Intracutaneous DNA Vaccination with the E8 Gene of Cottontail Rabbit Papillomavirus Induces Protective Immunity against Virus Challenge in Rabbits

Establishment of E8 mutant CRPV and DNA challenge in rabbits.

A CRPV genome previously described as Hershey CRPV (H-CRPV) and initially obtained from a Kansas cottontail rabbit (27) was cloned into pUC19 at the SalI site. A SacII restriction site at bp 169 to 174 (A171→G; C174→ G; E6 aa 6 and 7) was engineered into this genome by site-directed mutagenesis (Stratagene, La Jolla, Calif.), following the manufacturer's instructions. We next subcloned the CRPV E6/E8 region from SacII to EcoRI (bp 169 to 1064) into a modified pUC19 vector from which we had deleted the vector EcoRI site. This construct was subjected to site-directed mutagenesis to alter the E8 ATG start codon to ACG, which did not alter the corresponding E6 amino acid at this position. The mutated E6/E8 fragment was isolated by standard molecular biology techniques and replaced into the H-CRPV backbone. The mutation was confirmed by DNA sequence analysis of the entire fragment (bp 169 to 1064) prior to and after reinsertion into the H-CRPV genome. Plasmids containing the CRPV E8 knockout genome and the wild-type (wt) CRPV genome (containing the SacII site) were purified using a Qiagen Maxiprep kit followed by cesium chloride density gradient centrifugation. The final concentration of the plasmids was adjusted to 200 μg/ml in 1× TE buffer. Before DNA challenge, the back skin of the rabbits was pretreated with a mixture of turpentine and acetone (50:50 [vol/vol]) a total of four times, once every other day, to make the skin hyperplastic (13, 28). Several sites on the back were then scarified with a scalpel blade and inoculated with plasmid containing the CRPV genome at a concentration of 10 μg per site.

E8 gene expression vector and gene gun microparticle preparation.

The CRPV E8 gene was amplified by PCR and cloned into the V1Jns expression vector (a generous gift of M. A. Liu, Merck & Co., West Point, Pa.) at the BglII site (17). This plasmid was identified as E8V1Jns. E8V1Jns and V1Jns (empty vector) plasmid DNAs were purified with the Qiagen Maxiprep kit and by cesium chloride density gradient ultracentrifugation and then precipitated onto 1.6-mm-diameter gold microparticles (Bio-Rad, Hercules, Calif.) at a ratio of 1 μg of DNA/0.5 mg of gold particles as described by the manufacturer.

Rabbit vaccination and challenge.

Outbred NZW rabbits were purchased from Covance Research Products Inc. (Denver, Pa.). EIII/JC inbred rabbits were maintained in the animal facility of the Pennsylvania State University College of Medicine. These rabbits were obtained from an inbred audiogenic line of rabbits originally developed at the National Institutes of Health and do not reject skin grafts from inbred cagemates (data not shown). All animal care and handling procedures were approved by the Institutional Animal Care and Use Committee of the Pennsylvania State University. Inner ear skin sites were shaved and swabbed with 70% ethanol, and then the sites were bombarded with DNA-gold particles by a gene gun at 400 lb/in². The animals were immunized with E8V1Jns or V1Jns vector alone a total of three times at 3-week intervals. Each plasmid DNA was applied at a total dose of 20 μg per rabbit for each immunization. Two weeks after the final booster immunization, the rabbits were challenged with infectious CRPV (H-CRPV). All rabbits were challenged at four scarified dorsal sites with 100 μl of virus suspension diluted 1:100 in phosphate-buffered saline (PBS). Papilloma measurements began 3 weeks after virus challenge and continued weekly for 12 weeks.

Peptide synthesis.

