A combined nucleocapsid vaccine induces vigorous SARS-CD8+ T-cell immune responses

Chinese Hamster Ovary (CHO) cells were grown at 37°C, 5% CO2 in Iscove's Modified Dulbecco's Medium (IMDM: Sigma, St. Louis, MO) and supplemented with 10% Fetal Calf Serum (FCS: Life Technologies, Grand Island, NY), 100 U/ml penicillin and 100 μg/ml gentamyin.

Total RNA was purified using an RNeasy extraction kit (Qiagen, Mississauga, Ont) from the lung tissue of an autopsied patient who died from SARS. The full-length NC (1.2 kb) gene was amplified using specific primers (forward primer: 5'-ggatccatgtctgataatggaccc-3'; reverse primer: 5'-gaattcttatgcctgagttgaatc-3'). The amplicon was purified using the QIAquick gel extraction kit (Qiagen) and cloned into the PCR 2.1 TOPO-TA vector (Invitrogen, Burlington, Ont) according to the manufacturer's instructions. After plasmid digestion, the 1.2 kb band corresponding to the NC gene was sub-cloned into BamHI-and EcoRI sites of pVAX-1 which contains a CMV promoter for high level expression in vivo. The fragment was also sub-cloned into the pEF6-Myc/His (Invitrogen) and pQE (Qiagen) vectors. The pEF6 vector was designed to over produce recombinant proteins in mammalian cell lines and was used to establish a stable cell line by using a resistant blasticidine gene. The pQE Tri system vector was used for production of proteins in bacteria. These vectors ultimately allow for the purification of the protein by immobilized metal affinity chromatography. All expression constructs were confirmed and characterized by restriction enzymes and nucleotide sequence analysis.

CHO cells and JM109 bacteria were transfected with pEF6 and pQE vectors containing NC or vector alone. In order to increase and sustain expression of the NC protein, a stable NC expressing CHO cell line was established using blasticidine-supplemented medium. Cells were harvested, sonicated and lysed in lysis buffer (25 mM Tris base, 2.5 mM Mercaptoethanol, 1% Triton-X100 and a cocktail of protease inhibitors). Cell pellets were centrifuged and the supernatant was incubated with the TALON metal resin (Clontech, Palo Alto, CA) for one hour. After incubation, the mixture of protein-resin was added to the columns and washed three times with 20 bed volumes of Tris-Cl, NaCl (PH 8). The recombinant protein was eluted with 150 mM imidazole.

To confirm the proteins, samples were mixed with Laemmli loading buffer, boiled for 5 minutes and loaded on a 10% polyacrylamide gel. The proteins were then transferred to a nitrocellulose membrane by electrophoretic transfer. The membranes were blocked with 5% dried milk in PBS-Tween 20 (PBS-T) and incubated with 1/3000 dilution of a sera from a SARS patient for three hours at room temperature. After washing with PBS-Tween, the blots were incubated with anti-human IgG-HRP conjugate (BioRad, Hercules, CA) for one hour at room temperature. After incubation, the blots were washed and incubated with ECL reagent (Santa Cruz Biotechnology, Santa Cruz, CA) for one minute and exposed to X-ray film (Kodak).

CHO cells transfected with either the DNA vector expressing NC or DNA vector alone were harvested and washed with PBS. Pellets were then fixed with 2% glutaraldehyde. Cells were rinsed twice in 0.1 M sodium cacodylate buffer at 4°C. The cells were then fixed with 2% Osmium Tetroxide for 2 hr at 4°C. After washing with distilled water, the cells were dehydrated with increasing concentrations of ethanol and embedded in spur resin. Thin sections were stained with uranyl acetate and lead citrate. The sections were screened by using a JEOL 1010 Transmission Electron Microscope (TEM).

CpG oligodeoxynucleotide (5'-TCCATGACGTTCCTGACGTT-3') was provided by Coley (Ottawa, ON). Montanide ISA-51 mineral oil adjuvant was purchased from Seppic Inc. (Paris, France). The pcDNA3 construct expressing 1.5 kb XIAP gene encoding an anti-apoptotic gene product was a kind gift from Dr. R.G. Korneluk [24].

