Ubiquitin-Fused and/or Multiple Early Genes from Cottontail Rabbit Papillomavirus as DNA Vaccines

Cervical human papillomavirus (HPV) infection is an extremely common sexually transmitted disease that affects an estimated 15% of women in the United States (18). Persistent lesions can be treated, but available treatments do not necessarily prevent recurrence (from latently infected tissues) and reinfection (with the same or other HPV types). Cervical carcinogenesis is initiated by infection with high-risk types of HPV (3, 28, 39), the most common of which are HPV type 16 (HPV-16) and HPV-18. Worldwide, cervical cancer is the second or third most common cancer in women (23). Vaccination against HPV to prevent infection and to treat premalignant disease could substantially decrease morbidity and mortality from cervical cancer. The ideal HPV vaccine would not only prevent primary lesions from forming but also provide therapy for established lesions. While humoral antibodies can prevent infection, only cellular immune responses against the early (intracellular) papillomavirus proteins can mediate both functions, by providing the helper cell activities necessary for proliferation and differentiation of B cells and/or differentiation into cytotoxic T cells (CTLs) as well as by elaborating inhibitory cytokines, thereby participating in the responses that occur during the preclinical stage of infection as well as those that occur after the establishment of lesions. The only small-animal model of papillomavirus infection with long-term persistence and malignant progression of lesions is the cottontail rabbit papillomavirus (CRPV)-domestic rabbit model (4). We previously reported that a DNA vaccine encoding the CRPV early protein E6 induced significant protection against CRPV challenge (34). The E6 vaccine's efficacy was dramatically improved by priming the sites of vaccination with granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that stimulates the local recruitment and maturation of professional antigen-presenting cells (20), which initiate DNA vaccine-induced immune responses (6, 19). Another strategy for enhancing immune responses to vaccination is to increase the efficiency of antigen processing—for example, by using a gene that encodes a ubiquitin-fused version of the target protein (13). Ubiquitinated proteins enter the proteasomal pathway, where they are processed and presented through the major histocompatibility complex (MHC) class I pathway to stimulate differentiation and clonal expansion of MHC class I-restricted, typically CD8+, cytotoxic T cells (16, 29, 42). Rodriguez et al. showed that the fusion of a ubiquitin monomer to the nucleoprotein of lymphocytic choriomeningitis virus led to more rapid and complete degradation (25). When incorporated into a prophylactic vaccine for mice, the ubiquitin-fused nucleoprotein gene dramatically reduced the viral titer and the severity of lymphocytic choriomeningitis virus-induced disease. Similar ubiquitin-based strategies for other pathogens also have been effective (22, 36, 37). A different approach for generating broad immune responses is to vaccinate against multiple target proteins (2), thereby increasing the chances of inducing population-wide immunity in individuals, whose immune responses to different epitopes within a given protein may differ widely. The early papillomavirus genes most likely to be useful for prevention of disease and treatment of low-grade lesions are E1, E2, E6, and E7. Each of these genes performs critical functions for the virus life cycle (30, 38), is expressed in virtually all premalignant lesions (7, 31), and is required for papilloma formation (5, 21, 41). Another potential advantage of targeting the early proteins, particularly E1 and E2, is that they contain highly conserved regions (1), so immunity to an early protein of one HPV type might cross-react with other HPV types. Vaccines targeting E6 and E7 oncoproteins could additionally be used to treat high-grade lesions and cancer (7, 31). The first goal of the present study was to determine whether DNA vaccination with a ubiquitin-fused E6 gene could protect rabbits against papilloma formation following CRPV challenge and whether this strategy would be superior to GM-CSF priming. The second goal was to determine whether DNA vaccines encoding the E1, E2, E6, or E7 protein were capable of inducing prophylactic immunity and whether any combinations of the vaccines would help or hinder the development of protective immunity. Finally, we combined both strategies and tested the efficacy of a combination DNA vaccine encoding ubiquitin-fused versions of the E1, E2, and E7 proteins.