Three peptides encoded by the CRPV E8 gene and one peptide encoded by the rabbit oral papillomavirus E5 gene were synthesized and purified by Genemed Co. (South San Francisco, Calif.). Peptide 1 (MGPAETALYC; aa 1 to 10) and peptide 2 (RKYLAGSCVVQFAEEDC; aa 35 to 50) were dissolved in conjugation solution (0.83 M sodium phosphate, 0.9 M NaCl, 0.1 M EDTA, pH 7.2) to make stock solutions (20 mg/ml) and then diluted in sterile PBS prior to injection. The control for peptides 1 and 2 was a peptide (SMGVLECTLGVWEC) encoded by the rabbit oral papillomavirus E5 gene (6); peptide 3 (YCLVLWIFIVTLLLL; aa 9 to 23) was dissolved in dimethyl sulfoxide to make a stock solution (3.6 mg/ml) and then diluted in sterile PBS prior to injection. The control for peptide 3 consisted of dimethyl sulfoxide dissolved in PBS.

Skin test.

The skin test procedure consisted of the intracutaneous injection of 0.03 ml of test solution (10 μg of peptide) into the outer ear skin (22). Pilot studies were conducted to optimize the concentrations and volumes of antigen preparations. Peptide 1, peptide 2, and their control peptide were injected at different sites on one ear, and peptide 3 and its control solution were injected into different sites on the second ear. Elizabethan collars were fitted to all rabbits after ear injections to prevent nonspecific inflammation caused by scratching (22). In order to avoid antigen interference, the test was conducted several weeks after the virus challenge. Ear swelling was monitored over 5 days and measured to the nearest 0.01 mm with a constant-tension thickness gauge. In addition, the intensity of erythema was documented.

Papilloma size determination and statistical analysis.

Papilloma size was determined by calculating the cubic root of the product of length × width × height of individual papillomas in millimeters to obtain a geometric mean diameter (GMD). Data were represented as the means (± standard errors of the mean [SEM]) of the GMDs for each test group. Statistical significance was determined by unpaired t test comparison.

E8 mutant CRPV genomes induce small, slow-growing, but persistent papillomas in NZW rabbits.

Previous studies in our laboratory demonstrated that CRPV-E8 has weak oncogenic activity, analogous to those of several HPV E5 genes (14). To explore the role of E8 in the CRPV virus life cycle, an E8 ATG mutation was engineered into the CRPV genome and tested for infectivity. Five of both outbred and EIII/JC inbred rabbits were challenged with the wt CRPV DNA containing the introduced SacII site, and another five rabbits were challenged with DNA from the CRPV-E8 knockout genome at six dorsal sites. Three weeks after challenge, papilloma sizes were monitored and GMDs were calculated. Papillomas that developed from the wt CRPV DNA were significantly larger than those established from the DNA of the CRPV-E8 knockout genome (P < 0.01) (Fig. 1). Papillomas that developed from the wt CRPV DNA containing the introduced SacII site grew at rates comparable to those from wt H-CRPV DNA (data not shown). We conclude that the CRPV E8 gene plays a role in papilloma growth but is not essential for the formation of papillomas.

E8 immunization promotes papilloma regression in EIII/JC inbred rabbits.

The results of the above-mentioned studies indicated that CRPV E8 plays a functional role in the CRPV life cycle. We next wished to determine whether the E8 protein could represent an immunological target within developing papillomas established from wt CRPV virion and viral DNA infections. To examine the protective effect of E8 immunization, six EIII/JC inbred rabbits were separated into two groups of three rabbits each and vaccinated with E8V1Jns or V1Jns vector, respectively. Two weeks after the final immunizations, the rabbits were challenged with H-CRPV virus at four scarified dorsal sites per animal. Three weeks later, papillomas appeared in both groups. However, all rabbits in the control group developed papillomas at all four challenge sites, and these papillomas persisted throughout the course of the experiment. Of the three E8-vaccinated rabbits, one had two papillomas, the second had three papillomas, and the third had four papillomas. Between weeks 4 and 8, all papillomas on the E8-vaccinated rabbits regressed (Fig. 2 and Table 1). Therefore, E8 vaccination can provide partial protection against virus challenge and trigger regression of papillomas in inbred rabbits.

