Characterization of two new monoclonal antibodies against human papillomavirus type 16 L1 protein

Generation of recombinant HPV16 L1 protein was induced by isopropy-β-D-thiogalactoside (IPTG) from pQE31-HPV16L1/M15 constructed in our laboratory. Purified protein was used to immunize BALB/c mice, and two hybridoma cell lines (AE3 and AG7) were selected which stably expressed neutralizing antibodies against HPV16 L1 protein. The culture supernatant and ascites from mice carrying AE3 and AG7 hybridomas were purified by caprylic acid-ammonium sulfate methods.

HPV16 E6 and HPV16 E7 proteins were produced and purified using a baculovirus expression system. Fifty microgram aliquots of total protein were separated on 12% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were blocked with TBST buffer containing 5% skim milk and incubated with AE3 or AG7 mAb (1:1000) overnight. After washing, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA), and the protein bands were visualized by enhanced chemiluminescence (ECL).

Sf9 cells were infected with HPV recombinant baculovirus rBacV/HPV16 L1 on coverslips in 6-well plates. Three days later, the cells were fixed in 10% acetic acid, 50% ethanol, washed with phosphate-buffered saline (PBS) and then incubated with the mAbs of AE3 and AG7 for 1 h at 37°C followed by incubation with FITC-conjugated secondary antibody (1:80) (Invitrogen, USA). The fluorescence was detected under an Olympus AX70 epifluorescence microscope (Olympus, Tokyo, Japan).

HPV16 L1 VLP crude extract was incubated with purified mAb at 37°C for 1 h and then at 4°C overnight. The mixture was centrifuged at 12,000 g for 90 min, and then the supernatant was removed. In the negative control, 3% phosphotungstic acid and 400-mesh high-transmission nickel grids were used. VLPs were observed under a Hitachi H600 transmission electron microscope.

Red blood cells (RBCs) from BALB/c mice were diluted in 0.1% BSA-PBS to 1%. Ascites were mixed with the RBC precipitation of equal volume followed by incubation at 4°C overnight. The supernatant was collected after centrifugation at 1,000 g for 5 min and then incubated at 56˚C to inactivate the complements. The supernatant was mixed with HPV16 VLPs of equal volume and loaded into a 24-well plate followed by incubation at 37°C for 1 h. Next, 1% RBC of equal volume was added and the mixture was further incubated for 3 h at room temperature before evaluating the HI titer.

The titers of mAbs (AE3 and AG7) in the supernatants of cultured hybridoma cells and ascites were analyzed by indirect ELISA. Briefly, ELISA plates were coated with HPV16L1 protein at 4°C overnight. After washing with PBS and blocking with 1% BSA, the plates were incubated with serially diluted mAbs followed by incubation with horseradish peroxidase conjugated anti-mouse IgG for 1 h. After washing with PBS, the 3,3′,5,5′-Tetramethylbenzidine substrate (100 μl/well; Santa Cruz) was added followed by incubation for 15 min. The reaction was stopped by the addition of 1 M HCl (100 μl/well), and the absorbance was determined at 490 nm.

Two hybridoma cell lines (AE3 and AG7) in logarithmic growth phase were harvested, and total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. cDNA was synthesized with total RNA and labeled with polyG at the 3′ end of the RNA using TdT enzyme (Takara, Dalian, China). RT-PCR was performed using a RT-PCR kit (Takara) and primers were as follows: for VL gene P1 (5′-CCCCCCCCCCCCCCC-3′) and P2 (5′-GCGCCGTCTAGAATTAACACTCATTCCTGTTGAA-3′); for VH gene P1 (5′-CCCCCCCCCCCCCCC-3′) and H2 (5′-ACCTTAGGAGTCAGAGTAATGGTGAGCACATCC-3′). PCR conditions were denaturation at 95°C for 4 min, 35 cycles of denaturation at 94°C for 40 s, annealing at 55°C for 45 s and extension at 72°C for 1 min, and a final extension at 72°C for 7 min. PCR products were purified for sequencing. The nucleotide and amino acid sequences of AE3 and AG7 were compared and analyzed using the IgBLAST software (http://​www.​ncbi.​nlm.​nih.​gov). Three dimensional (3D) structure prediction was performed using Geno3D (http://​geno3d-pbil.​ibcp.​fr).

Western blot analysis showed that AE3 and AG7 mAbs could specially bind to HPV16 L1 VLP and HPV16 L1 proteins, but not to HPV16 E6 and HPV16 E7 proteins, indicating the specificity of two mAbs (Figure 1A). Immunostaining revealed that AE3 and AE7 could recognize the virus-infected Sf9 cells (Figure 1B). HPV16 L1 VLPs were a spherical particle with 55-nm in diameter (Figure 1C). The HI titer of AE3 and AG7 mAbs was 1:64 and 1:8, respectively. The titers of AE3 and AG7 mAb was 1:5120 and 1:80, respectively, in culture supernatant and 1:10⁶ and 1:10⁴, respectively, in ascites (Table 1).

The VL genes of AE3 and AG7 mAbs each included 336 nucleotides (nts) (Figure 2A), and encoded 112 amino acids (aas) (Figure 2B). The VL had 4 framework regions (FRs) and 3 complementary-determining regions (CDRs), and the VL-specific cysteine residues (aa23 and aa93) located in one of the FRs, in accordance with the characterization of mouse VL gene. The comparison of VL between two mAbs was shown in Figure 2C. The variation rate of VL in two mAbs was 27.7% at the nt level, including 29.2% (14/48) in the CDR1, 42.9% (9/21) in the CDR2, and 48.1% (13/27) in the CDR3. The variation rate of VL in two mAbs was 39.5% at the aa level, including 50% (8/16) in the CDR1, 57.1% (4/7) in the CDR2, and 66.7% (6/9) in the CDR3. In addition, the CDR2 and CDR3 regions had higher variation rate (Figure 2C).The VH genes of AE3 and AG7 mAbs each contained 354 nucleotides (Figure 3A), and encoded 118 amino acids (Figure3B). Similar to VL, each VH had 4 FRs and 3 CDRs. The VH protein contained 2 characteristic cysteine residues (aa22 and aa96), consistent with the characteristics of mouse VH gene. The difference in homology between two mAbs was shown in Figure 3C. The variation rate of VH regions was 33.3% (118/354) at the nt level, including 46.7% (7/15) in the CDR1, 27.5% (14/51) in the CDR2, and 77.8% (21/27) in the CDR3. At the aa level, the variation rate was 49.2% (58/118), including 100% (5/5) in the CDR1, 47.1% (8/17) in the CDR2, and 77.8% (7/9) in the CDR3. The highest variation rate at nt and aa levels was in the CDR3 for VH genes, but CDR1 also showed an increased variation rate between two mAbs (Figure 3C).3D structure predictions of VL of each mAb showed an alpha helix in the secondary structure of AE3, which was absent in AG7. For VH, two alpha helices were predicted in the secondary structure of AE3 located at two different sides of the protein, but two alpha helices were predicted in the secondary structure of AG7 located at at the same side of the protein (Figure 4).