In this study we used HPV16 variant strain isolated from a cervical cancer patient in East China (GenBank accession no. AF393502).

For the production of HPV16 L1 VLP, 1×109 sf9 cells were grown at 27°C in Grace's insect medium (Sigma) supplemented with 10% fetal calf serum, gentamycin and amphotericin. The cells were infected with recombinant baculovirus rBacV16 L1 at a MOI of 10. After 72h, the cells were harvested by low-speed centrifugation at 1000rpm (200×g) for 5min and washed once with ice-cold phosphate buffer saline (PBS). All subsequent procedures were carried out at 4°C. The cell pellet was resuspended in one volume of PBS to a total of 24mL and sonicated 30 times on ice at a setting of 200W. The total cell lysate was loaded onto a 40% (wt/vol) sucrose-PBS cushion and centrifuged at 25,000rpm (110,000×g) for 2.5h. The pellet was resuspended in 7mL of 35% (w/v) cesium chloride (CsCl)-PBS and centrifuged to equilibrium in 35% (wt/vol) CsCl-PBS for 20h at 35,000rpm (141,000×g). The visible band at a density of 1.29g/mL was harvested, dialyzed extensively against PBS, concentrated with PEG 2000, and VLP was examined with transmission electron microscopy (TEM) at 50,000 magnification. The prepared VLP was then stored at −80°C.

The plasmid pCAGGS-mIL-12 was prepared by standard alkaline lysis and DNA was further purified by banding CsCl2 gradient centrifugation as described previously.9

Six to eight week-old female BALB/c mice were used in all experiments. The protocols for animal experiments were approved by the Ethics Committee of Harbin Medical University. The immunization protocols are shown in Table 1. Plasmids were injected intramuscularly, while VLP and Freund's adjuvant (Gibcol, BRL) were injected subcutaneously. Mice serum was obtained by retrobulbar collection two weeks after each immunization, when mice were sacrificed with decapitation methods. The spleen was dissected and the size was measured. Single-cell suspensions were obtained from the spleen by gently pressing the spleen between two sterile slides; dissociated cells were then washed in DMEM containing 5% fetal bovine serum (Sigma).

Splenocytes were incubated for 30min at 4°C with PE- or FITC-labeled CD4+, CD8+ monoclonal antibody (Zhongshan Biotech), followed by washing three times with PBS, then analyzed by fluorescence-activated cell sorter (FACS).

The serum concentrations of interferon-γ (IFN-γ) were detected by using ELISA kit (Endogen) according to the manufacturer's instruction.

Serum IgG antibody titers were investigated using the HPV16VLP as the target antigen in a standard ELISA. Each well was coated with 100ng of purified VLP or denatured VLP (produced by the addition of 0.1M DTT and boiling) in 0.05M sodium carbonate buffer (pH 9.6) at 4°C overnight. Plates were then washed four times with TBS containing 0.1% Tween 20 (TBS/T). Non-specific binding sites in each well were blocked with 100μl of 1% BSA in TBS/T at 37°C for 1h. Then 100μl of serial 2-fold dilutions of serum were added to each well and incubated at 37°C for 1h. After washing, 100μl of 1:1000 HRP-conjugated goat anti-mouse IgG (Promega) were added and incubated at room temperature for 1h. Finally, the plates were developed by incubation with 100μl of OPD substrate reagent (2.43mL 0.1M citric acid, 2.57mL 0.2M Na2HPO4, 5mL H2O, 4mg OPD and 15μl 30% H2O2) for 5–15min in the dark, followed by quenching reaction with 100μl of 3M HCl. The absorbance was read at 490nm in an ELISA plate reader. Both HPV16 L1 monoclonal IgG CAMVIR-1 and HPV16 L1 polyclonal antibody M3 served as positive controls. CAMVIR-1 antibody was purchased from PharMingen. M3 antibody (kindly provided by Prof. Shang, Harbin Medical University) was collected from the serum after the mice were vaccinated with purified HPV16 L1 protein which was expressed in bacterial strains. All samples were examined in triplicate for each antibody class. The assay was considered valid only when the coefficient of variation of the triplicates was <10%. Sera with mean OD more than twice that of negative controls were considered positive for the respective IgG subclass.