Six to eight week-old female B6/C3/F1 mice (Charles River, St. Constant, PQ) were immunized subcutaneously at the base of the tail with 50 μg of DNA construct expressing the nucleocapsid gene, 5μg of nucleocapsid protein and 50 μl of montanide ISA-51 (Seppic)/30 μg CpG (Coley), or 50 μg pcDNA3-XIAP at each vaccination. Each mouse was boosted three times, at one month intervals. Fourteen days after the last boost, the mice were sacrificed and their spleens and blood was collected for further testing or for long-term storage in cryopreservation medium.

96 well ELISA plates were coated overnight at 4°C with NC protein, and the wells were washed with PBS containing 0.05% Tween 20 and then blocked with 1% BSA in PBS. Serially diluted sera was added and incubated for 2 h at 37°C. The plates were washed and incubated for 2 h with a 1/2000 dilution of a peroxidase-conjugated affinity-purified rabbit anti-mouse secondary antibody (Bio-Rad, Richmond, CA). The plates were washed three times and developed with O-phenylendiamine dihydrochloride (OPD) substrate (Sigma, St. Louis, MO). The color reaction was stopped with 1N HCl and absorbance was read at 490 nm with an ELISA plate reader (Bio-Rad).

Splenocytes from immunized mice were resuspended at 2 × 10⁶ cells/ml in RPMI 1640 containing 10% FCS, 50 μM β-mercaptoethanol and 100 U/ml penicillin/streptomycin. A 100 μl aliquot containing 2 × 10⁵ cells was added to each well of a 96 well plate. The NC protein (100 μl at 20μg/ml) was added to each well in triplicate. As a positive control, cells were also stimulated with phorbol 12-myristate 13-acetate and ionomycin (PMA/ION). After 72 h of culture, 1 μCi [³H] thymidine (Amersham, Arlington Heights, IL) was added to each well. Following 16 h of incubation, cells were harvested onto glass fibre filtermats and thymidine incorporation was measured with a Microbeta beta counter (Wallac, Turku, Finland).

Fresh blood and splenocytes from immunized mice were cultured in IMDM media in the presence of 10 μg/ml brefeldin A (Sigma) and stimulated in vitro with the NC protein (10 μg/ml) expressed in bacteria. In every experiment, a negative control (without stimulation), positive control (PMA/ION) and an irrelevant protein (HIV-1 gp120 protein) was included to control for spontaneous production of IFN-γ. Sixteen hours after incubation, the cells were washed once (1600 rpm for 5 min) with 3 ml PBS / 2% FCS / 0.01% Azide and surface-stained for 15 min with PE-labeled Ab to mouse CD3, TC-labeled Ab to mouse CD8α or CD4 (Caltag Laboratories, Hornby, ON). The cells were washed as above, fixed and permeabilized using 100 μl each of A and B fixation-permeabilization solution (Caltag Laboratories). The cells were stained intracellularly with anti-mouse IFN-γ FITC-labeled Ab and incubated for 30 min (in the dark) at 4°C. Following washing, cells were analyzed by FACScan (Becton Dickinson, Mississauga, ON). An increase of 0.1% of IFN-gamma producing cells over the unstimulated control was considered as positive response to vaccination.

Multiscreen-HTS plates (Millipore, Bedford, MA) were coated with 10 μg/ml of anti-mouse IFN-γ antibody (mAb AN18, Mabtech, Mariemont, OH) in PBS over night at 4°C. The plates were then washed with PBS and blocked with IMDM containing 10% FCS and 100 U/ml penicillin/streptomycin for 1 h at room temperature. The medium were removed and 4 × 10⁵ cell suspension (100 μl/well) including NC SARS protein expressed in bacteria (10 μg/ml) or irrelevant antigens at the same concentration were added and incubated for 30 h at 37°C. After incubation, cells were removed; plates were washed with PBS+0.05% Tween 20 and incubated with 1 μg/ml of biotinylated anti-mouse IFN-γ antibody (mAb R4-6A2-Biotin, Mabtech) for 2 hr at room temperature. After further washings, 100 μl/well of 1/2000 Streptavidin-ALP-PQ (Mabtech) in PBS+ 0.5% FCS was added and incubated for 1 hr at room temperature. The plates were washed as above and developed with 100 μl per well BCIP/NBT alkaline phosphatase (Moss Inc) for 20 minutes at room temperature. The reaction was stopped with rinsing the plates with tap water. The numbers of spots were analyzed with an ELISPOT reader.

