for 5 min and filtration over a 0.45 µm non-pyrogenic filter. The filtrate was overlaid onto a 2.5 ml 25% sucrose cushion in 1 mM Tris, 0.01 mM EDTA pH 8.0 (0.1 TE). Budded viruses (BVs) were pelleted by centrifugation at 25 000 r.p.m. for 90 min at 4 °C in a SW41 rotor. The pellet was suspended in 0.1 TE. Purified BVs were used for SDSPAGE and dot blot analysis. Western blots were incubated with either monoclonal AcV5 recognizing GP64 (Hohnmann and Faulkner, 1983) diluted 1:1000, or monoclonals ARIII 22.7 or ARIII 21.4, diluted 1:200 and recognizing p67 N- (amino acids 201215) and C-specific (609623) peptides, respectively (Nene et al., 1999). Rabbit anti-mouse immunoglobulins conjugated to horseradish peroxidase (Dako A/S, Glostrup, Denmark) were used as the second antibody.
For immunodot blot analysis, a sample of the budded virus preparation of Ac-gp64/p67N or Ac-gp64 equivalent to 5 µg of total protein was spotted onto a nitrocellulose membrane, either directly or after denaturation by boiling for 10 min in 10 mM TrisHCl pH 8.0, 1 mM EDTA, 2% SDS and 5% ß-mercaptoethanol. The filters obtained were incubated as described above using either monoclonal TpM12 or ARIII 22.7 (Nene et al., 1999). These blots were developed using ECL (Amersham Pharmacia Biotech, Roosendahl, The Netherlands). As a control, Sf21 cells were infected with an AcMNPV recombinant expressing non-fused p67 and 5 µg total cell protein was blotted under both denaturing and native conditions (data not shown).
For immunogold labelling, Sf21 cells were infected with the various recombinants in Graces medium without any supplements and the culture supernatant was replaced at 20 h p.i. The supernatant was collected at 36 h p.i. and cleared from cell debris by centrifugation at 1000 r.p.m. in a Labofuge with swing-out rotor. The virus suspension was attached to nickel grids. After blocking in 1% (w/v) BSA in PBS for 20 min, grids were incubated for 1.5 h with the monoclonal antibodies ARIII 22.7 (N-specific), ARIII 21.4 (C-specific) or AcV1 (specific for GP64), all in a 1:200 dilution. The grids were then washed on six drops of 20 µl PBSBSA for 5 min each and further incubated for 1 h with RAM coupled to 7 nm gold particles (Aurion Wagenigen, The Netherlands). The grids were washed with PBS, negatively stained with 2% (w/v) uranyl acetate pH 3.9 and examined in a Philips CM12 transmission electron microscope.
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Results and discussion |
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To avoid tedious rounds of plaque purification, recombinant viruses were generated using bacmid technology (Luckow et al., 1993). To this end, the gp64 sequences were amplified from the pBacSurf-1 vector (Novagen) and cloned downstream of the polyhedrin promoter between the EcoRI and HindIII sites of the pFastBacDUAL vector (Invitrogen). A KpnI site, situated downstream of the signal sequence, was used to introduce the p67 N- and C-terminal domains (Figure 1B and C
). The coding sequence for enhanced GFP was cloned downstream of the p10 promoter in order to follow transfection and infection processes, and to guarantee a simple read-out in virus titrations.
It has been reported that fusion of proteins to GP64 may adversely affect viral production or infectivity (Boublik et al., 1995). This appears to be dependent on the size of the protein, since larger proteins are more likely to interfere with GP64 trimer formation (Oomens et al., 1995
) and hence with incorporation into the virus particle. Apparently, since the titers obtained for the various viral stocks were within the normal range, the expression of GP64-p67 chimeric proteins did not significantly disturb virus budding and membrane fusion.
Immunofluorescence studies were performed in Graces medium on non-fixed, living Sf21 cells. In this way antisera could only reach p67 domains which were exposed on the surface of the cells. With monoclonals specific for the N- and C-domains (ARIII 22.7 and ARIII 21.4, respectively), and with a second antibody conjugated to rhodamine Red X, a strong red peripheral fluorescence was observed (Figure 2A and C), which surrounded the cytoplasmic green GFP fluorescence (Figure 2B and D
). In cells infected with the control Ac-gp64, only the green GFP fluorescence was observed with both monoclonal antibodies (data not shown). Cell surface expression has also been reported by Tami et al. (Tami et al., 2000
), when expressing FMDV structural proteins fused to GP64. To analyze the conformation of p67N on the cell surface, monoclonal TpM12 was used in immunofluorescence studies with recombinant-infected non-fixed cells. A red fluorescence was observed at the cell surface for cells infected with Ac-gp64/p67N (Figure 2E and F
), indicating that a conformational epitope in p67N was conserved. The amount of fluorescence observed was less with TpM12 than with the other monoclonals. Therefore, we showed confocal images of the cell surface (Figure 2E and F
) instead of optical slices through the middle of the cell. TpM12 did not react with cells infected with the Ac-gp64 control (Figure 2G and H
).
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The combination of surface display with bacmid technology as described in this paper has greatly accelerated the procedure of generating recombinant viruses, thereby facilitating the expression of antigenic domains for vaccine-related studies. The next step will be to test the immunogenic properties of the budded virions displaying the p67-GP64 chimeric proteins in cattle, and to determine whether it can protect against ECF and, if so, to evaluate the potential of p67-budded viruses under field conditions.
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Acknowledgments |
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References |
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Received April 23, 2002; revised October 8, 2002; accepted November 12, 2002.