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

Abstract

Background: Human papillomavirus type 16 (HPV16) infection is implicated in cervical carcinogenesis. This study aimed to characterize two new monoclonal antibodies (mAbs) against HPV L1 protein.

Methods: The immunocompetence of AE3 and AG7 mAbs for HPV L1 protein was evaluated by Western blot analysis, immunostaining, hemagglutination inhibition assay, and ELISA. The heavy chain variable region (VH) and light chain variable region (VL) of AE3 and AG7 mAbs were sequenced and analyzed.

Results: Both mAbs specifically recognized HPV16 L1 and virus-like particles (VLPs). Both the affinity and the titer of AE3 mAb were higher than that of AG7. There were differences in sequences in the complementary determining regions (CDR) 2 and 3 of VL, as well as in the CDR1 and CDR3 of VH. The two mAbs have distinct predicted three-dimensional structures.

Conclusions: We characterized two mAbs neutralizing antibodies for HPV L1 protein, which would help develop genetic-engineered neutralizing antibodies against HPV16 for diagnostic and therapeutic purposes.

Figure 1

Immunocompetence of AE3 and AG7 mAbs. A: Western blot analysis of the specificity of AE3 and AG7 mAbs (1:1000) to different antigens. B: Sf9 cells were inoculated with rBacV/HPV16 L1 in 6-well plates. The supernatant of cultured AE3 and AG7 was incubated for 1 h at 37°C followed by incubation with FITC-conjugated secondary antibody (1:80). Immunofluorescence was detected under an epifluorescence microscope (×200). C: The images of HPV16L1 VLPs under an immunoelectron microscope. The morphology was indicated by the arrows (×5,000).

Figure 2

Comparison of nucleotides and amino acids of VL between AE3 and AG7 mAbs. A: Comparison of nucleotides of VL between AE3 and AG7 mAbs. B: Comparison of amino acid of VL between AE3 and AG7 mAbs. C: Variation rate of VL in AE3 and AG7 mAbs. The variation rate of two selected sequences indicated the ratio of different nucleotide or amino acid to total nucleotide or amino acid.

Figure 3

Comparison of nucleotides and amino acids of VH between AE3 and AG7 mAbs. A: Comparison of nucleotides of VH between AE3 and AG7 mAbs. B: Comparison of amino acids of VH between AE3 and AG7 mAbs. C: Variation rate of VH of AE3 and AG7 mAbs.

Figure 4

Three-dimensional structure prediction of VL and VH of AE3 and AG7. The light chain of AE3 mAb had one α-helix (red), but the light chain of AG7 had no α-helix. There were two α-helies (red) at both sides of the heavy chain of AE3 mAb, while the two α-helies of heavy chain of AG7 (red) were located at the same side.