E8 immunization partially protects against CRPV virus challenge in outbred NZW rabbits.

Outbred rabbits were also immunized with CRPV E8 to determine whether protection could be achieved in outbred populations. One group of five outbred NZW rabbits was intracutaneously immunized with the CRPV E8 gene. A second group of five rabbits received vector alone as a control vaccine. The five control rabbits grew papillomas at all challenge sites, and these papillomas became large and persisted for the duration of the experiment. In contrast, E8-vaccinated rabbits showed strong protective immunity to CRPV infection (Fig. 2). One of the E8-vaccinated rabbits was completely protected against papilloma formation; three rabbits had either one or three tiny papillomas, of which two on two different rabbits regressed; and the remaining rabbit showed persistent papilloma growth (Fig. 2 and Table 1). The total sites protected against CRPV infection by E8 immunization in these rabbits were about 55% (9 of 20 challenge sites did not have papillomas [Table 1]). In addition, papilloma growth on vaccinated rabbits was significantly suppressed for all time points after week 5 compared to growth rates on control-vaccinated rabbits (P < 0.01; t test) (Fig. 2). These observations indicated that strong anti-papillomavirus immunity was induced in the E8-vaccinated rabbits but that it was insufficient to completely protect against infection and/or to eradicate developing papillomas on all outbred NZW rabbits.

E8 vaccination does not protect against E8 mutant (knockout) CRPV genomes.

We tested E8-vaccinated rabbits for protection against infection with E8 knockout CRPV DNA as an additional strategy to support our conclusion that specific immunity to E8 is responsible for the strong protections seen in the previous two experiments. One group of four inbred rabbits was intracutaneously immunized with the CRPV E8 gene. A second group of five rabbits received vector alone as a control vaccine. One week after the final booster immunizations, the back skin of the rabbits was challenged with E8 knockout CRPV DNA at six scarified dorsal sites for the E8-vaccinated rabbits and at four sites for rabbits immunized with vector alone. Small papillomas grew at approximately 70% of the challenge sites in both groups, and none of these papillomas regressed during the course of the experiment (Table 2). In addition, there was no significant difference between the mean papilloma sizes in these two groups (P > 0.05; unpaired t test) (data not shown). The results of these studies showed that E8-vaccinated rabbits were susceptible to infection by the E8 knockout CRPV DNA with no regressions of the lesions. We have demonstrated that rabbits vaccinated with E8 also generate strong responses to CRPV infections initiated with wt CRPV DNA (data not shown).

DTH response.

A second in vivo E8-specific cellular immune response was measured in rabbits by the DTH test. Three different peptides that covered the N- and C-terminal portions of CRPV E8 protein were synthesized and tested for DTH reactions in the ear skin as described in Materials and Methods. Inbred and outbred rabbits were tested at 9 and 3 months after virus challenge, respectively. Peptides 1 and 2 and their control peptide were injected intracutaneously into different sites of one ear, while peptide 3 and its control solution were injected into the second ear. Ear swellings were checked at 24-h intervals for a period of 120 h after peptide injection (Fig. 3).

Among the E8-vaccinated inbred rabbits, one rabbit showed positive skin test reactions to all three E8 peptides. A second vaccinated rabbit had positive reactions to peptides 1 and 2, while the remaining rabbit was reactive to peptide 1. In contrast, none of the rabbits in the control group showed reactivity to any of these E8 peptides (Table 3).

Among the E8-vaccinated outbred rabbits, one rabbit that was completely protected against CRPV infection showed positive DTH reactions to all three peptides. Erythema was seen in another of the E8-vaccinated rabbits, although no obvious swelling was observed. The rabbit that failed to show protection against papilloma growth also showed a positive skin test reaction to peptide 3 (Table 4). The remaining two rabbits in the E8 vaccination group had no DTH responses to the E8 peptides. These observations showed that strong skin test reactions were correlated with complete eradication of papillomas.