To increase the potency of the specific immune response, the full-length NC was amplified by RT-PCR and ligated into plasmid pVAX-1 under the control of the human cytomegalovirus promoter. For the expression and purification of the recombinant NC protein in CHO and bacteria cells, the amplified NC gene was also sub-cloned into pEF6-Myc/His and pQE-Tri system vectors. To express NC protein, CHO cells and E.coli (JM109) were transfected with pEF6 and pQE vectors encoding the NC gene, respectively. To increase the yield of the recombinant protein, a stable CHO cell line was created using a selective resistant blasticidine gene, allowing for efficient purification of the recombinant protein. Cells were harvested, lysed and the recombinant proteins were purified according to standard methods. The expression of the NC protein in transfected cells was verified by western blotting (Fig 1) and immunofluorescence staining of CHO cells infected with the vector-NC or the vector alone. Antibody raised in rabbits to the NC protein expressed in bacteria reacted strongly in the perinuclear region of the SARS-NC-CHO cell line (data not shown).

The CHO cells transfected with pEF6-NC or vector alone were examined by transmission electron microscopy. We observed bundles of VLP of the same morphology as wild type particles both inside and outside cells infected with pEF6-NC. However, neither the mock-transfected cells nor the cells transfected with the vector alone showed viral-like particles (Fig 2). These observations demonstrate that our construct expressing the NC protein synthesised sufficient protein within infected cells to facilitate the formation of VLPs.

In order to analyze the antibody titer against NC, five groups of mice were primed and boosted with SARS-nucleocapsid immunogen alone or in combination. Two weeks after the last boost, sera were collected and antibody titer was measured by ELISA. The group received protein and montanide/CpG showed a higher mean IgG antibody titer compared to the group receiving vector DNA+XIAP and DNA-NC alone. This group (NC protein + montanide/CpG) also showed a slightly higher antibody titer compared to the group received DNA-NC + NC protein and XIAP. However, the highest SARS-CoV-specific antibody response was detected in mice immunized with a combination of DNA-NC, protein and montanide/CpG (Fig 3).

To assess whether vaccination with nucleocapsid increases cell-mediated immune responses, splenocytes and fresh blood from immunized mice was retrieved, stimulated, and stained for surface CD4 and CD8+T cells as well as intracellular interferon gamma. The level of IFN-γ producing CD4+ T cell in fresh blood (Fig 4) and splenocytes (data not shown) from immunized mice did not demonstrate a significant CD4+T cell response against the SARS-NC protein. However, the group receiving DNA-NC, protein and montanide/CpG demonstrated higher levels of IFN-γ producing CD4+ T cells.

Splenocytes were also stimulated with NC protein, and CD4 lymphocyte proliferation was performed with tritiated thymidine. However, a high T-cell proliferation was not detected with this assay (data not shown).

Cell-mediated immune responses were evaluated by intracellular cytokine staining. The group receiving DNA-NC + NC protein and Montanide/CpG elicited higher levels of CD8+T-cells to nucleocapsid in comparison to groups receiveing DNA-NC or NC protein plus adjuvant. However, the highest NC-specific CD8+T-cell response was detected in both splenocytes (data not shown) and fresh blood (Fig 5) in mice that received the DNA construct, recombinant NC protein and adjuvant XIAP.

To confirm the results obtained by intracellular cytokine staining, we performed an IFN-γ ELISPOT assay to measure NC-specific T-cell responses of splenocytes from immunized mice. The groups DNA+XIAP, DNA-NC and NC protein + montanide/CpG did not show a high number of spot forming cells (SFC). Potent IFN-γ responses were observed in mice immunized with combination of DNA-NC+NC protein and adjuvants (Fig 6). However, following substitution of adjuvant montanide/CpG with XIAP, SFCs were more than two fold higher (p = 0.01). Although, IFN-γ may be produced by both antigen-stimulated CD4+ and CD8+ T cells, most likely the observed IFN-γ response was generated by effector CD8+ T-cells, since flow cytometry demonstrated CD8+ T cells as the main producers of IFN-γ in this study.