Papillomavirus-like particles (VLPs) are a promising prophylactic vaccine candidate to prevent human papillomavirus (HPV) infections and associated epithelial neoplasia. However, they are unlikely to have therapeutic effects because the virion capsid proteins are not detected in the proliferating cells of the infected epithelia or in cervical carcinomas. To increase the number of viral antigen targets for cell-mediated immune responses in a VLP-based vaccine, we have generated stable chimeric VLPs consisting of the L1 major capsid protein plus the entire E7 (11 kDa) or E2 (43 kDa) nonstructural papillomavirus protein fused to the L2 minor capsid protein. The chimeric VLPs are indistinguishable from the parental VLPs in their morphology and in their ability to agglutinate erythrocytes and elicit high titers of neutralizing antibodies. Protection from tumor challenge was tested in C57BL͞6 mice by using the tumor cell line TC-1, which expresses HPV16 E7, but not the virion structural proteins. Injection of HPV16 L1͞L2-HPV16 E7 chimeric VLPs, but not HPV16 L1͞L2 VLPs, protected the mice from tumor challenge, even in the absence of adjuvant. The chimeric VLPs also induced protection against tumor challenge in major histocompatibility class IIdeficient mice, but not in ␤ 2 -microglobulin or perforin knockout mice implying that protection was mediated by class I-restricted cytotoxic lymphocytes. These findings raise the possibility that VLPs may generally be efficient vehicles for generating cellmediated immune responses and that, specifically, chimeric VLPs containing papillomavirus nonstructural proteins may increase the therapeutic potential of VLP-based prophylactic vaccines in humans.Human papillomaviruses (HPVs) that infect the genital tract are associated with human anogenital tract cancer, particularly cervical cancer (reviewed in ref. 1). HPVs are thought to be the primary causative agent in Ͼ90% of cervical cancers (2), with HPV16 being the type most frequently found in these tumors. Approximately 500,000 women develop cervical cancer each year, and 200,000 women die from it, making this disease the second-most common cause of cancer deaths in women worldwide (3).Significant advances have been made recently in the development of a candidate prophylactic vaccine against papillomavirus infections (reviewed in ref. 4). Expression of the papillomavirus major capsid protein, L1, in eukaryotic cells leads to self-assembly into virus-like particles (VLPs) that are morphologically indistinguishable from native virions and present the conformational epitopes required for the induction of high titer neutralizing antisera (5). L2, the minor capsid protein, coassembles with L1 at a ratio of Ϸ1 L2 molecule to 30 L1 molecules (6). Although L2 presents some epitopes that induce the production of neutralizing antiserum (7), most neutralizing antibodies induced by L1͞L2 VLPs recognize L1 determinants (8). Several studies have shown that L1 and L1͞L2 VLP-based vaccines protect animals against high dose experimental pap...

Synthetic peptides have been used to sensitize target cells and thereby screen for epitopes recognized by T cells. Most epitopes of cytotoxic T lymphocytes can be mimicked by synthetic peptides of 12-15 amino acids. Although in specific cases, truncations of peptides improves sensitization of target cells, no optimum length for binding to major histocompatibility complex (MHC) class I molecules has been defined. We have now analysed synthetic peptide captured by empty MHC class I molecules of the mutant cell line RMA-S. We found that class I molecules preferentially bound short peptides (nine amino acids) and selectively bound these peptides even when they were a minor component in a mixture of longer peptides. These results may help to explain the difference in size restriction of T-cell epitopes between experiments with synthetic peptides and those with naturally processed peptides.

A single injection of HPV16 L1 virus-like particles induced potent CD8-mediated protection from tumor challenge by C3 cells, a line derived from embryonic mouse cells transfected with the HPV16 genome. L1 RNA, but not protein, was detected biochemically in C3 cells. These results indicate that low-level expression of HPV16 L1 can occur in proliferating cells and serve as a tumor vaccine target. Although L1 expression is generally thought to be restricted to terminally differentiated epithelial cells, these results suggest that additional analysis for low-level L1 expression in proliferating cells of HPV-induced lesions is warranted and might help in predicting the clinical potential of HPV L1 virus-like particle-based vaccines.

The antigen processing-defective mutant cell line RMA-S expresses at the cell surface major histocompatibility complex (MHC) class I molecules devoid of peptide that can be efficiently loaded with exogenous immunogenic peptides. We now report that viral peptide-loaded RMA-S cells, unlike parental RMA cells, can induce primary cytotoxic T lymphocyte (CTL) responses in vitro, in a T helper cell-independent fashion. This was shown for an H-2Kb-binding peptide of Sendai virus nucleoprotein and an H-2Db-binding peptide of adenovirus type 5 E1A protein with responding spleen cells of C57BL/6 mice, the strain of origin of RMA and RMA-S cells. Primary Sendai peptide-induced CTL lyse both peptide-loaded and virus-infected cells. Pre-culture of RMA-S cells at low temperature (22 degrees - 26 degrees C), which increases the amount of empty MHC class I molecules at the cell surface, decreases the peptide concentrations required for the induction of primary CTL responses. Primary peptide-specific CTL responses induced by peptide-loaded RMA-S cells are CD4+ cell- and MHC class II+ cell-independent. CTL response induction is blocked by the presence of anti-CD8 monoclonal antibody during culture. Direct peptide binding studies confirm the efficient loading of empty MHC molecules on RMA-S cells with peptide and show 2.5-fold more peptide bound per RMA-S cell compared to RMA cells. An additional factor explaining the difference in primary response induction between RMA and RMA-S cells is related to the CD8 dependence of these responses. MHC class I molecules occupied with irrelevant peptides (a majority present on RMA, largely absent on RMA-S) may interfere in the interaction of the CD8 molecule with relevant MHC/peptide complexes. The results delineate a novel strategy of peptide based in vitro immunization to elicit CD8+ cytotoxic T cell responses.