Preclinical model to test human papillomavirus virus (HPV) capsid vaccines in vivo using infectious HPV/cottontail rabbit papillomavirus chimeric papillomavirus particles

Abstract

A human papillomavirus (HPV) vaccine consisting of virus-like particles (VLPs) was recently approved for human use. It is generally assumed that VLP vaccines protect by inducing type-specific neutralizing antibodies. Preclinical animal models cannot be used to test for protection against HPV infections due to species restriction. We developed a model using chimeric HPV capsid/cottontail rabbit papillomavirus (CRPV) genome particles to permit the direct testing of HPV VLP vaccines in rabbits. Animals vaccinated with CRPV, HPV type 16 (HPV-16), or HPV-11 VLPs were challenged with both homologous (CRPV capsid) and chimeric (HPV-16 capsid) particles. Strong type-specific protection was observed, demonstrating the potential application of this approach.

FIG. 1.

Titration of antibody binding to L1-VLPs by enzyme-linked immunosorbent assay. All sera were tested against CRPV (a), HPV-16 (b), and HPV-11 (c) VLPs. The data shown are means ± the standard deviation (SD) for serum dilutions from each of six rabbits in the antigen-matched group. No cross-reactivity was seen with sera from rabbits in the other two vaccine groups (data not shown). Tenfold dilutions of sera shown on the abscissa (log₁₀) were tested in duplicate wells against VLPs preadhered to plates in PBS (pH 7.4) and blocked with 5% nonfat dry milk in PBS. Antibody binding was indicated by using an anti-rabbit secondary antibody conjugated to alkaline phosphatase and a standard colorimetric assay. (d) Mean optical density (O.D.) readings (± the SD) for each vaccine group against the specific antigen.

FIG. 2.

Titration of pseudovirus neutralization by rabbit sera. Serial 10-fold dilutions of antigen-specific rabbit sera (and a single dilution of sera from rabbits in the other two vaccine groups) were preincubated with CRPV (a), HPV-16 (b), and HPV-11 (c) pseudovirions produced in 293TT cells using a protocol described previously (3). The abscissa shows serum concentrations (log₁₀) during the 1-h preincubation with pseudovirions at 37°C, prior to infecting triplicate wells of 293TT cells seeded the previous day in 96-well plates at 3 × 10⁴ cells/well. At 3 days postinfection, spent media were analyzed for seAP levels by using a colorimetric assay. The data are presented relative to seAP in wells receiving pseudovirions incubated in the absence of rabbit serum. The horizontal gray line represents 50% less seAP than control wells and is interpreted as the successful neutralization of 50% input pseudovirions. All line scatter plots show data from individual rabbits in the antigen-specific group, while the single points plotted at 10⁻³ represent the sera of individual animals from the other two vaccine groups. (d) Mean concentrations (± the SD) of antiserum required to neutralize 50% of input pseudovirions.

FIG. 3.

Papilloma occurrence and growth at sites inoculated with papillomavirus particles. New Zealand White rabbits were vaccinated with CRPV (circles), HPV-16 (triangles), or HPV-11 (squares) L1 VLPs as described in the text. At 4 weeks after the third immunization, scarified sites on the dorsal skin of each rabbit were inoculated with 10 μl of a stock of homologous (CRPV capsid/CRPV genome [open symbols]) or chimeric (HPV-16 capsid/CRPV genome [solid symbols]) particles purified from DNase-treated 293TT cell lysates using an Optiprep density gradient (3, 3a). (a) The mean papilloma size (± the SD) is calculated by using the geometric mean diameters (GMD) for all sites receiving the inoculum. (b) The frequencies of papillomas induced at all sites inoculated with the homologous or chimeric infectious particles are shown separately for the three vaccine groups.

FIG. 4.

Papillomas on selected rabbits 4 weeks after challenge with infectious particles. Rabbits vaccinated with CRPV (a), HPV-16 (b), or HPV-11 (c) VLPs were inoculated at five sites each with homologous (CRPV capsid/CRPV genome) particles on the left side (L) and with chimeric (HPV-16 capsids/CRPV genomes) particles on the right side (R).