A papillomavirus-like particle (VLP) vaccine displaying HPV16 L2 epitopes induces cross-neutralizing antibodies to HPV11

Abstract

Peptides of the papillomavirus L2 minor capsid protein can induce antibodies (Ab) that neutralize a broad range of human papillomavirus (HPV) genotypes. Unfortunately, L2 is antigenically subdominant to L1 in the virus capsid. To induce a strong anti-L2 Ab response with cross-neutralizing activity to other mucosal types, chimeric virus-like particles (VLP) were generated in which HPV16 L2 neutralization epitopes (comprising L2 residues 69-81 or 108-120) are inserted within an immunodominant surface loop (between residues 133 and 134) of the L1 major capsid protein of bovine papillomavirus type 1 (BPV1). These chimeras self-assembled into pentameric capsomers, or complete VLP similar to wild type (wt) L1 protein. Immunization of rabbits with assembled particle preparations induced L2-specific serum Ab with titers 10-fold higher than those induced by cognate synthetic L2 peptides coupled to KLH. Antisera to both chimeric proteins partially neutralized HPV16 pseudovirions, confirming that both HPV16 L2 peptides define neutralization epitopes. When analyzed for the ability to cross-neutralize infection by authentic HPV11 virions, using detection of early viral RNA by RT-PCR-assays as the readout, immune serum to chimeric protein comprising L2 residues 69-81, but not 108-120, was partially neutralizing. In addition, mouse-antiserum induced by vaccinations with synthetic L2 peptide 108-120, but not 69-81, was partially neutralizing in this assay. Induction of cross-neutralization Ab by L2 epitopes displayed on chimeric VLP represents a possible strategy for the generation of broad-spectrum vaccines to protect against relevant mucosal HPV and associated neoplasia.

Fig. 1. Construction of recombinant BPV1-L1/HPV16-L2 baculovirus expression vectors.

Oligonucleotides encoding HPV16-L2 peptides 69–81 and 108–120 (indicated by gray bars) were engineered into the 505 amino acids encoding BPV1-L1 ORF (open bar), between residues 133 and 134. The HPV16 L2 peptides are underlined, and flanking BPV1-L1 sequences shown.

Fig. 2. Baculovirus expression of BPV1 L1-HPV16 L2 fusion proteins.

Sf-9 insect cells were infected for 3 days with wild type AcMNPV or recombinant baculoviruses expressing L1+69-81, L1+108-120, or wild type BPV1-L1 as control, lysed, separated by SDS-Page, and analyzed by Coomassie stain (A), and Western blotting using mAb AU-1 directed against BPV1 L1 (AU-1) (B), or immune serum raised against HPV16 L2 (anti-16L2) (C). Recombinant proteins migrated at about 55–60 kDa. Smaller Coomassie-stained and immunoreactive bands that are absent in wt baculovirus (AcMNPV) infected control lanes likely represent a protein degradation product. A slower migrating band present in all four lanes of panel C is unspecifically recognized by the polyclonal anti-16L2 antiserum.

Fig. 3. Transmission electron microscopy of baculovirus expressed fusion proteins.

Chimeric L1+69-81 (A) and L1+108-120 (B) fusion proteins were expressed in Sf-9 insect cells, purified on density gradients, negatively stained and analyzed by transmission electron microscopy at 30,000 magnification. (A) Inset shows L1+69-81 pentamers at three-fold higher magnification. (B) Inset shows BPV1 wild type L1 VLP for comparison.

Fig. 4. Analysis of baculovirus expressed chimeric L1+69-81 and L1+108-120 proteins by ELISA.

BPV1 wild type L1 (triangles), and chimeric L1+108-120 (squares) or L1+69-81 (diamonds) proteins were analyzed under native conditions by ELISA, using the indicated serial dilutions of mAb 5B6 directed to a conformation-dependent neutralizing epitope (A), or mAb AU-1 directed to a linear BPV1 L1 epitope under denaturing conditions (B). OD, absorbance at 405 nm, mean + S.D. of triplicate wells.

Fig. 5. Analysis of antisera raised against chimeric proteins L1+69-81 and L1+108-120, or synthetic peptides by ELISA.

Streptavidin-coated ELISA plates were satured with biotinylated synthetic HPV16 L2 peptides 69–81 (A) or 108–120 (B). Pre-immune and immune sera raised against multimeric chimeric proteins L1+69-81 (A) or L1+108-120 (B), or raised against synthetic KLH-coupled peptides were serially diluted as indicated and analyzed by ELISA. OD, absorbance at 405 nm, mean + S.D. of triplicate wells.

Fig. 6. Neutralization of HPV16 pseudovirions by antisera to chimeric VLP and pentamers.

HPV16 pseudovirions were incubated in the presence of indicated dilutions of pre-immune (black bars) or immune sera (grey bars) raised against chimeric proteins. (A) Antisera raised against fusion protein L1+69-81 neutralized with a titer of 240. (B) Antiserum raised against L1+108-120 neutralized with a titer of 60 (stars indicate a p-value 0.05 by Student’s t-test). Antisera raised against synthetic peptides 69–81≤or 108–120 did not neutralize (not shown).

Fig. 7. Cross-neutralization of HPV11 virions by RT-PCR assay.

Authentic HPV11 virions were incubated with sera at 1:50 dilution or left untreated for 1 h prior to infecting A431 cells. Transient HPV11-infection of A431 cells was analyzed by detection of spliced viral E1∧E4 mRNA by nested RT-PCR. Antisera raised against L1+69-81, L1+108-120, or pre-immune sera are indicated. H11.B2 is a neutralizing mAb raised against HPV11 virions. The negative control serum was raised against chimeric BPV1 L1 VLP containing an irrelevant beta-amyloid nona-peptide (Aβ-VLP).

Fig. 8. HPV11 virion neutralization assay by quantitative RT-PCR.

Authentic HPV11 virions were incubated with indicated pre-immune or immune sera at dilution 1:50 prior to infecting HaCaT cells. The ranges 69–81 and 108–120 indicate mouse (pre-)immune sera raised against synthetic L2 peptides; L1+69-81 and L1+108-120 indicate rabbit (pre-)immune sera raised against the respective chimeric proteins. H11.H3 is a neutralizing mAb raised against HPV11 virions that was used at excess concentration to ensure complete neutralization. Data are plotted as the mean + S.E. of five independent experiments as described in materials and methods. Pre-immune and immune sera were compared within groups using Student’s t-test, and p-values ≤ 0.05 (indicated by stars) are considered significant.