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A cottontail rabbit papillomavirus (CRPV) E6 DNA vaccine that induces significant protection against CRPV challenge was used in a superior vaccination regimen in which the cutaneous sites of vaccination were primed with an expression vector encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that induces differentiation and local recruitment of professional antigen-presenting cells. This treatment induced a massive influx of major histocompatibility complex class II-positive cells. In a vaccinationchallenge experiment, rabbit groups were treated by E6 DNA vaccination, GM-CSF DNA inoculation, or a combination of both treatments. After two immunizations, rabbits were challenged with CRPV at low, moderate, and high stringencies and monitored for papilloma formation. As expected, all clinical outcomes were monotonically related to the stringency of the viral challenge. The results demonstrate that GM-CSF priming greatly augmented the effects of CRPV E6 vaccination. First, challenge sites in control rabbits (at the moderate challenge stringency) had a 0% probability of remaining disease free, versus a 50% probability in E6-vaccinated rabbits, and whereas GM-CSF alone had no effect, the interaction between GM-CSF priming and E6 vaccination increased disease-free survival to 67%. Second, the incubation period before papilloma onset was lengthened by E6 DNA vaccination alone or to some extent by GM-CSF DNA inoculation alone, and the combination of treatments induced additive effects. Third, the rate of papilloma growth was reduced by E6 vaccination and, to a lesser extent, by GM-CSF treatment. In addition, the interaction between the E6 and GM-CSF treatments was synergistic and yielded more than a 99% reduction in papilloma volume. Finally, regression occurred among the papillomas that formed in rabbits treated with the E6 vaccine and/or with GM-CSF, with the highest regression frequency occurring in rabbits that received the combination treatment.

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Previously we showed that intracutaneous vaccination of rabbits with DNA vectors encoding ubiquitin-fused versions of the cottontail rabbit papillomavirus (CRPV) early proteins E1, E2, E6 and E7 protected against subsequent challenge with CRPV. Here we tested the immunotherapeutic activity of a vaccine composed of the four CRPV DNA vectors (designated UbE1267) in rabbits. The results show that the UbE1267 DNA vaccine, relative to empty vector DNA, virtually eliminated papilloma growth in rabbits with subclinical infection and greatly reduced papilloma volumes in rabbits bearing papillomas at the time of vaccination. These results in a physiologically relevant animal model of high-risk human papillomavirus (HPV) infection indicate that DNA vaccines targeting the early papillomavirus proteins may have a role in the treatment of HPV-associated lesions in humans.

Cervical cancer arises from lesions caused by infection with high-risk types of human papillomavirus (HPV).Therefore, vaccination against HPV could prevent carcinogenesis by preventing HPV infection or inducing lesion regression. HPV E2 protein is an attractive candidate for vaccine development because it is required for papilloma formation, is involved in all stages of the virus life cycle, and is expressed in all premalignant lesions as well as some cancers. This study reports vaccination against E2 protein using a rabbit model of papillomavirus infection. A recombinant adenovirus (Ad) vector expressing the E2 protein of cottontail rabbit papillomavirus (CRPV) was tested for therapeutic efficacy in CRPV-infected rabbits. Primary immunization with the Ad-E2 vaccine, compared to immunization with a control Ad vector, reduced the number of papillomaforming sites from 17 of 45 to 4 of 45. After booster immunization, vaccinated rabbits formed no new papillomas versus an additional 23 papillomas in rabbits that received the control vector. Papillomas in the Ad-E2 vaccinees were significantly smaller than those in the control rabbits, and all four papillomas in the Ad-E2 vaccinated rabbits regressed. No CRPV DNA was detected either in the regression sites or in sites that did not form papillomas, indicating that the vaccination led to clearance of CRPV from all infected sites.Human papillomaviruses (HPVs) cause cervical cancer, which affects about one-half million women worldwide annually (37, 51, 56). HPV-associated disease includes anal, vulvar (56), oral and other respiratory tract cancers (20), and nearly all skin cancers in patients with epidermodysplasia verruciformis (13). HPV infection is also implicated in nonmelanoma skin cancers in immunocompetent as well as immunodeficient patients (4-6, 13, 26, 29, 40). It has been estimated that 10% of the world's tumor burden is attributable to HPV infection (57).Cervical carcinogenesis begins with benign epithelial lesions induced by HPV. Because genital HPV infection is highly prevalent, many women are at risk (41). Progression to cervical cancer typically takes more than a decade, so cytological screening can detect high-grade lesions in time for treatment. However, present treatments do not cure all lesions in all patients, and recurrence is a common sequela. Furthermore, cervical cancer has a mortality rate of 33%, clearly indicating the need for better therapy.Because premalignant lesions caused by HPV can be detected early, vaccination against HPV antigens could provide an effective therapy to induce lesion regression and prevent cancer (9-12, 45). A therapeutic vaccine could also eliminate residual HPV infection after surgical removal of a lesion (9)(10)(11)(12)45). The viral E6 and E7 oncoproteins are presently popular targets for a therapeutic HPV vaccine. These proteins stimulate cellular proliferation, promote genetic instability, and transform cells, in large part by perturbing the p53 and retinoblastoma tumor suppressor pathways (reviewed in reference 38)...

Persistent infection with high-risk human papillomaviruses (HPVs) is the greatest risk factor for the development of HPV-associated cancers. In this study rabbits bearing persistent and potentially malignant papillomas were used to test the efficacy of vaccination with a recombinant DNA and/or vesicular stomatitis virus (VSV) targeting the cottontail rabbit papillomavirus (CRPV) E6 protein. Immune responses were primed with either vector and boosted twice with the homologous or heterologous E6 vector. Over the course of 18 weeks, E6 vaccination reduced papilloma volumes to one third the volume in the controls, and the rabbits boosted with an heterologous vector tended to mount stronger responses. Small and medium-sized papillomas responded significantly but only slightly better than large papillomas. Finally the initial papilloma burden per rabbit, ranging from <100 mm3 to >1000 mm3, was not prognostic of antitumor efficacy. In summary both E6 vaccines elicited significant therapeutic immunity, and their sequential use tended to be advantageous.

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Sendai virus irradiated by UV light, the dose of which is sufficient for inactivation of infectivity, is capable of inducing synthesis of early protein only. The synthesis of early virus-specific proteins was observed in the absence of virusspecific RNA-synthesis. Consequently, RNA of virion (plus-strand) is the m-RNA for the synthesis of early virus-specific proteins of Sendal virus. Since viral structural proteins do not synthesize under these conditions, it is assumed, that the synthesis of virus capsid proteins is coded by a minus-strand. The possible significance of such processes for an understanding of the nature of viral oncogenesis and stable antiviral immunity is discussed. 21"