1993;67:4154C4162. (30). Development and screening of papillomavirus vaccines have been conducted extensively in animal models, such as bovine papillomavirus (BPV) contamination of cattle (3) and cottontail rabbit papillomavirus (CRPV) contamination of domestic rabbits (16). Currently, several strategies have been utilized to develop papillomavirus vaccines. One strategy entails the induction DDX3-IN-1 of neutralizing antibodies by immunization with the viral structural protein L1 or L2, particularly, virus-like particles (VLPs) put together from L1 or L1/L2. Another strategy involves the induction of cell-mediated immunity by papillomavirus early gene/protein-based vaccination. Recently, VLPs made up of L1 and chimeric molecules of L1 and early proteins were used as immunogens. This strategy has been applied to elicit concurrent viral neutralizing antibodies and cell-mediated immunity specific for viral early proteins (11). A number of studies have exhibited that VLP immunization guarded animals against experimental computer virus challenge in the CRPV-infected rabbit model (2, 6, 13), in the BPV-infected cow model (15), and in the canine oral papillomavirus (COPV)-infected beagle doggie model (28). Furthermore, genetic vaccination with CRPV L1 (9, 26) or immunization with CRPV L1 proteins expressed as bacterial fusion proteins (18) also guarded rabbits from viral challenge. Protection has also been achieved by immunization with L2 proteins (5, 10, 19). One caveat of these animal model systems is that protection from natural papillomavirus infection has not been decided. In experimental contamination models, sites to be infected are vigorously scarified or wounded, resulting DDX3-IN-1 in damage to local blood vessels and the release of circulating neutralizing antibodies at the sites of infection. In contrast, natural infection may occur following microtrauma to the epithelium without significant damage to blood vessels and subsequent direct exposure of computer virus Mmp2 to circulating neutralizing antibodies. Thus, circulating neutralizing antibodies may be unable to protect against natural papillomavirus contamination. Protective vaccines targeting DDX3-IN-1 virus-infected epithelium via cell-mediated immunity would overcome these potential limitations. Induction of protective cell-mediated immunity by immunization with papillomavirus early gene/proteins is usually expected to prevent the establishment of new lesions (immunoprophylaxis) as well as to eliminate existing lesions (immunotherapy). However, early studies disclosed variable results. In the CRPV-infected rabbit model, different papillomavirus early antigens and several methods of antigen delivery have been applied in an attempt to elicit protective antipapillomavirus immunity: (i) immunization with bacterial fusion proteins of CRPV E1 and/or E2 (24); (ii) immunization with recombinant expressing CRPV E1 (14); (iii) intracutaneous genetic vaccination of rabbits with CRPV E6 (27); and (iv) intramuscular injection of plasmid DNA encoding CRPV E1, E2, E6, or E7 (12). The immunity so induced stimulated papilloma regression in a portion of vaccinated rabbits (14, 24) and partially guarded rabbits from subsequent virus challenge (27). However, none of these studies revealed total protection. In the BPV-infected cow model, immunization with BPV E6 and E7 proteins delayed papilloma formation, reduced papilloma size, and promoted DDX3-IN-1 papilloma regression but also did not lead to total protection (3, 4). BPV E2 immunizations were ineffective (4). In the present study, we immunized rabbits by gene gun-mediated intracutaneous vaccination with individual CRPV E1, E2, E6, and E7 genes or with a combination of all four genes. We statement that vaccination with the combination of CRPV E1, E2, E6, and E7 genes provided strong and total protection of outbred rabbits against CRPV challenge. Preparation of DNA vectors and gene gun-mediated immunization.CRPV E1, E2, E6, and E7 DNA genes were amplified by PCR and cloned into V1Jns expression vector (generous gift of M. A. Liu, Merck & Co, Westpoint, Pa.) at the test, 0.05; vector group versus E2 group, test, 0.05). Papilloma size per site for E6-vaccinated rabbits was somewhat smaller than that for vector-vaccinated control rabbits, but size differences were not statistically significant at 14 weeks postchallenge with computer virus (Fig. ?(Fig.1).1). E7 vaccination has little effect on.