Chimeric human papillomavirus type 16 (HPV-16) L1 particles presenting the common neutralizing epitope for the L2 minor capsid protein of HPV-6 and HPV-16

Abstract

Both the Human papillomavirus (HPV) major (L1) and minor (L2) capsid proteins have been well investigated as potential vaccine candidates. The L1 protein first oligomerizes into pentamers, and these capsomers assemble into virus-like particles (VLPs) that are highly immunogenic. Here we examine the potential of using HPV type 16 (HPV-16) L1 subunits to display a well-characterized HPV-16 L2 epitope (LVEETSFIDAGAP), which is a common-neutralizing epitope for HPV types 6 and 16, in various regions of the L1 structure. The L2 sequence was introduced by PCR (by replacing 13 codons) into sequences coding for L1 surface loops D-E (chideltaC-L2), E-F (chideltaA-L2), and an internal loop C-D (chideltaH-L2); into the h4 helix (chideltaF-L2); and between h4 and beta-J structural regions (chideltaE-L2). The chimeric protein product was characterized using a panel of monoclonal antibodies (MAbs) that bind to conformational and linear epitopes, as well as a polyclonal antiserum raised to the L2 epitope. All five chimeras reacted with the L2 serum. ChideltaA-L2, chideltaE-L2, and chideltaF-L2 reacted with all the L1 antibodies, chideltaC-L2 did not bind H16:V5 and H16:E70, and chideltaH-L2 did not bind any conformation-dependent MAb. The chimeric particles elicited high-titer anti-L1 immune responses in BALB/c mice. Of the five chimeras tested only chideltaH-L2 did not elicit an L2 response, while chideltaF-L2 elicited the highest L2 response. This study provides support for the use of PV particles as vectors to deliver various epitopes in a number of locations internal to the L1 protein and for the potential of using chimeric PV particles as multivalent vaccines. Moreover, it contributes to knowledge of the structure of HPV-16 L1 VLPs and their derivatives.

FIG. 1.

Western blot analysis of chimeric products using H16:J4 MAb. This MAb binds a linear epitope in the region aa 261 to 280. A 55-kDa product was detected for all the chimeras, similar to that of native HPV-16 L1.

FIG. 2.

EM of chimeric particles trapped with MAb H16:J4 onto carbon-coated copper grids and negatively stained with 2% uranyl acetate (bar = 50 nm). Enlargements on the right show individual particles for ChiΔC-L2 and ChiΔE-L2, VLPs in state of disassembly for ChiΔF-L2, and pentameric aggregates for ChiΔA-L2 and ChiΔH-L2.

FIG. 3.

Antibody-binding characterization of the purified chimeric product using a panel of MAbs raised to HPV-16 L1 VLPs and a polyclonal antibody raised to the L2 epitope. H16:V5 and H16:E70 are conformation-specific neutralizing MAbs, and residues Phe-50, Ala-266, and Ser-282 are critical for their binding (41). MAbs H16:U4 (binding epitope not characterized) and H16:9A (binds in the region aa 1 to 172) are both conformation-specific and neutralizing, whereas H16:J4 (binds between aa 261 to 280) and H16:I23 (binds between aa 111 and 130) are weakly neutralizing and recognize linear epitopes. H16:D9 has a high affinity for denatured L1.

FIG. 4.

End point titrations of sera from BALB/c mice immunized with chimeric products against HPV-16 L1 VLPs.

FIG. 5.

Western blot analysis of immune responses to L2 epitopes presented by various chimeras. The sera were analyzed against HPV-16 L2 protein expressed in E. coli.