Intranasal vaccination with a recombinant vesicular stomatitis virus expressing cottontail rabbit papillomavirus L1 protein provides complete protection against papillomavirus-induced disease

Abstract

Immunizations with live recombinant vesicular stomatitis viruses (rVSV) expressing foreign viral proteins have successfully protected animals from challenges with several heterologous viruses. We developed an rVSV expressing the major capsid protein (L1) of cottontail rabbit papillomavirus (CRPV) and tested the efficacy of protection following CRPV challenge. An rVSV expressing L1 of CRPV (VSV-L1) was characterized for the protective ability afforded by intranasal, intradermal, or intramuscular vaccination in rabbits subsequently challenged with CRPV. Protein expression of L1 in VSV-L1 was confirmed by radioimmunoprecipitation assays. Nuclear localization of L1 was demonstrated by indirect immunofluorescence assays. Immunized rabbits elicited significant VSV neutralization and VLP-L1 enzyme-linked immunosorbent assay titers. VSV-L1 vaccination was not associated with weight loss or any other adverse clinical signs in the rabbit model. VSV shedding in nasal secretions occurred in some rabbits, peaking at 4 to 6 days after intranasal vaccination, with no further shedding after day 6. Specific humoral immunity to the L1 protein was consistently seen after a single VSV-L1 vaccination when administered through an intradermal or intramuscular route or after a boost via the intranasal route. Rabbits were completely protected from CRPV-induced papillomas after VSV-L1 vaccination and boost given intranasally or intramuscularly. Vaccination with VSV-L1 is a novel approach to prevent papillomavirus-induced disease and demonstrates a potential strategy for developing a human papillomavirus vaccine that can be given without injection.

FIG. 1.

(A) Schematic representation of the VSV-L1 genome. The genome of VSV-L1 is shown in 3′ → 5′ orientation, and the open reading frames of the major VSV proteins are indicated. The CRPV-L1 gene was inserted between the VSV G and L genes. The GFP gene in the control VSV-GFP is also inserted between the VSV G and L genes. (B) Immunofluorescence of L1 expression from VSV-L1. VSV-G protein expression is seen in both VSV-L1- and rwtVSV-infected cells and is primarily localized at the plasma membrane (lower two panels). In contrast, L1 protein expression is highly localized to the nuclear compartment and is seen only in VSV-L1-infected cells (upper left panel). (C) Immunoprecipitation of L1 expression from VSV-L1. VSV proteins (L, G, N, P, and M) are present in both rwtVSV and VSV-L1 extracts (left panel). In contrast, L1 is present only in VSV-L1 extracts (right panel).

FIG. 2.

Protection from CRPV challenge in experiment 1. Average percentages of papilloma-free sites and average papilloma volumes (natural log) after CRPV challenge were determined in rabbits immunized once with intranasal VSV-L1 vaccine (gray bar and ▴), rabbits immunized once with intranasal VSV-GFP vaccine (diagonal line bar and ▪), and unimmunized control rabbits (black bar and ×). The results for high-challenge (A), moderate-challenge (B), and low-challenge (C) CRPV doses are shown. Error bars represent standard errors of the mean.

FIG. 3.

Antibody to VLP-L1 in rabbits in experiment 2. Rabbits were immunized through the intradermal (A), intranasal (B), or intramuscular (C) route, and antibody titers were determined by ELISA using VLP-L1 protein. Results are shown for rabbits tested prior to immunization (pre-immune), 8 weeks after the primary injection (post-primary), and 13 weeks after the primary injection (i.e., 5 weeks after the homologous VSV-L1 booster injection).

FIG. 4.

Protection from CRPV challenge in experiment 2. Average percentages of papilloma-free sites and average papilloma volumes (natural log) are shown for rabbits immunized with VSV-L1 vaccine through intradermal (light gray bar and ♦), intranasal (dark gray bar and ▪), and intramuscular (diagonal line bar and ▴) routes and for unvaccinated control rabbits (black bar and ×). The results for high-challenge (A), moderate-challenge (B), and low-challenge (C) CRPV doses are shown. Error bars represent standard errors of the mean.