The difference of splenic lymphocyte percentages and serum IFN-γ level were analyzed for statistical significance (p<0.05) using SigmaStat version 10.

To determine whether the variant HPV16 L1 protein could self-assemble efficiently, lysates from insect cells infected with L1 recombinant baculovirus rBacV16 L1 were subjected to CsCl2 density gradient centrifugation and the visible band was analyzed by TEM. The predominant structure consisted of spherical particles about 50nm in diameter with a regular array of capsomeres, although smaller, larger, and irregular spheres were also seen (Fig. 1). These results demonstrated that HPV16 L1 protein could efficiently self-assemble into VLP.

Fig. 1.

Purified HPV16 L1 VLP. VLP were purified from recombinant baculovirus-infected Sf9 cells on CsCl gradients, stained with uranyl acetate, and observed by TEM. Magnification, ×50,000.

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To monitor the development of an immune response after the last immunization, spleen T lymphocytes were collected and subjected to FACS. The percentage of CD4+ T lymphocytes was increased in all three immunized groups compared with the control group. In addition, the percentage of CD8+ T lymphocytes was higher in L1P group than the other experimental groups and L1P group had the lowest CD4+/CD8+ ratio (Table 2).

Purified HPV16 L1 VLP induced specific antibody in vaccinated mice (Fig. 2). When mice were given three consecutive booster doses, specific serum IgG responses to VLP16 increased. After the third immunization, IgG titers were 1:120,000 in L1N group and 1:360,000 in L1A and L1P groups. In contrast, by ELISA we determined that the titers of HPV16 L1 monoclonal IgG CAMVIR-1 (PharMingen) and polyclonal antibody M3 to purified VLP were only 1:13,500. No specific IgG was detected in control group.

Fig. 2.

Titers of serum IgG antibodies from vaccinated mice. L1N, L1A, L1P, control indicated sera from the corresponding group of mice. -1, -2, -3 indicated the first, second and third immunization. M3 indicated sera antibodies from mice immunized with HPV16 L1 expressed in Escherichia coli. CAMVIR-1 indicated commercial HPV16 L1 monoclonal antibody.

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Considering the intrinsic capacity of HPV L1 to self-assemble into VLP in sf9 cells, the lysates of sf9 infected with rBacV16 L1 were used as VLP antigen to analyze the specific IgG produced in L1A group (Fig. 3). ELISA results showed that the titers of specific IgG to the lysates of infected sf9 cells were the same as purified VLP. No positive results were found for the lysates of normal sf9 cells. Similar results were observed in the other two immunized groups.

Fig. 3.

Titers of sera IgG antibodies to purified VLP and lysates of infected Sf9 cell. L1A indicated sera from the L1A group of mice. -VLP indicated ELISA plate coated with VLP. -sfvlp indicated ELISA plate coated with lysates of Sf9 cells infected by rBacV16 L1. -sf. indicated ELISA plate coated with lysates of normal Sf9 cells.

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Next we used denatured VLP to develop ELISA for L1A group serum (Fig. 4). Compared to the high titers to the purified VLP (1:360,000), the titers of specific IgG to denatured VLP (1:13,500) were significantly decreased. Similar results were observed in the other two immunized groups.

Fig. 4.

Antibodies from vaccinated mice recognize confirmation-dependent epitopes. ELISA plates were coated with HPV16 L1 VLP or the denatured VLP (-denat.) and anti-sera in L1A group, M3 and CAMVIR-1 were used for the detection.

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Serum from immunized mice was examined for the concentration of Th1-type cytokine IFN-γ (Table 3). Only a slight increase in serum IFN-γ level was observed in L1N group after three boosts, but serum IFN-γ level was significantly boosted after three consecutive immunizations in L1P group (799.7pg/mL). In L1A group, after the first immunization serum IFN-γ level increased to 190pg/mL, but then decreased slowly with continued immunization. Serum IFN-γ level in every vaccination group was higher than that in control mice (p<0.05).